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- Exogenous WNT5A and WNT11 proteins rescue CITED2 dysfunction in mouse embryonic stem cells and zebrafish morphantsPublication . Santos, João; Mendes-Silva, Leonardo; Afonso,Vanessa; Martins, Gil; Machado, Rui; Lopes, Joao; Cancela, M. Leonor; Futschik, Matthias; Sachinidis, Agapios; Gavaia, Paulo; Bragança, JoséMutations and inadequate methylation profiles of CITED2 are associated with human congenital heart disease (CHD). In mouse, Cited2 is necessary for embryogenesis, particularly for heart development, and its depletion in embryonic stem cells (ESC) impairs cardiac differentiation. We have now determined that Cited2 depletion in ESC affects the expression of transcription factors and cardiopoietic genes involved in early mesoderm and cardiac specification. Interestingly, the supplementation of the secretome prepared from ESC overexpressing CITED2, during the onset of differentiation, rescued the cardiogenic defects of Cited2-depleted ESC. In addition, we demonstrate that the proteins WNT5A and WNT11 held the potential for rescue. We also validated the zebrafish as a model to investigate cited2 function during development. Indeed, the microinjection of morpholinos targeting cited2 transcripts caused developmental defects recapitulating those of mice knockout models, including the increased propensity for cardiac defects and severe death rate. Importantly, the co-injection of anti-cited2 morpholinos with either CITED2 or WNT5A and WNT11 recombinant proteins corrected the developmental defects of Cited2-morphants. This study argues that defects caused by the dysfunction of Cited2 at early stages of development, including heart anomalies, may be remediable by supplementation of exogenous molecules, offering the opportunity to develop novel therapeutic strategies aiming to prevent CHD.
- Spatiotemporal modelling of the quality and ripening of two cultivars of "Algarve Citrus" orchards at different edaphoclimatic conditionsPublication . Cavaco, Ana M.; Cruz, Sandra P.; Antunes, M. Dulce; