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- Development of a stable melanoma dual reporter cell line expressing Luciferase and GFPPublication . Aguiar, Gonçalo; Torres, Sílvia; Prudêncio, Cristina; Soares, Raquel; Coelho, Pedro; Prudêncio, Cristina; Coelho, PedroMelanoma is the most aggressive and lethal form of skin cancer, with a high risk of metastatic spread. Obesity is recognized as a risk factor for various types of cancer. However, regarding melanoma, this association remains controversial. Obesity might act as a double-edged sword in melanoma, promoting primary tumour growth but at the same time limiting metastatic spread - the "obesity paradox”. Herein, we aimed to create a stable murine B16F10 melanoma cell line expressing both firefly luciferase (Luc) and green fluorescent protein (GFP), which will later be engrafted into diet induced-obesity animal model for future in vivo studies. B16F10-Luc-GFP cells were generated by transfection with premade lentiviral particles, featuring a construct with Luc and GFP under a cytomegalovirus promoter and mediated by a F2A element. The antibiotic selection marker (puromycin) is expressed under a Rous sarcoma virus promoter. Afterwards, the transfected cells were selected with 1 μg/ml of puromycin. The clones with the highest levels of GFP-positive cells and GFP fluorescence were purified by two rounds of cell sorting and submitted to fluorescence and bioluminescence quantification, morphology, injury, BrdU incorporation, 7-AAD, and PI cell cycle assays and compared to the parental cell line. B16F10-Luc-GFP were successfully generated, and both GFP fluorescence and D-luciferin bioluminescence are present and proportional to cell density. As expected, the parental cell line didn’t display GFP or Luc activities. Moreover, transduced cells exhibit similar morphology, motility, proliferation, viability, and cell cycle progression as B16F10 cells. Conclusions: Altogether, the future engraftment of B16F10-LucGFP in obese mice, will improve melanoma research models, enabling the in vivo and ex vivo visualization of primary tumours and metastasis, providing a better understanding of the underlying molecular mechanisms, to clarify the “obesity paradox” in melanoma.
- Creation of a fungal library and screening of antimicrobial and anticancer activityPublication . Ferreira, Diogo; Hermida, Lara Areal; Rocha, Ana Catarina; Baylina, Pilar; Sieiro, Carmen; Fernandes, Rúben; BAYLINA MACHADO, PILARAccording to the World Health Organization, cancer and infectious diseases are two of the most problematic diseases nowadays. Cancer kills 10 million people every year and the emergence of resistance to antitumoral drugs is an important medical challenge. At the same time, antimicrobial resistance (AMR) is also a serious threat to human and environmental health. Besides mortality, AMR burdens healthcare services and dampens medical procedures such as surgeries, cancer treatments and other invasive procedures. The development of new drug therapies to fight drug resistance is essential to contest the rising of resistant bacteria and reduction of the effectiveness of antitumoral drugs. Microorganisms have been a major source for natural compounds throughout the years. Fungi, renowned for their ability to produce an array of broad and diverse secondary metabolites, offer a rich resource for drug discovery. We built a collection of fungal species, isolated from chestnuts, sunflower seeds, and chestnut flour, and explored their extracts for potential antimicrobial and anticancer activity. Fungi cultures for secondary metabolite biosynthesis were done in submerged fermentation in Malt Extract broth for 15 days at 26 °C. Liquid-liquid extraction techniques, with ethyl acetate as a solvent, were applied to obtain crude secondary metabolite extracts. Clinical resistant bacteria, yeasts, and prostate cell lines (human prostate epithelial cells – HpepiC; human caucasian prostate adenocarcinoma cells - PC3) were exposed to fungal extracts at a single concentration of 100 µg/mL. Our results so far show several extracts with antimicrobial and/or anticancer activity without decreasing cell viability of non-tumoral cells, showing their potential as therapeutic drugs without possible secondary effects. Although, more studies should be done, and pending fungal identification will allow us to select which extracts will be further investigated to find if the displayed bioactivity could be happening due to unknown natural compounds
- Avaliação do potencial antimicrobiano de fungos filamentosos: Um estudo promissor na abordagem do pé diabético e da resistência bacterianaPublication . Ferreira, D.; BAYLINA MACHADO, PILAR; Sá, S.; Areal Hermida, L.; Rocha, A. C.; Baylina, Pilar; Fernandes, R.; Sieiro, C.Prevê-se que até 2045, cerca de 700 milhões de pessoas possam ser afetadas pela Diabetes Mellitus (DM). Uma complicação frequente em indivíduos com DM é o pé diabético, que se manifesta por feridas nos pés causadas por danos nos nervos e vasos sanguíneos, resultando frequentemente na amputação dos membros inferiores. Esta situação é agravada por infeções bacterianas, causadas por estirpes resistentes, como Staphylococcus aureus resistente à meticilina (MRSA), Pseudomonas aeruginosa e Klebsiella spp. produtoras de beta-lactamases de espectro estendido (ESBL), dificultando o tratamento destas lesões. A resistência antibiótica, impulsionada pelo uso excessivo e indiscriminado de antibióticos, destaca a necessidade urgente de novos fármacos e terapias mais eficazes. Neste contexto, os fungos apresentam-se como uma fonte promissora de novos agentes antimicrobianos, devido à vasta gama e diversidade de compostos bioativos que conseguem sintetizar.
- Avaliação do potenicial antimicrobiano do extrato de Gnomoniopsis sp. contra agentes infeciosos do pé diabéticoPublication . Rocha, A. C.; Areal Hermida, L.; Baylina, Pilar; Fernandes, R.; Sieiro, C.; BAYLINA MACHADO, PILAREstima-se que até ao ano de 2045, aproximadamente 700 milhões de pessoas sofram de Diabetes mellitus (DM). O pé diabético é uma complicação comum em pacientes com DM e caracteriza-se por lesões nos pés devido a danos nos nervos e vasos sanguíneos, levando muitas vezes à amputação dos membros inferiores. A infeção causada por bactérias resistentes, como Staphyloccus aureus resistente a meticilina (MRSA), Pseudomonas aeruginosa e espécies de Klebsiella beta-lactamases de espetro estendido (ESBL), acentuam a gravidade destas lesões, tornando o seu tratamento mais complexo. A resistência a antibióticos resulta do uso exagerado e indiscriminado de antibióticos e o desenvolvimento de medicamentos inovadores e de terapias mais eficazes é urgente. Assim, os fungos, nomeadamente fungos filamentosos, surgem como um potencial reservatório para novos compostos antimicrobianos, devido à grande quantidade e diversidade de compostos bioativos produzidos por estes organismos.
- The effects of 24-hour sleep deprivation on the human brain: a multimodal neurophysiological approachPublication . Gonçalves, Alice; Pinto, Sara; Ferreira, Simão; Borges, Daniel FilipeIntroduction: Sleep is an important aspect of human health and well-being and influences various physiological and cognitive functions such as learning and attention. On the other hand, sleep deprivation activates the sympathetic nervous system, negatively impacting blood pressure, heart rate, glucose metabolism, cortisol, and hormones. It alters mood, behaviour, and reduces awareness leading to a poor performance, hence the importance of studying the neurophysiological and neurocognitive effects of 24h sleep deprivation. Objective: Our main goal is to investigate the effects of acute sleep deprivation on the cerebral cortex in healthy university students using a multimodal approach and neurocognitive scales. Methods: This study will use a magnetic stimulator to assess neurophysiological changes, while actigraphy will verify sleep deprivation in the intervention group. Neuropsychological assessments include the Trail Making Test (TMT), measuring visual processing speed and cognitive flexibility, the Psychomotor Vigilance Task (PVT), assessing sustained attention through reaction times to unpredictable stimuli and N-Back test measuring working memory by requiring participants to recall objects presented a few steps earlier. EEG data will be recorded using a cap with 32 channels during. This integrated approach provides a comprehensive analysis of the effects of sleep deprivation on cognitive and neurophysiological functions. Expected Results: Hypothetically, there will be a significant decline in cognitive performance, with impairments in key areas such as visual attention, processing speed and cognitive flexibility. These results will likely manifest in longer reaction times, more frequent errors and overall lower task execution.
