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- Unzippers, resolvers and sensors: a structural and functional biochemistry tale of RNA helicasesPublication . Leitão, Ana; Costa, Marina C.; Enguita, Francisco J.The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.
- The circulating non-coding RNA landscape for biomarker research : lessons and prospects from cardiovascular diseasesPublication . Stępień, Ewa; Costa, Marina C.; Kurc, Szczepan; Drożdż, Anna; Cortez-Dias, Nuno; Enguita, Francisco J.Pervasive transcription of the human genome is responsible for the production of a myriad of non-coding RNA molecules (ncRNAs) some of them with regulatory functions. The pivotal role of ncRNAs in cardiovascular biology has been unveiled in the last decade, starting from the characterization of the involvement of micro-RNAs in cardiovascular development and function, and followed by the use of circulating ncRNAs as biomarkers of cardiovascular diseases. The human non-coding secretome is composed by several RNA species that circulate in body fluids and could be used as biomarkers for diagnosis and outcome prediction. In cardiovascular diseases, secreted ncRNAs have been described as biomarkers of several conditions including myocardial infarction, cardiac failure, and atrial fibrillation. Among circulating ncRNAs, micro-RNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have been proposed as biomarkers in different cardiovascular diseases. In comparison with standard biomarkers, the biochemical nature of ncRNAs offers better stability and flexible storage conditions of the samples, and increased sensitivity and specificity. In this review we describe the current trends and future prospects of the use of the ncRNA secretome components as biomarkers of cardiovascular diseases, including the opening questions related with their secretion mechanisms and regulatory actions.
- Transcriptomic crosstalk between fungal invasive pathogens and their host cells: opportunities and challenges for next-generation sequencing methodsPublication . Enguita, Francisco J.; Costa, Marina C.; Fusco-Almeida, Ana; Mendes-Giannini, Maria; Leitão, AnaFungal invasive infections are an increasing health problem. The intrinsic complexity of pathogenic fungi and the unmet clinical need for new and more effective treatments requires a detailed knowledge of the infection process. During infection, fungal pathogens are able to trigger a specific transcriptional program in their host cells. The detailed knowledge of this transcriptional program will allow for a better understanding of the infection process and consequently will help in the future design of more efficient therapeutic strategies. Simultaneous transcriptomic studies of pathogen and host by high-throughput sequencing (dual RNA-seq) is an unbiased protocol to understand the intricate regulatory networks underlying the infectious process. This protocol is starting to be applied to the study of the interactions between fungal pathogens and their hosts. To date, our knowledge of the molecular basis of infection for fungal pathogens is still very limited, and the putative role of regulatory players such as non-coding RNAs or epigenetic factors remains elusive. The wider application of high-throughput transcriptomics in the near future will help to understand the fungal mechanisms for colonization and survival, as well as to characterize the molecular responses of the host cell against a fungal infection.
- Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesisPublication . Ribeiro-Rodrigues, Teresa M.; Laundos, Tiago L.; Pereira-Carvalho, Rita; Batista-Almeida, Daniela; Pereira, Ricardo; Coelho-Santos, Vanessa; Silva, Ana P.; Fernandes, Rosa; Zuzarte, Monica; Enguita, Francisco J.; Costa, Marina C.; Pinto-do-Ó, Perpetua; Pinto, Marta T.; Gouveia, Pedro; Ferreira, Lino; Mason, Justin C.; Pereira, Paulo; Kwak, Brenda R.; Nascimento, Diana S.; Girão, HenriqueAims: Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs). Methods and results: Exosomes secreted by H9c2 myocardial cells and primary cardiomyocytes, cultured either in control or ischaemic conditions were isolated and added to ECs. We show that ischaemic exosomes, in comparison with control exosomes, confer protection against oxidative-induced lesion, promote proliferation, and sprouting of ECs, stimulate the formation of capillary-like structures and strengthen adhesion complexes and barrier properties. Moreover, ischaemic exosomes display higher levels of metalloproteases (MMP) and promote the secretion of MMP by ECs. We demonstrate that miR-222 and miR-143, the relatively most abundant miRs in ischaemic exosomes, partially recapitulate the angiogenic effect of exosomes. Additionally, we show that ischaemic exosomes stimulate the formation of new functional vessels in vivo using in ovo and Matrigel plug assays. Finally, we demonstrate that intramyocardial delivery of ischaemic exosomes improves neovascularization following MI. Conclusions: This study establishes that exosomes secreted by cardiomyocytes under ischaemic conditions promote heart angiogenesis, which may pave the way towards the development of add-on therapies to enhance myocardial blood supply.
- Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolitesPublication . Leitão, Ana Lúcia; Costa, Marina C.; Enguita, Francisco J.Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity.
- circRNA-miRNA cross-talk in the transition from paroxysmal to permanent atrial fibrillationPublication . Costa, Marina C.; Cortez-Dias, Nuno; Gabriel, André F.; De Sousa, João; Fiuza, Manuela; Gallego, Javier; Nobre, Ângelo; Pinto, Fausto J.; Enguita, Francisco J.Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in western countries. The factors governing the progression of AF to a permanent chronic condition are still not well characterized. Among epigenetic factors, non-coding RNAs (ncRNAs) such as miRNAs and lncRNAs have been recently described as important players involved in the AF progression. We hypothesize about the existence of additional regulatory layers in AF involving an intricate cross-talk between different ncRNA species, namely miRNAs and circRNAs for the establishment of a chronic AF condition. Methods and results: We have performed an unbiased study analyzing the expression profile for miRNAs and circRNAs in left-atrial biopsies from patients with paroxysmal and permanent AF by RNA-seq. The transition from paroxysmal to permanent AF is characterized by a pattern of down-regulated miRNAs, concomitant to the appearance of specific circRNA species. The analysis of the sponging activities of the circRNAs exclusively expressed in permanent AF samples, allowed us to determine that they could be responsible for the downregulation of specific miRNAs in establishment of a permanent AF condition. Conclusion: Sponging activity of circRNAs sequestering specific miRNAs is an important factor to be considered for the determination of the molecular mechanisms involved in AF progression.
- miRNAtools: advanced training using the miRNA web of knowledgePublication . Stępień, Ewa; Costa, Marina C.; Enguita, Francisco J.Micro-RNAs (miRNAs) are small non-coding RNAs that act as negative regulators of the genomic output. Their intrinsic importance within cell biology and human disease is well known. Their mechanism of action based on the base pairing binding to their cognate targets have helped the development not only of many computer applications for the prediction of miRNA target recognition but also of specific applications for functional assessment and analysis. Learning about miRNA function requires practical training in the use of specific computer and web-based applications that are complementary to wet-lab studies. In order to guide the learning process about miRNAs, we have created miRNAtools (http://mirnatools.eu), a web repository of miRNA tools and tutorials. This article compiles tools with which miRNAs and their regulatory action can be analyzed and that function to collect and organize information dispersed on the web. The miRNAtools website contains a collection of tutorials that can be used by students and tutors engaged in advanced training courses. The tutorials engage in analyses of the functions of selected miRNAs, starting with their nomenclature and genomic localization and finishing with their involvement in specific cellular functions.
- Genomic characterization in dilated cardiomyopathyPublication . Costa, Marina C.Dilated cardiomyopathy (DCM), a leading cause of heart failure and sudden cardiac death, is characterized by ventricular dilatation and impaired systolic function in the absence of abnormal loading conditions or coronary artery disease. Its prevalence is approximately 1 in 2500 individuals, and 30-50% of cases are familial.