Browsing by Author "Fernandes, Alexandra R."
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- Cation-mediated gelation of the fucose-rich polysaccharide FucoPol: preparation and characterization of hydrogel beads and their cytotoxicity assessmentPublication . Fialho, Letícia; Araújo, Diana; Delgado Alves, Vitor; Roma-Rodrigues, Catarina; Baptista, Pedro V.; Fernandes, Alexandra R.; Freitas, Filomena; Reis, Maria A.M.This study describes for the first time the iron- and copper-mediated gelation of FucoPol, fucoserich bacterial polysaccharide. The ability of FucoPol to gel in the presence of metal cations, including iron(III) and copper(II), was used for the preparation of hydrogel beads. Iron mediated the formation of stable and not cytotoxic gel beads, while copper resulted in fragile and cytotoxic ones. Copper-mediated beads coated with an iron-mediated gel layer were more stable and had reduced cytotoxicity. The resulting polymeric structures had differing morphology, physical properties and cytotoxicity, which support their use in several applications, including biomedicine, agriculture and bioremediation
- Chitin-Glucan complex hydrogels: physical-chemical characterization, stability, in vitro drug permeation, and biological assessment in primary cellsPublication . Araújo, Diana; Rodrigues, Thomas; Roma-Rodrigues, Catarina; Alves, Vitor D.; Fernandes, Alexandra R.; Freitas, FilomenaChitin-glucan complex (CGC) hydrogels were fabricated by coagulation of the biopolymer from an aqueous alkaline solution, and their morphology, swelling behavior, mechanical, rheological, and biological properties were studied. In addition, their in vitro drug loading/release ability and permeation through mimic-skin artificial membranes (Strat-M) were assessed. The CGC hydrogels prepared from 4 and 6 wt% CGC suspensions (Na51*4 and Na51*6 hydrogels, respectively) had polymer contents of 2.40 0.15 and 3.09 0.22 wt%, respectively, and displayed a highly porous microstructure, characterized by compressive moduli of 39.36 and 47.30 kPa and storage moduli of 523.20 and 7012.25 Pa, respectively. Both hydrogels had a spontaneous and almost immediate swelling in aqueous media, and a high-water retention capacity (>80%), after 30 min incubation at 37 C. Nevertheless, the Na51*4 hydrogels had higher fatigue resistance and slightly higher-water retention capacity. These hydrogels were loaded with caffeine, ibuprofen, diclofenac, or salicylic acid, reaching entrapment efficiency values ranging between 13.11 0.49% for caffeine, and 15.15 1.54% for salicylic acid. Similar release profiles in PBS were observed for all tested APIs, comprising an initial fast release followed by a steady slower release. In vitro permeation experiments through Strat-M membranes using Franz diffusion cells showed considerably higher permeation fluxes for caffeine (33.09 g/cm2/h) and salicylic acid (19.53 g/cm2/h), compared to ibuprofen sodium and diclofenac sodium (4.26 and 0.44 g/cm2/h, respectively). Analysis in normal human dermal fibroblasts revealed that CGC hydrogels have no major effects on the viability, migration ability, and morphology of the cells. Given their demonstrated features, CGC hydrogels are very promising structures, displaying tunable physical properties, which support their future development into novel transdermal drug delivery platforms.
- Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samplesPublication . Oliveira, Beatriz B.; Costa, Beatriz; Morão, Barbara; Faias, Sandra; Veigas, Bruno; Pereira, Lucília Pebre; Albuquerque, Cristina; Maio, Rui; Cravo, Marília; Fernandes, Alexandra R.; Baptista, Pedro VianaThe success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor's temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.
