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- Development of an antisense-mediated exon skipping approach as a therapeutic option for the ML II-causing mutation c.3503_3504delTCPublication . Matos, Liliana; Vilela, Regina; Coutinho, Maria Francisca; Gaspar, Paulo; Alves, SandraLysosomal storage disorders (LSDs) are a group of a rare inherited metabolic disorders.
- Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and TreatmentPublication . Isidro, Joana; Mendes, Aristides L.; Serrano, Mónica; Henriques, Adriano O.; Oleastro, MónicaThe use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of Clostridium difficile. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life-cycle of C. difficile involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of C. difficile. Finally, we review recent developments in treatment and prevention of C. difficile infection.
- Ixodes ventalloi: a vector role to be exploredPublication . Santos, A.S.; de Bruin, A.; Veloso, R.; Marques, C.; Pereira da Fonseca, I.; de Sousa, R.; Sprong, H.; Santos-Silva, M.M.Ixodes ventalloi is still a poorly studied tick and its public health relevance largely unknown. In Portugal it is known to share habitats with Ixodes ricinus in mainland littoral areas and to parasitize a variety of wild and domesticated animals and also humans. The potential involvement of I. ventalloi in the natural cycle of tick‐borne agents was first suggested by the detection of a new variant of Anaplasma phagocytophilum in questing and cat‐feeding ticks from Setúbal and Santarém, and is again questioned here. In this study we present the detection of A. phagocytophilum, Candidatus Neoehrlichia mikurensis and Coxiella burnetii in questing I. ventalloi collected from Parque Florestal de Monsanto (PFM), a Lisbon recreational park.
- Whole-genome-based characterization of invasive Haemophilus influenzae isolates from a pre- and post-vaccine era in PortugalPublication . Gonzalez Diaz, Aida; Pinto, Miguel; Bettencourt, Célia; Duarte, Silvia; Marti, Sara; Gomes, João Paulo; Bajanca-Lavado, PaulaIntroduction: Haemophilus influenzae (Hi) is responsible for severe invasive infections in both adults and children. Since the introduction in the year 2000 of the Hib vaccine, the incidence of disease has substantially declined, even though it doesn’t protect against non-typeable Hi (NTHi) isolates. Although not all NTHi are pathogenic, these are known to possess important virulence factors to promote colonization and host cells interactions, ultimately leading to disease. The application of WGS technology allows the uncovering of Hi population structure, including novel insights into its genomic features. Aims: This study aims to fully characterize, by WGS, Hi isolates from a pre- and post-vaccine era, from 1992 to 2015, in Portugal. Materials and Methods: Ninety invasive Hi isolates from the Portuguese NIH collection were selected for WGS. More than half were NTHi (63.3%) and 32.2% of the strains belong to a pre-vaccine era. Genomes were assembled and both sequence type (ST) and serotype were determined by PCR and confirmed in silico. A core-single nucleotide polymorphism-based phylogenetic tree was reconstructed to analyze overall genomic diversity between strains. Strains were further characterized by identifying the presence and genetic profile of genes related to antibiotic resistance and virulence factors, namely genes involved in adherence, host immune evasion, iron acquisition and lipooligosaccharides (LOS). Results: Preliminary results show high ST heterogeneity among NTHi, contrasting with the homogeneity of ST for Hib strains (all ST6, except one). Core-SNP-based analysis revealed that all strains were distinguishable by more than 140,000 single nucleotide variant sites, with a highest genetic diversity observed between NTHi (overall ~35,000 nucleotide differences). Interestingly, although all Hib segregated together, the ST282 Hib strain possessed a distinct genome profile, diverging by ~17,200 nucleotide differences from ST6, while these overall diverged between them by ~2,480. Differential presence of important virulence factors was observed among strains, namely for hia/hsf, hmw1/hmw2, hap and iga, with distinct genomic profiles observed between strains, requiring in-depth analysis. Curiously, 90% of NTHi had the lgtA LOS-coding gene which was absent in all Hib. Additionally, five genes coding for other LOS were found to be simultaneously present or absent among NTHi strains, most belonging to a post-vaccine era, indicating a potential cluster of circulating strains. Conclusions: Overall, we expect that the integrative analysis of all Hi isolates will strengthen the characterization of the genomic features in pre- and post-vaccine era, ultimately contributing to the understanding of the scenario of strains circulating in Portugal throughout more than 20 years.
- A abundância de selenometionina, selenito e selenato em alimentos como consumidosPublication . Sanches, Diana; Gueifão, Sandra; Ventura, Marta; Delgado, Inês; Coelho, Inês; Castanheira, IsabelEste ano (2017), celebra-se o aniversário da descoberta do micronutriente selénio (Se), que foi pela primeira vez identificado pelo químico sueco Jacob Berzelius. Posteriormente, Klaus Schwarz with Foltz provaram que o Se é um nutriente essencial para o normal crescimento e reprodução dos animais e do homem. A maioria das enzimas dependentes de Se, já foram identificadas em várias funções biológicas, tal como regulação da resposta inflamatória, propriedades antioxidantes, através da regulação das espécies reativas de oxigénio (ROS) e do estado redox, e promoção da proliferação/diferenciação das células imunes. A deficiência de Se foi associada, a uma miríade de patologias, sendo de referir o aumento do risco de doenças não transmissíveis como o cancro – incluindo da tiróide – infeções e estados de imunodepressão, infertilidade masculina, diabetes, doenças de Alzheimer e Parkinson, perturbações do humor, doença de Keshan e doença de Kashin-Beck. A carência grave de Se associa-se a disfunção muscular e cardiomiopatia. Estudos no âmbito da relação do Se com o controlo glicémico, Diabetes Mellitus gestacional e hipertrofia adipocitária ainda têm resultados controversos e necessitam de mais estudos para provar a sua correlação. A interdependência entre os teores de selénio presentes nos alimentos e a redução de toxicidade do arsénio são também descritos na literatura em particular nos últimos anos. Segundo o IOM, nos EUA, e outros artigos da literatura referem que a ingestão alimentar recomendada é de 55μg/dia para um adulto. E a dose máxima admissível (UL) é o nível mais alto de ingestão diária de nutrientes suscetível de não representar qualquer risco de efeitos adversos, em quase todos os indivíduos, sendo importante que todos os indivíduos da população sejam aconselhados para não exceder esta dose – 400 μg/dia. Os alimentos são a fonte principal de selénio. Porém a biodisponibilidade deste oligoelemento está dependente da espécie química presente no alimento. Em Portugal existem poucos dados sobre o perfil de selénio nos alimentos. O objetivo deste estudo foi determinar a abundância de selenometionina (SeMet), selenito (Se IV) e selenato (SeVI) em leite e pescado como consumido pelos portugueses. As amostras foram recolhidas seguindo a metodologia adotada no projeto TDS – Exposure. Recorreu-se à técnica hifenada de HPLC-ICP-MS precedida da extração das espécies, por métodos enzimáticos, da matriz alimentar. Os ensaios foram realizados em condições de controlo da qualidade que refletem os requisitos da Norma 17025:2005. A SeMet foi a espécie predominante, em todos os alimentos analisados. No leite foram encontradas ambas as espécies inorgânicas. No pescado o Se (VI) foi a única espécie inorgânica encontrada. Este estudo foi muito importante porque possibilitou conhecer o perfil das espécies de selénio presentes em alimentos como consumidos, sendo um parâmetro que irá permitir estimar o aporte de selénio, com maior rigor. A importância das estratégias analíticas aplicadas para a quantificação e identificação das espécies de selénio serão objeto de estudos futuros.
- Farmacogenética na Hepatite CPublication . Alves, HelenaA prevalência da Hepatite C na população em geral estima-se entre 0,4 % e 1,5% Segundo dados publicados, no final de 2015, 14,4% da população reclusa em Portugal estava infectada com o vírus da Hepatite C. O genótipo IL28B pode ser usado para individualizar estratégias de tratamento, de tal forma que os pacientes que possuem o genótipo IL28B de boa resposta possam ser elegíveis para regimes mais curtos, mais simples ou mais baratos, enquanto que pacientes com má resposta podem exigir uma terapia mais longa e/ou com múltiplas drogas DAAs (direct-acting antiviral), podendo mesmo vir a criar -se algoritmos de tratamento individualizados. A genotipagem da IL28B foi apontada como útil na medicina personalizada do tratamento da Hepatite C Crónica na era dos antivirais de acção directa (DAAs). O genótipo IL28b CT / TT correlaciona-se fortemente com a não resposta do tratamento em doentes infectados com o genótipo 3 de HCV e o genótipo CC de IL28b está associado com uma resposta viral rápida mais elevada. Como objectivo terminal pretende-se contribuir para o melhor tratamento, com o mínimo de gastos ao SNS, no âmbito do protocolo de tratamento entre o Ministério da Saúde e o Ministério da Justiça e ainda estreitar e melhorar a colaboração inter-institucional.
