Applied Molecular Biosciences Unit

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Publications

Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

Publication . 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 Viana

The 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.

2023Journal article

Open access

Exploring the multifaceted potential of a peptide fraction derived from Saccharomyces cerevisiae metabolism: antimicrobial, antioxidant, antidiabetic, and anti-inflammatory properties

Publication . Branco, Patrícia; Maurício, Elisabete Muchagato; Costa, Ana; Ventura, Diogo; Roma-Rodrigues, Catarina; Duarte, Maria Paula; Fernandes, Alexandra R.; Prista, Catarina

The 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.

2023-08Journal article

Open access

Metabolic background, not photosynthetic physiology, determines drought and drought recovery responses in C3 and C2 moricandias

Publication . Pinheiro, Carla; Emiliani, Giovanni; Marino, Giovanni; Fortunato, Ana S.; Haworth, Matthew; De Carlo, Anna; Chaves, Maria Manuela; Loreto, Francesco; Centritto, Mauro

Distinct photosynthetic physiologies are found within the Moricandia genus, both C3-type and C2-type representatives being known. As C2-physiology is an adaptation to drier environments, a study of physiology, biochemistry and transcriptomics was conducted to investigate whether plants with C2-physiology are more tolerant of low water availability and recover better from drought. Our data on Moricandia moricandioides (Mmo, C3), M. arvensis (Mav, C2) and M. suffruticosa (Msu, C2) show that C3 and C2-type Moricandias are metabolically distinct under all conditions tested (well-watered, severe drought, early drought recovery). Photosynthetic activity was found to be largely dependent upon the stomatal opening. The C2-type M. arvensis was able to secure 25–50% of photosynthesis under severe drought as compared to the C3-type M. moricandioides. Nevertheless, the C2-physiology does not seem to play a central role in M. arvensis drought responses and drought recovery. Instead, our biochemical data indicated metabolic differences in carbon and redox-related metabolism under the examined conditions. The cell wall dynamics and glucosinolate metabolism regulations were found to be major discriminators between M. arvensis and M. moricandioides at the transcription level.

2023-02Journal article

Open access

Tackling Humidity with Designer Ionic Liquid-Based Gas Sensing Soft Materials

Publication . Esteves, C.; Palma, S.I.C.J.; Costa, H.M.A.; Alves, C.; Santos, G.M.C.; Ramou, E.; Carvalho, A.L.; Delgado Alves, Vitor; Roque, A.C.A.

Relative humidity is simultaneously a sensing target and a contaminant in gas and volatile organic compound (VOC) sensing systems, where strategies to control humidity interference are required. An unmet challenge is the creation of gas-sensitive materials where the response to humidity is controlled by the material itself. Here, humidity effects are controlled through the design of gelatin formulations in ionic liquids without and with liquid crystals as electrical and optical sensors, respectively. In this design, the anions [DCA]− and [Cl]− of room temperature ionic liquids from the 1-butyl-3-methylimidazolium family tailor the response to humidity and, subsequently, sensing of VOCs in dry and humid conditions. Due to the combined effect of the materials formulations and sensing mechanisms, changing the anion from [DCA]− to the much more hygroscopic [Cl]−, leads to stronger electrical responses and much weaker optical responses to humidity. Thus, either humidity sensors or humidity-tolerant VOC sensors that do not require sample preconditioning or signal processing to correct humidity impact are obtained. With the wide spread of 3D- and 4D-printing and intelligent devices, the monitoring and tuning of humidity in sustainable biobased materials offers excellent opportunities in e-nose sensing arrays and wearable devices compatible with operation at room conditions

2022Journal article

Open access

Novel Hydrogel Membranes Based on the Bacterial Polysaccharide FucoPol: Design, Characterization and Biological Properties

Publication . Araújo, Diana; Martins, Matilde; Concórdio-Reis, Patrícia; Roma-Rodrigues, Catarina; Morais, Maria; Delgado Alves, Vitor; Fernandes, Alexandra; Freitas, Filomena

FucoPol, a fucose-rich polyanionic polysaccharide, was used for the first time for the preparation of hydrogel membranes (HMs) using Fe3+ as a crosslinking agent. This study evaluated the impact of Fe3+ and FucoPol concentrations on the HMs’ strength. The results show that, above 1.5 g/L, Fe3+ concentration had a limited influence on the HMs’ strength, and varying the FucoPol concentration had a more significant effect. Three different FucoPol concentrations (1.0, 1.75 and 2.5 wt.%) were combined with Fe3+ (1.5 g/L), resulting in HMs with a water content above 97 wt.% and an Fe3+ content up to 0.16 wt.%. HMs with lower FucoPol content exhibited a denser porous microstructure as the polymer concentration increased. Moreover, the low polymer content HM presented the highest swelling ratio (22.3 1.8 g/g) and a lower hardness value (32.4 5.8 kPa). However, improved mechanical properties (221.9 10.2 kPa) along with a decrease in the swelling ratio (11.9 1.6 g/g) were obtained for HMs with a higher polymer content. Furthermore, all HMs were non-cytotoxic and revealed anti-inflammatory activity. The incorporation of FucoPol as a structuring agent and bioactive ingredient in the development of HMs opens up new possibilities for its use in tissue engineering, drug delivery and wound care management.

2023-07Journal article

Open access