- Exploring the multifaceted potential of a peptide fraction derived from Saccharomyces cerevisiae metabolism: antimicrobial, antioxidant, antidiabetic, and anti-inflammatory propertiesPublication . Branco, Patrícia; Maurício, Elisabete Muchagato; Costa, Ana; Ventura, Diogo; Roma-Rodrigues, Catarina; Duarte, Maria Paula; Fernandes, Alexandra R.; Prista, CatarinaThe rising demand for minimally processed, natural, and healthier food products has led to the search for alternative and multifunctional bioactive food components. Therefore, the present study focuses on the functional proprieties of a peptide fraction derived from Saccharomyces cerevisiae metabolism. The antimicrobial activity of the peptide fraction is evaluated against various foodborne pathogens, including Candida albicans, Candida krusei, Escherichia coli, Listeria monocytogenes, and Salmonella sp. The peptide fraction antioxidant properties are assessed using FRAP and DPPH scavenging capacity assays. Furthermore, the peptide fraction’s cytotoxicity is evaluated in colorectal carcinoma and normal colon epithelial cells while its potential as an antidiabetic agent is investigated through -amylase and -glucosidase inhibitory assays. The results demonstrate that the 2–10 kDa peptide fraction exhibits antimicrobial effects against all tested microorganisms, except C. krusei. The minimal inhibitory concentration for E. coli, L. monocytogenes, and Salmonella sp. remains consistently low, at 0.25 mg/mL, while C. albicans requires a higher concentration of 1.0 mg/mL. Furthermore, the peptide fraction displays antioxidant activity, as evidenced by DPPH radical scavenging activity of 81.03%, and FRAP values of 1042.50 32.5 M TE/mL at 1.0 mg/mL. The peptide fraction exhibits no cytotoxicity in both tumor and non-tumoral human cells at a concentration up to 0.3 mg/mL. Moreover, the peptide fraction presents anti-inflammatory activity, significantly reducing the expression of the TNF gene by more than 29.7% in non-stimulated colon cells and by 50% in lipopolysaccharide-stimulated colon cells. It also inhibits the activity of the carbohydrate digestive enzymes -amylase (IC50 of 199.3 0.9 g/mL) and -glucosidase (IC20 of 270.6 6.0 g/mL). Overall, the findings showed that the peptide fraction exhibits antibacterial, antioxidant, anti-inflammatory, and antidiabetic activity. This study represents a step forward in the evaluation of the functional biological properties of S. cerevisiae bioactive peptides.
- Genetic diagnosis of hypertrophic cardiomyopathy using mass spectrometry DNA arrays and high resolution meltingPublication . Santos, Susana; Lança, Vasco; Oliveira, Helena; Branco, Patrícia; Silveira, Leonor; Marques, Vanda; Brito, Dulce; Madeira, Hugo; Bicho, Manuel; Fernandes, Alexandra R.Hypertrophic cardiomyopathy (HCM), a complex myocardial disorder with an autosomal dominant genetic pattern and prevalence of 1:500, is the most frequent cause of sudden death in apparently healthy young people. The benefits of gene-based diagnosis of HCME for both basic research and clinical medicine are limited by the considerable costs of current genetic testing due to the large number of genes and mutations involved in this pathology. However, coupling two high-throughput techniques--mass spectrometry genotyping (MSG) and high resolution melting (HRM)--is an encouraging new strategy for HCM diagnosis. Our aim was to evaluate the diagnostic efficacy of both techniques in this pathology by studying 13 individuals with a clinical phenotype of HCM. Methods: Peripheral blood samples were collected from: (i) seven subjects with a clinical diagnosis of HCM, all bearing known mutations previously identified by dideoxy sequencing and thus being used as blinded samples (sample type 1); (ii) one individual with a clinical diagnosis of HCM negative for mutations after dideoxy sequencing of the five most common HCM genes, MYH7, MYBPC3, TNNI3, TNNT2 and MYL2 (sample type 2); and (iii) five individuals individual with a clinical diagnosis of HCM who had not previously been genetically studied (sample type 3). Results: The 13 samples were analyzed by MSG for 534 known mutations in 32 genes associated with HCM phenotypes and for all coding regions and exon-intron boundaries of the same HCM genes by HRM. The 32 studied genes include the most frequent HCM-associated sarcomere genes, as well as 27 genes with lower reported HCM phenotype association. This coupled genotyping strategy enabled us to identify a c.128delC (p.A43Vfs165) frame-shift mutation in the CSRP3 gene, a gene not usually studied in current HCM genetics. The heterozygous CSRP3 mutation was found in two patients (sample types 2 and 3) aged 50 and 52 years, respectively, both with diffuse left ventricular hypertrophy. Furthermore, this coupled strategy enabled us to find a novel mutation, c.817C>T (p.Arg273Cys), in MYBPC3 in an individual from sample type 3, subsequently confirmed by dideoxy sequencing. This novel mutation in MYBPC3, not present in 200 chromosomes from 200 healthy individuals, affects a codon known to harbor an HCM-causing mutation – p.Arg253His. Conclusion: In conclusion, in the cohort used in this work coupling two technologies, MSG and HRM, with high sensitivity and low false positive results, enabled rapid, innovative and lowcost genotyping of HCM patients, which may in the short term be suitable for accurate genetic diagnosis of HCM.
- High resolution melting : improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohortPublication . Santos, Susana; Marques, Vanda; Pires, Marina; Silveira, Leonor; Oliveira, Helena; Lança, Vasco; Brito, Dulce; Madeira, Hugo; Esteves, J. Fonseca; Freitas, António; Carreira, Isabel M.; Gaspar, Isabel M.; Monteiro, Carolino; Fernandes, Alexandra R.Background: Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology: In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results: HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5’UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions: HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations.