CBR - Contribuições em Revistas Científicas / Contribution to Journals
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- 3DCellPol: joint detection and pairing of cell structures to compute cell polarityPublication . Narotamo, Hemaxi; Franco, Cláudio A.; Silveira, MargaridaCell polarity is essential for tissue structure and cell migration, and its dysregulation is linked to diseases such as cancer and vascular disorders. Understanding the associations between molecular mechanisms, such as genetic defects, and abnormal cell polarization can provide clinicians with valuable biomarkers for early disease diagnosis and lead to more targeted therapeutic interventions. Here, we present a deep-learning framework for cell polarity computation based on the association between pairs of objects. Our approach, named 3DCellPol, is trained to detect and group the centroids of two distinct objects. To demonstrate the potential of 3DCellPol, we use it to compute cell polarity by pairing two cell organelles: nuclei and Golgi. The vectors between nuclei and Golgi define the front-rear polarity axis in endothelial cells. 3DCellPol was evaluated on 3D microscopy images of mouse retinas. It detected 71% of the nucleus–Golgi vectors and outperformed previous methods while requiring much less supervision. Moreover, incorporating synthetic data generated by a generative adversarial network further improved detection to 78%. We additionally demonstrated our model's adaptability to 2D images by applying it to a public dataset of cervical cytology images, where polarity is defined based on the cytoplasm-nucleus vectors. In this dataset, our model detected over 90% of vectors. 3DCellPol's ability to robustly compute cell polarity is crucial for understanding mechanisms of diseases where abnormal polarity plays a key role, and it may contribute to improved diagnostics and enable targeted therapies. Hence, it is a valuable open-source tool for both biomedical research and clinical practice.
- 3DVascNet: an automated software for segmentation and quantification of mouse vascular networks in 3DPublication . Narotamo, Hemaxi; Silveira, Margarida; Franco, Cláudio A.BACKGROUND: Analysis of vascular networks is an essential step to unravel the mechanisms regulating the physiological and pathological organization of blood vessels. So far, most of the analyses are performed using 2-dimensional projections of 3-dimensional (3D) networks, a strategy that has several obvious shortcomings. For instance, it does not capture the true geometry of the vasculature and generates artifacts on vessel connectivity. These limitations are accepted in the field because manual analysis of 3D vascular networks is a laborious and complex process that is often prohibitive for large volumes. METHODS: To overcome these issues, we developed 3DVascNet, a deep learning–based software for automated segmentation and quantification of 3D retinal vascular networks. 3DVascNet performs segmentation based on a deep learning model, and it quantifies vascular morphometric parameters such as vessel density, branch length, vessel radius, and branching point density. We tested the performance of 3DVascNet using a large data set of 3D microscopy images of mouse retinal blood vessels. RESULTS: We demonstrated that 3DVascNet efficiently segments vascular networks in 3D and that vascular morphometric parameters capture phenotypes detected by using manual segmentation and quantification in 2 dimension. In addition, we showed that, despite being trained on retinal images, 3DVascNet has high generalization capability and successfully segments images originating from other data sets and organs. CONCLUSIONS: Overall, we present 3DVascNet, a freely available software that includes a user-friendly graphical interface for researchers with no programming experience, which will greatly facilitate the ability to study vascular networks in 3D in health and disease. Moreover, the source code of 3DVascNet is publicly available, thus it can be easily extended for the analysis of other 3D vascular networks by other users.
- A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditionsPublication . Fonseca, Catarina Gonçalves; Silvério, Vânia; Barata, David; Giese, Wolfgang; Gerhardt, Holger; Cardoso, Susana; Franco, Cláudio AreiasThe ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress. [Figure not available: see fulltext.].
- Aedes albopictus arrives in Lisbon: an emerging public health threatPublication . Nazareth, Teresa; Seixas, Gonçalo; Lourenço, José; Bettencourt, Paulo J. G.
- Aerocyte specification and lung adaptation to breathing is dependent on alternative splicing changesPublication . Fidalgo, Marta F.; Fonseca, Catarina G.; Caldas, Paulo; Raposo, Alexandre A. S. F.; Balboni, Tania; Henao-Mišíková, Lenka; Grosso, Ana R.; Vasconcelos, Francisca F.; Franco, Cláudio A.Adaptation to breathing is a critical step in lung function and it is crucial for organismal survival. Alveoli are the lung gas exchange units and their development, from late embryonic to early postnatal stages, requires feedbacks between multiple cell types. However, how the crosstalk between the alveolar cell types is modulated to anticipate lung adaptation to breathing is still unclear. Here, we uncovered a synchronous alternative splicing switch in multiple genes in the developing mouse lungs at the transition to birth, and we identified hnRNP A1, Cpeb4, and Elavl2/HuB as putative splicing regulators of this transition. Notably, we found that Vegfa switches from the Vegfa 164 isoform to the longer Vegfa 188 isoform exclusively in lung alveolar epithelial AT1 cells. Functional analysis revealed that VEGFA 188 (and not VEGFA 164) drives the specification of Car4-positive aerocytes, a subtype of alveolar endothelial cells specialized in gas exchanges. Our results reveal that the cell type-specific regulation of Vegfa alternative splicing just before birth modulates the epithelial-endothelial crosstalk in the developing alveoli to promote lung adaptation to breathing.