- Wearable sleep staging technology as an alternative to polysomnography: a systematic review and meta-analysisPublication . Borges, Maria; Pereira, Telmo; Borges, Daniel Filipe; Soares, Joana IsabelIntroduction:Sleep is vital for health as it has regenerative and protective functions. During sleep, there is a cyclicity of different phases that are analysed and classified for clinical purposes using polysomnography (PSG), a costly and technically demanding method. The tremendous growth of sleep medicine, where demand for studies far outstrips supply, opens a window for the development of accurate, low-threshold sleep monitoring solutions that can be self-administered at home and could help avoid these issues of convenience, accessibility and reproducibility. Objective:This study aims to analyse the existing literature on the feasibility of wearable devices as an alternative to PSG for the classification of sleep stages. Methods: The literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). All studies published in English or Portuguese with healthy adults who used wearables to record sleep were included. A meta-analysis was also performed to assess the mean values of the sleep metrics: total sleep time (TST), sleep latency (SL) and wake after sleep onset (WASO), all in minutes and time per stage (in minutes and as relative frequency of TST), as well as sleep efficiency (SE) (in %) and the corresponding statistics between the wearables used and the PSG. Results: Given the high variability of wearables, the analysed metrics whose values were closest to the PSG came from different devices. Nevertheless, the meta-analysis revealed that most wearables tend to overestimate these variables Conclusions: The performance of wearables demonstrates remarkable accuracy in sleep staging, rivalling the gold standard PSG in some variables while providing a more convenient and unobtrusive alternative. This review enriches our global knowledge of sleep measurement and summarizes the limitations that need to be overcome, as informed decision making depends on understanding the different device options, validation contexts and cost implications.
- Potential anticancer activity from food-isolated fungi extractsPublication . Ferreira, Diogo; Rocha, Ana Catarina; Baylina, Pilar; Sieiro, Carmen; Fernandes, RúbenFungal species have demonstrated great potential to produce a wide range of metabolites, including enzymes, antibiotics, and other bioactive compounds with therapeutic interest. Prostate cancer (PCa) is one of the most frequent cancers in men. This type of tumors have high levels of heterogeneity, leading to therapeutic failures and increasing resistance against chemotherapeutic drugs. Hence, is essential to research new therapeutic agents against PCa. Exploring the rich reservoir of fungal diversity, this study aims to uncover bioactive compounds that may serve as valuable candidates for developing novel therapeutics against prostate cancer. Isolation from chestnuts, chestnut flour and sunflower seeds led to the creation of a fungal collection of 165 isolates. Fungi isolates grew in flask cultures for 15 days, and culture broths were extracted with ethyl acetate. Human prostate epithelial cells (HPepiC) and the human prostate cancer cell line (PC3) were exposed to the fungal extracts at a concentration 100 μg/mL, and cell viability was evaluated by MTT assay. Results show that several fungal extracts significantly reduce the viability of tumor cells, with some showing little to no effect on healthy human cells, however, species identification is essential to carry on our studies.
- Antimicrobial activity of food-isolated fungi extractsPublication . Ferreira, Diogo; Areal-Hermida, Lara; Baylina, Pilar; Fernandes, Rúben; Sieiro, CarmenOne major source for drug discovery are microbial metabolites. Fungi, renowned for their ability to produce an array of broad and diverse secondary metabolites, due to their extensive dispersion and diversity, offer a rich resource for drug discovery. Antibiotic resistance is a major concern. Rapid increase of resistant bacteria worldwide, dampens antibiotic efficiency, burdens healthcare services and increase morbidity and mortality. Antibiotic misuse and lack of new drug development are the main responsible for this health crisis. So, the creation of fungal libraries to find and study new compounds is essential to tackle the rising of antimicrobial resistance and continue with industrial efforts of drug discovery and production. Isolation from chestnuts, chestnut flour and sunflower seeds allowed us to obtain a collection of 165 fungal isolates. Bioactivity of fungal extracts were screened against different antibiotic resistant bacteria. Bacteria grown overnight, adjusted to 1.5 x 108 CFU/mL was exposed to fungal extracts, at a concentration of 100 μg/mL for 24 hours and inhibition rates were calculated. Several extracts showed activity against antimicrobial resistant bacteria and further studies should be made in order to find if new molecules could be responsible for our fungi antimicrobial activity.
- Combining natural bile acids with old basic drugs affords new triple stage antimalarial surface-active ionic liquidsPublication . Silva, Ana Teresa; Oliveira, Isabel; Duarte, Denise; Moita, Diana; Prudêncio, Miguel; Nogueira, Fátima; Ferraz, Ricardo; Marques, Eduardo F.; Gomes, PaulaIonic liquids (ILs) are special organic salts that have been gaining momentum in medicinal chemistry. Despite their simple and cost-effective synthesis, ILs offer an easy access to structures of biological interest by combining bioactive molecules with opposite polarities, e.g., via simple ionic pairing of an acid with a base. This makes ILs of special interest for treating malaria. Since this disease is prevalent mainly in low-to-middle income countries, novel chemotherapeutic strategies must be kept affordable. Malaria is caused by Plasmodium parasites, whose complex life cycle includes three developmental stages in the host: the blood stage, the liver stage, and the gametocyte stage. This complexity turns the development of new effective drugs quite difficult, which is aggravated by the fast emergence of drug-resistant strains. This fact has often led to the disuse of several antimalarials, driving the need to find new ones with multiple-stage action. In this context, we have been working on new antimalarial ILs by mixing antimalarial aminoquinolines—chloroquine and primaquine—with natural lipids. Two new families of salts derived from those antimalarial drugs and naturally-occurring bile acids were now produced by acid-base neutralization, and evaluated for their antiplasmodial action. The chloroquine-derived bile salts were found active against all the three stages of parasite development in the host. Their behavior as surface-active ionic liquids (SAILs), i.e. their interfacial and self-aggregation properties, were also investigated, as they may contribute critically to their delivery and therapeutic action.
- Synthesis and physicochemical characterization of antimalarial surface-active ionic liquidsPublication . Silva, Ana Teresa; Oliveira, Isabel; Ferraz, Ricardo; Marques, Eduardo F.; Gomes, PaulaIonic liquids are a particular class of compounds that attract interest in medicinal chemistry due to the simplicity of their preparation. Novel structures with biological activity can be achieved through simple, cost-effective reactions.1 Reusing old ionizable drugs and improving their characteristics can be achieved economically and simply by mixing them with molecules of opposite charge. This approach is attractive for reviving old antimalarials, not only because of the prevalence of malaria in low- to middle-income countries, but also because several of these drugs are associated with malaria parasite resistance. In this context, our work has been focusing on synthesizing ionic liquids with potential antimalarial activity by mixing antimalarial aminoquinolines, specifically chloroquine, and primaquine, with natural lipids.2, 3 More recently, using an acid-base reaction between chloroquine and bile acids (Figure 1), we synthesized surface-active ionic liquids (SAILs), which proved to possess significant antiplasmodial activity in vitro. The presence of an amphiphilic anion in the ionic pair confers surfaceactive and self-aggregation properties to the ionic liquids. The interfacial and aggregations properties of these SAILs have been characterized by surface tension, electric conductivity, dynamic light scattering, and differential scanning microcalorimetry. Moreover, the interactions of SAILs with micelles of the block copolymer F127 have been studied with the aim of designing an efficient, robust, and biocompatible nanocarrier system for the encapsulation and in vivo release of these antimalarial ionic liquids.