DGH - Artigos em revistas internacionais
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- Development of Engineered-U1 snRNA Therapies: Current StatusPublication . Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Matos, Liliana; Alves, SandraSplicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.
- Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step towards Understanding and Treating MucopolysaccharidosesPublication . Carvalho, Sofia; Santos, Juliana Inês; Moreira, Luciana; Gonçalves, Mariana; David, Hugo; Matos, Liliana; Encarnação, Marisa; Alves, Sandra; Coutinho, Maria FranciscaDespite extensive research, the links between the accumulation of glycosaminoglycans (GAGs) and the clinical features seen in patients suffering from various forms of mucopolysaccharidoses (MPSs) have yet to be further elucidated. This is particularly true for the neuropathology of these disorders; the neurological symptoms are currently incurable, even in the cases where a disease-specific therapeutic approach does exist. One of the best ways to get insights on the molecular mechanisms driving that pathogenesis is the analysis of patient-derived cells. Yet, not every patient-derived cell recapitulates relevant disease features. For the neuronopathic forms of MPSs, for example, this is particularly evident because of the obvious inability to access live neurons. This scenario changed significantly with the advent of induced pluripotent stem cell (iPSC) technologies. From then on, a series of differentiation protocols to generate neurons from iPSC was developed and extensively used for disease modeling. Currently, human iPSC and iPSC-derived cell models have been generated for several MPSs and numerous lessons were learnt from their analysis. Here we review most of those studies, not only listing the currently available MPS iPSC lines and their derived models, but also summarizing how they were generated and the major information different groups have gathered from their analyses. Finally, and taking into account that iPSC generation is a laborious/expensive protocol that holds significant limitations, we also hypothesize on a tempting alternative to establish MPS patient-derived neuronal cells in a much more expedite way, by taking advantage of the existence of a population of multipotent stem cells in human dental pulp to establish mixed neuronal and glial cultures.
- Leukocyte Imbalances in Mucopolysaccharidoses PatientsPublication . Lopes, Nuno; Maia, Maria L.; Pereira, Cátia S.; Mondragão-Rodrigues, Inês; Martins, Esmeralda; Ribeiro, Rosa; Gaspar, Ana; Aguiar, Patrício; Garcia, Paula; Cardoso, Maria Teresa; Rodrigues, Esmeralda; Leão-Teles, Elisa; Giugliani, Roberto; Coutinho, Maria F.; Alves, Sandra; Macedo, M. FátimaMucopolysaccharidoses (MPSs) are rare inherited lysosomal storage diseases (LSDs) caused by deficient activity in one of the enzymes responsible for glycosaminoglycans lysosomal degradation. MPS II is caused by pathogenic mutations in the IDS gene, leading to deficient activity of the enzyme iduronate-2-sulfatase, which causes dermatan and heparan sulfate storage in the lysosomes. In MPS VI, there is dermatan sulfate lysosomal accumulation due to pathogenic mutations in the ARSB gene, leading to arylsulfatase B deficiency. Alterations in the immune system of MPS mouse models have already been described, but data concerning MPSs patients is still scarce. Herein, we study different leukocyte populations in MPS II and VI disease patients. MPS VI, but not MPS II patients, have a decrease percentage of natural killer (NK) cells and monocytes when compared with controls. No alterations were identified in the percentage of T, invariant NKT, and B cells in both groups of MPS disease patients. However, we discovered alterations in the naïve versus memory status of both helper and cytotoxic T cells in MPS VI disease patients compared to control group. Indeed, MPS VI disease patients have a higher frequency of naïve T cells and, consequently, lower memory T cell frequency than control subjects. Altogether, these results reveal MPS VI disease-specific alterations in some leukocyte populations, suggesting that the type of substrate accumulated and/or enzyme deficiency in the lysosome may have a particular effect on the normal cellular composition of the immune system.
- From Stress to Sick(le) and Back Again–Oxidative/Antioxidant Mechanisms, Genetic Modulation, and Cerebrovascular Disease in Children with Sickle Cell AnemiaPublication . Silva, Marisa; Faustino, PaulaSickle cell anemia (SCA) is a genetic disease caused by the homozygosity of the HBB:c.20A>T mutation, which results in the production of hemoglobin S (HbS). In hypoxic conditions, HbS suffers autoxidation and polymerizes inside red blood cells, altering their morphology into a sickle shape, with increased rigidity and fragility. This triggers complex pathophysiological mechanisms, including inflammation, cell adhesion, oxidative stress, and vaso-occlusion, along with metabolic alterations and endocrine complications. SCA is phenotypically heterogeneous due to the modulation of both environmental and genetic factors. Pediatric cerebrovascular disease (CVD), namely ischemic stroke and silent cerebral infarctions, is one of the most impactful manifestations. In this review, we highlight the role of oxidative stress in the pathophysiology of pediatric CVD. Since oxidative stress is an interdependent mechanism in vasculopathy, occurring alongside (or as result of) endothelial dysfunction, cell adhesion, inflammation, chronic hemolysis, ischemia-reperfusion injury, and vaso-occlusion, a brief overview of the main mechanisms involved is included. Moreover, the genetic modulation of CVD in SCA is discussed. The knowledge of the intricate network of altered mechanisms in SCA, and how it is affected by different genetic factors, is fundamental for the identification of potential therapeutic targets, drug development, and patient-specific treatment alternatives.
- MicroRNA Profile, Putative Diagnostic Biomarkers and RNA-Based Therapies in the Inherited Lipid Storage Disease Niemann-Pick Type CPublication . Encarnação, Marisa; David, Hugo; Coutinho, Maria Francisca; Moreira, Luciana; Alves, SandraLipids are essential for cellular function and are tightly controlled at the transcriptional and post-transcriptional levels. Dysregulation of these pathways is associated with vascular diseases, diabetes, cancer, and several inherited metabolic disorders. MicroRNAs (miRNAs), in particular, are a family of post-transcriptional gene repressors associated with the regulation of many genes that encode proteins involved in multiple lipid metabolism pathways, thereby influencing their homeostasis. Thus, this class of non-coding RNAs (ncRNAs) has emerged as a promising therapeutic target for the treatment of lipid-related metabolic alterations. Most of these miRNAs act at an intracellular level, but in the past few years, a role for miRNAs as intercellular signaling molecules has also been uncovered since they can be transported in bodily fluids and used as potential biomarkers of lipid metabolic alterations. In this review, we point out the current knowledge on the miRNA signature in a lysosomal storage disorder associated with lipid dysfunction, Niemann-Pick type C, and discuss the potential use of miRNAs as biomarkers and therapeutic targets for RNA-based therapies.
- Challenges in the Definitive Diagnosis of Niemann–Pick Type C—Leaky Variants and Alternative TranscriptsPublication . Encarnação, Marisa; Ribeiro, Isaura; David, Hugo; Coutinho, Maria Francisca; Quelhas, Dulce; Alves, SandraNiemann-Pick type C (NPC, ORPHA: 646) is a neuro-visceral, psychiatric disease caused predominantly by pathogenic variants in the NPC1 gene or seldom in NPC2. The rarity of the disease, and its wide range of clinical phenotypes and ages of onset, turn the diagnosis into a significant challenge. Other than the detailed clinical history, the typical diagnostic work-up for NPC includes the quantification of pathognomonic metabolites. However, the molecular basis diagnosis is still of utmost importance to fully characterize the disorder. Here, the authors provide an overview of splicing variants in the NPC1 and NPC2 genes and propose a new workflow for NPC diagnosis. Splicing variants cover a significant part of the disease-causing variants in NPC. The authors used cDNA analysis to study the impact of such variants, including the collection of data to classify them as leaky or non-leaky pathogenic variants. However, the presence of naturally occurring spliced transcripts can misdiagnose or mask a pathogenic variant and make the analysis even more difficult. Analysis of the NPC1 cDNA in NPC patients in parallel with controls is vital to assess and detect alternatively spliced forms. Moreover, nonsense-mediated mRNA decay (NMD) analysis plays an essential role in evaluating the naturally occurring transcripts during cDNA analysis and distinguishing them from other pathogenic variants' associated transcripts.
- Candida auris in Intensive Care Setting: The First Case Reported in PortugalPublication . Henriques, João; Mixão, Verónica; Cabrita, Joana; Duarte, Tiago Isidoro; Sequeira, Tânia; Cardoso, Sofia; Germano, Nuno; Dias, Liliana; Bento, Luís; Duarte, Sílvia; Veríssimo, Cristina; Gomes, João Paulo; Sabino, RaquelCandida auris is an opportunistic human pathogen that has rapidly spread to multiple countries and continents and has been associated with a high number of nosocomial outbreaks. Herein, we report the first case of C. auris in Portugal, which was associated with a patient transferred from Angola to an ICU in Portugal for liver transplantation after a SARS-CoV-2 infection. C. auris was isolated during the course of bronchoalveolar lavage, and it was subjected to antifungal susceptibility testing and whole-genome sequence analysis. This isolate presents low susceptibility to azoles and belongs to the genetic clade III with a phylogenetic placement close to African isolates. Although clade III has already been reported in Europe, taking into account the patient’s clinical history, we cannot discard the possibility that the patient’s colonization/infection occurred in Angola, prior to admission in the Portuguese hospital. Considering that C. auris is a fungal pathogen referenced by WHO as a critical priority, this case reinforces the need for continuous surveillance in a hospital setting
- Predictive Value for Increased Red Blood Cell Distribution Width in Unprovoked Acute Venous Thromboembolism at the Emergency DepartmentPublication . Febra, Cláudia; Spinu, Verónica; Ferreira, Filipa; Gil, Victor; Maio, Rui; Penque, Deborah; Macedo, AnaAcute venous thromboembolism (VTE) is a common worldwide disease admitted to emergency departments (ED), usually presenting as pulmonary embolism or lower limb deep vein thrombosis (DVT). Due to the lack of typical clinical and biomarker diagnostic features of unprovoked VTE, early identification is challenging and has direct consequences on correct treatment delay. Longitudinal, prospective, observational study. Patients admitted to ED with a suspicion of unprovoked acute VTE between October 2020 and January 2021 were included. Clinical and laboratorial variables were compared between VTE positive and negative diagnoses. Red cell distribution width (RDW) cut point was determinate through a receiver operating characteristic analysis. RDW accuracy, sensitivity, and specificity were calculated. Fifty-eight patients were analyzed. And 82.8% of suspected patients with VTE were diagnosed with an acute thrombotic event confirmed by imaging examination. In patients with VTE, RDW at admission in ED was higher than with other diagnosis, respectively, 14.3% (13.2-15.1) and 13.5% (13.0-13.8). Platelet count was the only additional characteristic that revealed difference between the 2 groups (264×109/L for VTE and 209×109/L for non-VTE). Logistic regression models showed good discriminatory values for RDW≥14%, with an area under the curve (AUC) = 0.685 (95% confidence interval, 0.535-0.834). These findings were more pronounced in isolated DVT, with a sensitivity of 76.9%, specificity 100%, and accuracy 85.7%. Our study demonstrated a significant association between an early high RDW and the diagnosis of acute unprovoked DVT. RDW ≥ 14% has an independent predictor of unprovoked VTE in adult patients
- Genome-Scale Characterization of Mycobacterium abscessus Complex Isolates from PortugalPublication . Carneiro, Sofia; Pinto, Miguel; Silva, Sónia; Santos, Andrea; Rodrigues, Irene; Santos, Daniela; Duarte, Sílvia; Vieira, Luís; Gomes, João Paulo; Macedo, RitaThe Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.
- Epidemiology and genetic diversity of invasive Neisseria meningitidis strains circulating in Portugal from 2003 to 2020Publication . Bettencourt, Célia; Nunes, Alexandra; Nogueira, Paulo; Duarte, Sílvia; Silva, Catarina; Gomes, João Paulo; Simões, Maria JoãoInvasive meningococcal disease (IMD) continues to be a public health problem due to its epidemic potential, affecting mostly children. We aimed to present a detailed description of the epidemiology of IMD in Portugal, including insights into the genetic diversity of Neisseria meningitidis strains. Epidemiological analysis included data from the Portuguese National Reference Laboratory of Neisseria meningitidis during 2003 to 2020. Since 2012, N. meningitidis isolates have also been assessed for their susceptibility to antibiotics and were characterized by whole genome sequencing. During 2003–2020, 1392 confirmed cases of IMD were analyzed. A decrease in the annual incidence rate was observed, ranging from 1.99 (2003) to 0.39 (2020), with an average case fatality rate of 7.1%. Serogroup B was the most frequent (69.7%), followed by serogroups C (9.7%), Y (5.7%), and W (2.6%). Genomic characterization of 329 isolates identified 20 clonal complexes (cc), with the most prevalent belonging to serogroup B cc41/44 (26.3%) and cc213 (16.3%). Isolates belonging to cc11 were predominantly from serogroups W (77.3%) and C (76.5%), whereas cc23 was dominant from serogroup Y (65.7%). Over the past 4 years (2017–2020), we observed an increasing trend of cases assigned to cc213, cc32, and cc11. Regarding antimicrobial susceptibility, all isolates were susceptible to ceftriaxone and 61.8% were penicillin-nonsusceptible, whereas 1.4% and 1.0% were resistant to ciprofloxacin and rifampicin. This is the first detailed study on the epidemiology and genomics of invasive N. meningitidis infections in Portugal, providing relevant data to public health policy makers for a more effective control of this disease.