- Artificial intelligence-powered microscopy: transforming the landscape of parasitologyPublication . Niz, Mariana De; Pereira, Sara Silva; Kirchenbuechler, David; Lemgruber, Leandro; Arvanitis, ConstadinaMicroscopy and image analysis play a vital role in parasitology research; they are critical for identifying parasitic organisms and elucidating their complex life cycles. Despite major advancements in imaging and analysis, several challenges remain. These include the integration of interdisciplinary data; information derived from various model organisms; and data acquired from clinical research. In our view, artificial intelligence—with the latest advances in machine and deep learning—holds enormous potential to address many of these challenges. This review addresses how artificial intelligence, machine learning and deep learning have been used in the field of parasitology—mainly focused on Apicomplexan, Diplomonad, and Kinetoplastid groups. We explore how gaps in our understanding could be filled by AI in future parasitology research and diagnosis in the field. Moreover, it addresses challenges and limitations currently faced in implementing and expanding the use of artificial intelligence across biomedical fields. The necessary increased collaboration between biologists and computational scientists will facilitate understanding, development, and implementation of the latest advances for both scientific discovery and clinical impact. Current and future AI tools hold the potential to revolutionise parasitology and expand One Health principles.
- ATG9A regulates the dissociation of recycling endosomes from microtubules to form liquid influenza A virus inclusionsPublication . Vale-Costa, Sílvia; Etibor, Temitope Akhigbe; Brás, Daniela; Sousa, Ana Laura; Ferreira, Mariana; Martins, Gabriel G.; Mello, Victor Hugo; Amorim, Maria JoãoAU It is:now Pleaseconfirmthatallheadinglevelsarerepresentedcorrectly established that many viruses that threaten public health : establish condensates via phase transitions to complete their lifecycles, and knowledge on such processes may offer new strategies for antiviral therapy. In the case of influenza A virus (IAV), liquid condensates known as viral inclusions, concentrate the 8 distinct viral ribonucleoproteins (vRNPs) that form IAV genome and are viewed as sites dedicated to the assembly of the 8-partite genomic complex. Despite not being delimited by host membranes, IAV liquid inclusions accumulate host membranes inside as a result of vRNP binding to the recycling endocytic marker Rab11a, a driver of the biogenesis of these structures. We lack molecular understanding on how Rab11a-recycling endosomes condensate specifically near the endoplasmic reticulum (ER) exit sites upon IAV infection. We show here that liquid viral inclusions interact with the ER to fuse, divide, and slide. We uncover that, contrary to previous indications, the reported reduction in recycling endocytic activity is a regulated process rather than a competition for cellular resources involving a novel role for the host factor ATG9A. In infection, ATG9A mediates the removal of Rab11a-recycling endosomes carrying vRNPs from microtubules. We observe that the recycling endocytic usage of microtubules is rescued when ATG9A is depleted, which prevents condensation of Rab11a endosomes near the ER. The failure to produce viral inclusions accumulates vRNPs in the cytosol andAU reduces: Pleasecheckandconfirmthattheeditst genome assembly and the release of infectious virions. We propose that the ER supports the dynamics of liquid IAV inclusions, with ATG9A facilitating their formation. This work advances our understanding on how epidemic and pandemic influenza genomes are formed. It also reveals the plasticity of recycling pathway endosomes to undergo condensation in response to infection, disclosing new roles for ATG9A beyond its classical involvement in autophagy.
