FCT: DCV - Artigos em revista internacional com arbitragem científica
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- Isothermal Amplification of Nucleic AcidsPublication . Oliveira, Beatriz B.; Veigas, Bruno; Baptista, Pedro Viana; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Frontiers MediaNucleic acid amplification technologies (NAATs) have become fundamental tools in molecular diagnostics, due to their ability to detect small amounts of target molecules. Since its development, Polymerase Chain Reaction (PCR) has been the most exploited method, being stablished as the “gold standard” technique for DNA amplification. However, the requirement for different working temperatures leads to the need of a thermocycler machine or complex thermal apparatus, which have been preventing its application in novel integrated devices for single workflow and high throughput analysis. Conversely, isothermal amplification methods have been gaining attention, especially for point-of-care diagnosis and applications. These non-PCR based methods have been developed by mimicking the in vivo amplification mechanisms, while performing the amplification with high sensitivity, selectivity and allowing for high-throughput analysis. These favorable capabilities have pushed forward the implementation and commercialization of several platforms that exploit isothermal amplification methods, mostly against virus, bacteria and other pathogens in water, food, environmental and clinical samples. Nevertheless, the future of isothermal amplification methods is still dependent on achieving technical maturity and broader commercialization of enzymes and reagents.
- Identification of homologs of the Chlamydia trachomatis effector CteG reveals a family of Chlamydiaceae type III secreted proteins that can be delivered into host cellsPublication . Pereira, Inês Serrano; da Cunha, Maria; Leal, Inês Pacheco; Luís, Maria Pequito; Gonçalves, Paula; Gonçalves, Carla; Mota, Luís Jaime; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Springer VerlagChlamydiae are a large group of obligate endosymbionts of eukaryotes that includes the Chlamydiaceae family, comprising several animal pathogens. Among Chlamydiaceae, Chlamydia trachomatis causes widespread ocular and urogenital infections in humans. Like many bacterial pathogens, all Chlamydiae manipulate host cells by injecting them with type III secretion effector proteins. We previously characterized the C. trachomatis effector CteG, which localizes at the host cell Golgi and plasma membrane during distinct phases of the chlamydial infectious cycle. Here, we show that CteG is a Chlamydiaceae-specific effector with over 60 homologs phylogenetically categorized into two distinct clades (CteG I and CteG II) and exhibiting several inparalogs and outparalogs. Notably, cteG I homologs are syntenic to C. trachomatis cteG, whereas cteG II homologs are syntenic among themselves but not with C. trachomatis cteG. This indicates a complex evolution of cteG homologs, which is unique among C. trachomatis effectors, marked by numerous events of gene duplication and loss. Despite relatively modest sequence conservation, nearly all tested CteG I and CteG II proteins were identified as type III secretion substrates using Yersinia as a heterologous bacterial host. Moreover, most of the type III secreted CteG I and CteG II homologs were delivered by C. trachomatis into host cells, where they localized at the Golgi region and cell periphery. Overall, this provided insights into the evolution of bacterial effectors and revealed a Chlamydiaceae family of type III secreted proteins that underwent substantial divergence during evolution while conserving the capacity to localize at specific host cell compartments.
- Detecting mir-155-3p through a Molecular Beacon Bead-Based AssayPublication . Moreira, David; Alexandre, Daniela; Miranda, André; Lourenço, Pedro; Baptista, Pedro V.; Tomaz, Cândida; Lu, Yi; Cruz, Carla; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; MDPI - Multidisciplinary Digital Publishing InstituteLung cancer (LC) is recognized as one of the most prevalent and lethal cancers worldwide, underscoring an urgent need for innovative diagnostic and therapeutic approaches. MicroRNAs (miRNAs) have emerged as promising biomarkers for several diseases and their progression, such as LC. However, traditional methods for detecting and quantifying miRNAs, such as PCR, are time-consuming and expensive. Herein, we used a molecular beacon (MB) bead-based assay immobilized in a microfluidic device to detect miR-155-3p, which is frequently overexpressed in LC. The assay relies on the fluorescence enhancement of the MB upon binding to the target miRNA via Watson and Crick complementarity, resulting in a conformational change from a stem–loop to a linear structure, thereby bringing apart the fluorophores at each end. This assay was performed on a microfluidic platform enabling rapid and straightforward target detection. We successfully detected miR-155-3p in a saline solution, obtaining a limit of detection (LOD) of 42 nM. Furthermore, we evaluated the method’s performance in more complex biological samples, including A549 cells’ total RNA and peripheral blood mononuclear cells (PBMCs) spiked with the target miRNA. We achieved satisfactory recovery rates, especially in A549 cells’ total RNA.
- Membrane-localized magnetic hyperthermia promotes intracellular delivery of cell-impermeant probesPublication . Idiago-López, Javier; Ferreira, Daniela; Asín, Laura; Moros, María; Armenia, Ilaria; Grazú, Valeria; Fernandes, Alexandra R.; de la Fuente, Jesús M.; Baptista, Pedro V.; Fratila, Raluca M.; DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; RSC - Royal Society of ChemistryIn this work, we report the disruptive use of membrane-localized magnetic hyperthermia to promote the internalization of cell-impermeant probes. Under an alternating magnetic field, magnetic nanoparticles (MNPs) immobilized on the cell membrane via bioorthogonal click chemistry act as nanoheaters and lead to the thermal disruption of the plasma membrane, which can be used for internalization of different types of molecules, such as small fluorescent probes and nucleic acids. Noteworthily, no cell death, oxidative stress and alterations of the cell cycle are detected after the thermal stimulus, although cells are able to sense and respond to the thermal stimulus through the expression of different types of heat shock proteins (HSPs). Finally, we demonstrate the utility of this approach for the transfection of cells with a small interference RNA (siRNA), revealing a similar efficacy to a standard transfection method based on the use of cationic lipid-based reagents (such as Lipofectamine), but with lower cell toxicity. These results open the possibility of developing new procedures for “opening and closing” cellular membranes with minimal disturbance of cellular integrity. This on-demand modification of cell membrane permeability could allow the direct intracellular delivery of biologically relevant (bio)molecules, drugs and nanomaterials, thus overcoming traditional endocytosis pathways and avoiding endosomal entrapment.
- SRRM2 splicing factor modulates cell fate in early developmentPublication . Carvalho, Silvia; Zea-Redondo, Luna; Tang, Tsz Ching Chloe; Stachel-Braum, Philipp; Miller, Duncan; Caldas, Paulo; Kukalev, Alexander; Diecke, Sebastian; Grosswendt, Stefanie; Grosso, Ana Rita; Pombo, Ana; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Company of BiologistsEmbryo development is an orchestrated process that relies on tight regulation of gene expression to guide cell differentiation and fate decisions. The Srrm2 splicing factor has recently been implicated in developmental disorders and diseases, but its role in early mammalian development remains unexplored. Here, we show that Srrm2 dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness, characterised by the coexistence of cells expressing naive and formative pluripotency markers, together with extensive changes in gene expression, including genes regulated by serum-response transcription factor (SRF) and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells shows that the earliest effects of Srrm2 heterozygosity are specific alternative splicing events on a small number of genes, followed by expression changes in metabolism and differentiation-related genes. Our findings unveil molecular and cellular roles of Srrm2 in stemness and lineage commitment, shedding light on the roles of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.
- Primary Metabolism Is Distinctly Modulated by Plant Resistance Inducers in Coffea arabica Leaves Infected by Hemileia vastatrixPublication . Possa, Kátia Ferreira; Silva, Joyce Alves Goulart; Resende, Mário Lúcio Vilela; Tenente, Rita; Pinheiro, Carla; Chaves, Inês; Planchon, Sebastien; Monteiro, Ana Cristina Andrade; Renaut, Jenny; Carvalho, Milene Alves Figueiredo; Ricardo, Cândido Pinto; Guerra-Guimarães, Leonor; DCV - Departamento de Ciências da Vida; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Frontiers MediaEpidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.
- Microevolution of a Mycobacteroides abscessus subsp. bolletii strain in a clinical persistent infectionPublication . Santos, Andrea; Pinto, Miguel; Carneiro, Sofia; Silva, Sónia; Rodrigues, Irene; Munhá, João; Gomes, João Paulo; Macedo, Rita; DCV - Departamento de Ciências da Vida; ElsevierMycobacteroides abscessus complex (MAB), a fast-growing nontuberculous mycobacterium, is emerging as a significant infectious disease threat, due to both intrinsic and acquired resistance mechanisms to antibiotics and disinfectants and the need for extensive and multidrug regimens for treatment. Despite the prolonged regimens, outcomes are poor and persistence cases have been reported. Here, we describe clinical, microbiologic and genomic features of a M. abscessus subsp. bolletii (M. bolletii) strain consecutively isolated from a patient within an eight-year infection period. From April 2014 to September 2021, the National Reference Laboratory for Mycobacteria received eight strains isolated from a male patient. Species identification, molecular resistance profile and phenotypic drug susceptibility were determined. Five of these isolates were recovered for further in-depth genomic analysis. Genomic analysis confirmed the multidrug resistant pattern of the strain and also other genetic changes associated with adaptation to environment and defence mechanisms. We highlight the identification of new mutations in locus MAB_1881c and in locus MAB_4099c (mps1 gene), already described as associated with macrolides resistance and morphotype switching, respectively. Additionally, we also observed the emergence and fixation of a mutation in locus MAB_0364c that appeared at a frequency of 36% for the 2014 isolate, 57% for the 2015 isolate and 100% for the 2017 and 2021 isolates, clearly illustrating a fixation process underlying a microevolution of the MAB strain within the patient. Altogether these results suggest that the observed genetic alterations are a reflection of the bacterial population's continuous adaptation and survival to the host environment during infection, contributing to persistence and treatment failure.
- Exploring the Multifaceted Potential of a Peptide Fraction Derived from Saccharomyces cerevisiae MetabolismPublication . Branco, Patrícia; Maurício, Elisabete Muchagato; Costa, Ana; Ventura, Diogo; Roma-Rodrigues, Catarina; Duarte, Maria Paula; Fernandes, Alexandra R.; Prista, Catarina; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; DQ - Departamento de Química; MEtRICS - Centro de Engenharia Mecânica e Sustentabilidade de Recursos; MDPI - Multidisciplinary Digital Publishing InstituteThe 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 α-amylase and α-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 µM 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 TNFα 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 α-amylase (IC50 of 199.3 ± 0.9 µg/mL) and α-glucosidase (IC20 of 270.6 ± 6.0 µg/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.
- A Participatory Framework for Plain Language Clinical Management Guideline DevelopmentPublication . Francisco, Rita; Alves, Susana; Gomes, Catarina; Granjo, Pedro; Pascoal, Carlota; Brasil, Sandra; Neves, Alice; Santos, Inês; Miller, Andrea; Krasnewich, Donna; Morava, Eva; Lam, Christina; Jaeken, Jaak; Videira, Paula A.; dos Reis Ferreira, Vanessa; UCIBIO - Applied Molecular Biosciences Unit; DCV - Departamento de Ciências da Vida; Molecular Diversity Preservation International (MDPI)Background: Clinical management guidelines (CMGs) are decision support tools for patient care used by professionals, patients, and family caregivers. Since clinical experts develop numerous CMGs, their technical language hinders comprehension and access by nonmedical stakeholders. Additionally, the views of affected individuals and their families are often not incorporated into treatment guidelines. We developed an adequate methodology for addressing the needs and preferences of family and professional stakeholders regarding CMGs, a recently developed protocol for managing congenital disorders of glycosylation (CDG), a family of rare metabolic diseases. We used the CDG community and phosphomannomutase 2 (PMM2)-CDG CMGs as a pilot to test and implement our methodology. Results: We listened to 89 PMM2-CDG families and 35 professional stakeholders and quantified their CMG-related needs and preferences through an electronic questionnaire. Most families and professionals rated CMGs as relevant (86.5% and 94.3%, respectively), and valuable (84.3% and 94.3%, respectively) in CDG management. The most identified challenges were the lack of CMG awareness (50.6% of families) and the lack of plain language CMG (39.3% of professionals). Concordantly, among families, the most suggested solution was involving them in CMG development (55.1%), while professionals proposed adapting CMGs to include plain language (71.4%). Based on these results, a participatory framework built upon health literacy principles was created to improve CMG comprehension and accessibility. The outputs are six complementary CMG-related resources differentially adapted to the CDG community’s needs and preferences, with a plain language PMM2-CDG CMG as the primary outcome. Additionally, the participants established a distribution plan to ensure wider access to all resources. Conclusions: This empowering, people-centric methodology accelerates CMG development and accessibility to all stakeholders, ultimately improving the quality of life of individuals living with a specific condition and raising the possibility of application to other clinical guidelines.
- A Community-Based Participatory Framework to Co-Develop Patient Education Materials (PEMs) for Rare DiseasesPublication . Falcão, Marta; Allocca, Mariateresa; Rodrigues, Ana Sofia; Granjo, Pedro; Francisco, Rita; Pascoal, Carlota; Rossi, Maria Grazia; Marques-da-Silva, Dorinda; Magrinho, Salvador C. M.; Jaeken, Jaak; Castro, Larisa Aragon; de Freitas, Cláudia; Videira, Paula A.; de Andrés-Aguayo, Luísa; dos Reis Ferreira, Vanessa; Instituto de Higiene e Medicina Tropical (IHMT); DCV - Departamento de Ciências da Vida; UCIBIO - Applied Molecular Biosciences Unit; Instituto de Filosofia da NOVA (IFILNOVA); LAQV@REQUIMTE; DQ - Departamento de Química; Molecular Diversity Preservation International (MDPI)At least 50% of chronic disease patients don’t follow their care plans, leading to lower health outcomes and higher medical costs. Providing Patient Education Materials (PEMs) to individuals living with a disease can help to overcome these problems. PEMs are especially beneficial for people suffering from multisystemic and underrecognized diseases, such as rare diseases. Congenital disorders of glycosylation (CDG) are ultra-rare diseases, where a need was identified for PEMs in plain language that can clearly explain complex information. Community involvement in the design of PEMs is extremely important for diseases whose needs are underserved, such as rare diseases; however, attempts to involve lay and professional stakeholders are lacking. This paper presents a community-based participatory framework to co-create PEMs for CDG, that is transferable to other diseases. A literature review and questionnaire were performed, and only four articles describing the development of PEMS for rare diseases have been found, which demonstrates a lack of standardized approaches. The framework and PEMs were co-developed with CDG families and will be crucial in increasing health literacy and empowering families. We will close a gap in the creation of PEMs for CDG by delivering these resources in lay language in several languages.