Browsing by Issue Date, starting with "2020-04"
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- Biofilms and catheter related bloodstream infection: a tale of two kigdomsPublication . Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel; Faria, Isabel; Pessanha, Maria Ana; Verissimo, Cristina; Sabino, Raquel; Rodrigues, Joao; Matias, Rui; Martins, Filomena; Carvalho, Patricia; Gomes, Joao Paulo; Jordão, LuísaBackground: Biofilm-associated infections are a public health concern in the context of healthcare-associated infections (HAI) such as catheter-related bloodstream infections (CRBSI). Here, we studied two top ten CRBS etiological agents, Enterobacter cloacae and Candida parapsilosis, isolated from a patient with CRBSI in order to understand the role played by biofilms on this HAI. Materials/methods: E.cloacae and C.parapsilosis were isolated from CVC and peripheral blood by standard procedures. EUCAST guidelines were followed for antimicrobial susceptibility evaluation. Single and/or mixed biofilms were assembled on different materials in Mueller-Hinton broth with 2% glucose. Biofilm assembly was assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification and to assess microorganisms distribution within the biofilm (3D reconstruction). In addition, Focus Ion Beam (FIB)-SEM was used to assess biofilms assembled on inner and outer surfaces of CVCs and construct tomograms. CVC and hemoculture (HC) isolates were subjected to whole-genome sequencing (WGS). Results: All Enterobacter and Candida isolates were antimicrobial resistant. Of note, E. cloacae-CVC revealed an additional resistance (ceftolozame-tazobactam) in comparison to the HC- isolate. Both microorganisms assembled biofilms on glass, polystyrene and polyurethane. Mixed biofilms were denser when both microorganisms were present from the beginning. Biofilm phenotype was not dependent of biofilm initiation by E.cloacae or C.parapsilosis. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Environmental isolates of the same species were also tested, showing that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), we observed denser mixed biofilms richer in EPS. FIB-SEM preliminary results suggest that biofilms assembled on inner and outer catheter surface might differ on microorganisms’ distribution. WGS confirmed the genetic identity of the CVC/HC pairs while corroborating the virulence potential and antimicrobial resistant character of the CRBSI-driving pathogens. Conclusions: The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Commercial Red Seaweed in Portugal (Gelidium sesquipedale and Pterocladiella capillacea, Florideophyceae): Going beyond a Single-Purpose Product Approach by Valorizing BioactivityPublication . Matos, J.; Gomes, A,; Cardoso, C.; Afonso, c.; Campos, A.M.; Gomes, R.; Falé, P.; Delgado, Inês; coelho, Inês; Castanheira, Isabel; Bandarra, N.M.The red seaweed species Gelidium sesquipedale and Pterocladiella capillacea are commercially explored as one of the main seaweed resources in Portugal. However, they are essentially harvested for extraction of agar, leaving a large biomass share needing an adequate valorization. The two studied red seaweed species were characterized by a large share of saturated fatty acids (SFA) in the vicinity of 60% (of the total FAs). Concerning ω3 highly unsaturated FAs, only EPA reached a significant percentage in P. capillacea and G. sesquipedale, 13.0 ± 0.5% and 7.7 ± 0.1%, respectively. In comparison with other seaweeds, the phenolic content was low for both species and aqueous and ethanolic extracts. The antioxidant activity was also low or even undetected. Regarding anti-inflammatory activity, as measured by inhibition of cyclooxygenase-2, it was not detected in the aqueous extracts of the seaweed, but was significant in the ethanolic extracts, 69 ± 3% and 54 ± 6%, for P. capillacea and G. sesquipedale, respectively. Concerning cytotoxicity, while ethanolic extracts did not cause any detectable cytotoxicity, the biomass and the aqueous extracts reduced HeLa cell viability. Finally, the elemental composition showed differences between the two seaweed species. In particular, G. sesquipedale contained a higher I level than P. capillacea, 807 ± 51 mg/kg dw vs 435 ± 18 mg/kg dw. On the whole, attained results were promising and warrant further study.
- Alergénios e alergias alimentaresPublication . Batista, RitaPalestra sobre alergénios e alergias alimentares, no âmbito de "Processos em Nutrição Ambiente e Saúde".