- Balance between maternal antiviral response and placental transfer of protection in gestational SARS-CoV-2 infectionPublication . Gonçalves, Juliana; Melro, Magda; Alenquer, Marta; Araújo, Catarina; Castro-Neves, Júlia; Amaral-Silva, Daniela; Ferreira, Filipe; Ramalho, José S.; Charepe, Nádia; Serrano, Fátima; Pontinha, Carlos; Amorim, Maria João; Soares, HelenaThe intricate interplay between maternal immune response to SARS-CoV-2 and the transfer of protective factors to the fetus remains unclear. By analyzing mother-neonate dyads from second and third trimester SARS-CoV-2 infections, our study shows that neutralizing antibodies (NAbs) are infrequently detected in cord blood. We uncovered that this is due to impaired IgG-NAb placental transfer in symptomatic infection and to the predominance of maternal SARS-CoV-2 NAbs of the IgA and IgM isotypes, which are prevented from crossing the placenta. Crucially, the balance between maternal antiviral response and transplacental transfer of IgG-NAbs appears to hinge on IL-6 and IL-10 produced in response to SARS-CoV-2 infection. In addition, asymptomatic maternal infection was associated with expansion of anti-SARS-CoV-2 IgM and NK cell frequency. Our findings identify a protective role for IgA/IgM-NAbs in gestational SARS-CoV-2 infection and open the possibility that the maternal immune response to SARS-CoV-2 infection might benefit the neonate in 2 ways, first by skewing maternal immune response toward immediate viral clearance, and second by endowing the neonate with protective mechanisms to curtail horizontal viral transmission in the critical postnatal period, via the priming of IgA/IgM-NAbs to be transferred by the breast milk and via NK cell expansion in the neonate.
- Beyond chemicals: opportunities and challenges of integrating non-chemical stressors in adverse outcome pathwaysPublication . Clerbaux, Laure Alix; Filipovska, Julija; Nymark, Penny; Chauhan, Vinita; Sewald, Katherina; Alb, Miriam; Sachana, Madgalini; Beronius, Anna; Amorim, Maria João; Wittwehr, ClemensThe adverse outcome pathways (AOPs) were developed to accelerate evidence-based chemical risk assessment by leveraging data from new approach methodologies. Thanks to their stressor-agnostic approach, AOPs were seen as instrumental in other fields. Here, we present AOPs that report non-chemical stressors along with the challenges encountered for their development. Challenges regarding AOPs linked to nanomaterials include non-specific molecular initiating events, limited understanding of nanomaterial biodistribution, and needs for adaptations of the in silico modeling and testing systems. Development of AOPs for radiation face challenges in how to incorporate ionizing events type, dose rate, energy deposition, and how to account for targeting multiple macromolecules. AOPs for COVID-19 required the inclusion of SARS-CoV-2-specific replicative steps to capture the essential events driving the disease. Developing AOPs to evaluate efficacy and toxicity of cell therapies necessitates addressing the cellular nature and the therapeutic function of the stressor. Finally, addressing toxicity of emerging biological stressors like microbial pesticides can learn from COVID-19 AOPs. We further discuss that the adaptations needed to expand AOP applicability beyond chemicals are mainly at the molecular and cellular levels while downstream key events at tissue or organ level, such as inflammation, are shared by many AOPs initiated by various stressors. In conclusion, although it is challenging to integrate non-chemical stressors within AOPs, this expands opportunities to account for real-world scenarios, to identify vulnerable individuals, and to bridge knowledge on mechanisms of adversity.
- Bioengineered 3D microvessels and complementary animal models reveal mechanisms of Trypanosoma congolense sequestrationPublication . Porqueddu, Teresa; Zorrinho-Almeida, Maria; Niz, Mariana De; Casas-Sánchez, Aitor; Introini, Viola; Sender, Silvia Sanz; Carrasqueira, Diana; Figueiredo, Luísa M.; Bernabeu, Maria; Pereira, Sara SilvaIn the mammalian host, Trypanosoma congolense cytoadheres, or sequesters, to the vascular endothelium. Although sequestration influences clinical outcome, disease severity and organ pathology, its determinants and mediators remain unknown. Challenges such as the variability of animal models, the only-recently developed tools to genetically manipulate the parasite, and the lack of physiologically-relevant in vitro models have hindered progress. Here, we engineered brain and cardiac 3D bovine endothelial microvessel models that mimic the bovine brain microvasculature and the bovine aorta, respectively. By perfusing these models with two T. congolense strains, we investigated the roles of flow for parasite sequestration and tropism for different endothelial beds. We discovered that sequestration is dependent on cyclic adenosine monophosphate (cAMP) signalling, closely linked to parasite proliferation, but not associated with parasite transmission to the tsetse fly vector. Finally, by comparing the expression profiles of sequestered and non-sequestered parasites collected from a rodent model, we showed gene expression changes in sequestered parasites, including of surface variant antigens. This work presents a physiologically-relevant platform to study trypanosome interactions with the vasculature and provides a deeper understanding of the molecular and biophysical mechanisms underlying T. congolense sequestration.