- Genetically modulated Substrate Reduction Therapy for Mucopolysaccharidoses – in vitro studiesPublication . Coutinho, Maria Francisca; Santos, Juliana Inês; Gaspar, Paulo; Alves, SandraMucopolysaccharidoses (MPSs) are caused by dysfunction in enzymes responsible for the intralysosomal degradation of glycosaminoglycans (GAGs). We have designed an RNA-based strategy based upon the selective downregulation of genes involved in the biosynthesis of GAGs, which is currently under evaluation. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPSs’ symptoms. Taking advantage of the RNA interference (RNAi) technology potential, we have designed and assayed specific siRNAs targeting genes on those biosynthetic cascades to decrease the levels of production of each one of the four substrates: dermatan sulphate (DS), heparan sulphate (HS), keratan sulphate (KS), and chondroitin sulphate (CS). MPSs were divided into two major sub-groups: (1) those that accumulate DS/CS and (2) those that accumulate HS. ‘Group 1’ included MPS types I, II, VI and VII, while ‘group 2’ includes the Sanfilippo syndrome, or MPS III, which subdivides into four different diseases: IIIA, IIIB, IIIC and IIID. Proof of principle on the effect of siRNAs targeting CHSY1 and XYLT1 was achieved for two independent control cell lines, with 8-12 fold decreases on the target mRNA levels, after 24h of incubation with concentrations of each siRNA as low as 20nM. Subsequent analysis on the effect of those same siRNAs on patients’ cell lines resulted in significant CHSY1 expression decrease in MPS I/MPS VI cell lines (‘group 1’), as well as that of XYLT1 in MPS IIIA and IIID fibroblasts (‘group 2’). Initial studies evaluated mRNA levels after 24-48h incubation with each siRNA. Even though relevant decreases were observed for all tested cell lines, it became evident that the treatment efficacy may depend on the features of each specific MPS cell line, with some lines requiring higher siRNA concentrations to promote similar inhibition levels. In order to assess the effect of that treatment on substrate reduction, we have used both the routine Alcian blue and a modified, more sensitive 1,9-dimethylmethylene blue assay on the culture media collected after seeding and incubation, at different time points. Nevertheless, the low confluency levels required for siRNA transfection did not allow detection of GAGs excreted to the culture media. Similar problems have been noted by other authors, particularly in small samples, like the ones we used. Thus, we are currently quantifying GAGs’ storage by direct measurement of tissue samples after papain extraction. By doing so, we can access the intralysosomal levels of GAGs instead of their excretion. Here we present an overview of the preliminary results of this project and unveil its next steps towards a full characterization/evaluation of its potential therapeutic effect.
- Ticks collected from humans within the framework of the national vector surveillance program, PortugalPublication . Santos-Silva, M.M.; de Sousa, R.; Lopes de Carvalho, I.; Santos, A.S.; Núncio, M.S.; REVIVE WorkgroupTicks collected from humans within the framework of the Portuguese National Vector Surveillance National Program (REVIVE).
- Regulation of glucose transporters by protein kinases in cancer cellsPublication . Henriques, Andreia; Matos, Paulo; Jordan, PeterBackground: Cancer cells require increased glucose supply to sustain proliferation. One mechanism involves increased expression of glucose transporter (GLUT) genes. But insulin has revealed that protein phosphorylation is another key mechanism in glucose uptake regulation: insulin binding to responsive cells triggers a signalling cascade with phosphorylation of TBC1D4, a negative regulator of endosomal GLUT trafficking, so that more transporters are inserted into the plasma membrane. Previous work from the host lab has identified the family of WNK protein kinases and shown that WNK1 can also phosphorylate TBC1D4 and promote GLUT translocation to the cell surface. Our objective is to understand the contribution of WNK1 to glucose uptake in colorectal cancer cells. Our objective is to understand the contribution of WNK1 to glucose uptake in colorectal cancer cells.
- Adenoids, friends or foes?Publication . Subtil, João; Lavado, Paula; Rodrigues, João; Rodrigues, Lúcia; Nogueira, Isabel; Duarte, Aida; Jordao, LuisaThe main goal of this study is to evaluate the existence of a link between biofilm assembly on adenoids and the incidence of recurrent infections within a paediatric population. Thirty-three different bacterial genera were isolated from 186 samples (nasal/adenoid scrubs and adenoid biopsies) being Haemophilus, Neisseria, Streptococcus and Staphylococcus the most frequent. Biofilms were present in 27.4% of the adenoid samples as assessed by SEM. Since the aim of the study is to assess the role of biofilms on adenoid colonization/ invasion and onset of infection a group of samples harbouring clinically relevant bacteria (H.influenzae, S. aureus, S. pyogenes, S. pneumococcus and Moraxella) both on the adenoid surface and core were selected for further studies. Based on biofilm assembly in vitro, assessed by crystal violet assay, bacteria were classified as weak, moderate and strong biofilm assemblers. No direct relation between the ability to assemble biofilms in vitro and the presence of biofilms on the adenoid (biofilm in vivo) was found. A similar result was obtained for antibiotic susceptibility with the majority of bacteria being antibiotic susceptible independently of its origin (sample with or without biofilms). This result might be explained at least partially, by the nature of the sample since the adenoidectomy can only be performed in individuals without infection. H. influenzae, the most isolated bacterium, is an opportunistic pathogen, highly adapted to colonize the upper respiratory tract and easily progresses to infection, especially in children. For this reason, virulence factors such as the capsular type were investigated by PCR. However, all strains were characterized as non-capsulated, which might explain adenoid colonization and biofilm formation, as have been also described in the literature. Further studies must be performed to validate the thesis that adenoids function as a reservoir of etiologic agents of respiratory and ear infections.