- Differential gene expression in cells from Fabry and Gaucher diseases: cell reprogramming and culture agingPublication . Amaral, Olga; Santos, Renato; Ribeiro, DiogoGene expression varies deeply, even in the same individual’s cells, depending on stress factors, cell aging, gene variants or gene and cell manipulation In this work, gene expression can be seen to shift in many genes, using fibroblasts from donors with Fabry and Gaucher type 3 genetic diseases Tests were also made to compare the impact of aging cultures Real time qRT PCR protocols for genes of interest were used for expression comparison after cell induction and reprogramming.
- On nontuberculous mycobacteria in human alveolar macrophagesPublication . João, Inês; Borges, Vítor; Sousa, Sara; Gomes, João Paulo; Jordão, LuísaBackground: Despite growing relevance of nontuberculous mycobacteria (NTM) infections, namely pulmonary infections, accurate data on incidence and pathogenesis is lacking. Iintracellular persistence of three NTM species in a human alveolar macrophages model. The contribution of particular innate immune mechanisms and NTM genomes to mycobacteria intracellular fate were investigated. Materials/methods: M.smegmatis mc2155, reference strains (M.avium ATCC25291; M.fortuitum ATCC6841) and clinical isolates (M.avium 60/08; M. fortuitum 747/08) were used. Mycobacteria intracellular persistence was assessed via: 4x104 THP-1 cells platted/well and incubated for 72h with 100nM PMA. Then fresh medium without PMA was added and the cells incubated for further 24h. The cells were infected for 1h (fast) or 3h (slow growers). Intracellular persistence was evaluated by CFU enumeration at different time points. NO production was evaluated using the Griess reagent, phagosome acidification and apoptosis was followed by confocal microscopy. Persistence ability of mycobacteria at different pHs was evaluated using BACTEC-MGIT960. NTMs virulence factors were characterized by whole-genome sequencing.
- Genetic Substrate Reduction Therapy for Mucopolysaccharidoses type III: toward a siRNA-containing nanoparticle targeted to brain cellsPublication . Santos, Juliana Inês; Coutinho, Maria Francisca; Gaspar, Paulo; Prata, Maria João; Alves, SandraThe classical therapeutic approach for LSD, enzyme replacement therapy, would hardly rise as a potentially successful tool to reduce the disease burden in MPS III patients, as it is long known to have no impact on neuropathology. A tempting alternative, however, would be to block substrate accumulation upstream, by decreasing its synthesis. That concept is known as substrate reduction therapy (SRT). Having this in mind, we designed an RNA-based strategy based upon the selective downregulation of one gene involved in the very early stages of the glycosaminoglycans’ (GAG) biosynthethic cascade. Our goal is to promote an effective reduction of the accumulating substrate, ultimately decreasing or delaying MPS’ symptoms. As tools to achieve substrate reduction, we are evaluating a specific type of antisense oligonucleotides, able to trigger a naturally-occurring post-transcriptional gene silencing process called RNA interference: the small interfering RNAs (siRNAs). So far, the obtained results are quite promising with marked decreases of the target mRNA levels in all tested cell lines (MPS IIIA, IIIC and IIID patients’ fibroblasts). Currently, we are evaluating the effect of that decrease on the overall storage of GAGs 7 days post-transfection, also with promising results. Here we present an overview on the current results of this project, while discussing its next steps, namely the development and evaluation of vectors for in vivo delivery. Our goal is to develop targeted stable nucleic acid lipid particles (t-SNALPs) coupled with different ligands, which promote cell uptake of the ‘anti-GAG’ siRNAs in a variety of cells, including neurons.
- Alimentos geneticamente modificadosPublication . Batista, RitaPalestra sobre alimentos geneticamente modificados
- Analytical techniques for multiplex analysis of protein biomarkersPublication . Van Gool, Alain; Corrales, Fernado; Čolović, Mirjana; Krstić, Danijela; Oliver-Martos, Begona; Martínez-Cáceres, Eva; Jakasa, Ivone; Gajski, Goran; Brun, Virginie; Kyriacou, Kyriacos; Burzynska-Pedziwiatr, Izabela; Wozniak, Lucyna Alicja; Nierkens, Stephan; Pascual García, César; Katrlik, Jaroslav; Bojic-Trbojevic, Zanka; Vacek, Jan; Llorente, Alicia; Antohe, Felicia; Suica, Viorel; Suarez, Guillaume; t’Kindt, Ruben; Martin, Petra; Penque, Deborah; Martins, Ines Lanca; Bodoki, Ede; Iacob, Bogdan-Cezar; Aydindogan, Eda; Timur, Suna; Allinson, John; Sutton, Christopher; Luider, Theo; Wittfooth, Saara; Sammar, MareiThe importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research.
- From bench to bioterium and back again: development of a U1snRNA-based therapeutic strategy for mucopolysaccharidosis IIICPublication . Santos, Juliana Inês Pinto; Matos, Liliana; Oliveira, Paula Alexandra; Gonçalves, Mariana; Pires, Maria João; Coutinho, Maria Francisca; Prata, Maria João; Alves, SandraSplicing is an essential cellular process to generate mature transcripts from pre-mRNA. One of the most important factors for mRNA transcription is the U1snRNA, a spliceosomal component that recognizes 5’ splicing donor sites (SDS) at specific regions in pre-mRNA. Splicing mutations represent one of the most frequent (~20%) genetic defects in Mucopolysaccharidosis IIIC (MPS IIIC), a Lysosomal Storage Disorder (LSD) caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. Exon-skipping has been demonstrated as, probably, the most frequent aberrant splicing defect, and occurs due to mutations in the 5’ SDS. Application of modified U1snRNAs to improve recognition of mutated 5’ SDS represent a potential therapeutic strategy to recover the normal splicing process. The c.234+1G>A is a frequent mutation among patients of countries around the Mediterranean basin (Portugal, Spain, Morocco and Tunisia). It’s located in the + 1 position of intron 2 of HGSNAT gene and leads to the skipping of exon 2. We demonstrated in fibroblast cells that a modified U1snRNA vector (comprising exon 1 to exon 3) designed to improve the definition of exon 2 5’ SDS of the HGSNAT can restore the splicing defect caused by the mutation c.234+1G>A (Matos et al., 2014). Our goal is to evaluate in vivo the therapeutic potential of the modified U1snRNA by testing it in mice expressing the human splicing defect.
- Comprehensive clinically oriented workflow for nucleotide level resolution and interpretation in prenatal diagnosis of de novo apparently balanced chromosomal translocations in their genomic landscapePublication . David, Dezső; Freixo, João P.; Fino, Joana; Carvalho, Inês; Marques, Mariana; Cardoso, Manuela; Piña-Aguilar, Raul E.; Morton, Cynthia C.We present a comprehensive clinically oriented workflow for large-insert genome sequencing (liGS)-based nucleotide level resolution and interpretation of de novo (dn) apparently balanced chromosomal abnormalities (BCA) in prenatal diagnosis (PND). Retrospective or concomitant with conventional PND and liGS, molecular and newly developed clinically inspired bioinformatic tools (TAD-GConTool and CNV-ConTool) are applied to analyze and assess the functional and phenotypic outcome of dn structural variants (dnSVs). Retrospective analysis of four phenotype-associated dnSVs identified during conventional PND precisely reveal the genomic elements disrupted by the translocation breakpoints. Identification of autosomal dominant disease due to the disruption of ANKS1B and WDR26 by t(12;17)(q23.1;q21.33)dn and t(1;3)(q24.11;p25.3)dn breakpoints, respectively, substantiated the proposed workflow. We then applied this workflow to two ongoing prenatal cases with apparently balanced dnBCAs: 46,XX,t(16;17)(q24;q21.3)dn referred for increased risk on combined first trimester screening and 46,XY,t(2;19)(p13;q13.1)dn referred due to a previous trisomy 21 pregnancy. Translocation breakpoints in the t(16;17) involve ANKRD11 and WNT3 and disruption of ANKRD11 resulted in KBG syndrome confirmed in postnatal follow-up. Breakpoints in the t(2;19) are within ATP6V1B1 and the 3' UTR of CEP89, and are not interpreted to cause disease. Genotype-phenotype correlation confirms the causative role of WDR26 in the Skraban-Deardorff and 1q41q42 microdeletion phenocopy syndromes, and that disruption of ANKS1B causes ANKS1B haploinsufficiency syndrome. In sum, we show that an liGS-based approach can be realized in PND care providing additional information concerning clinical outcomes of dnBCAs in patients with such rearrangements.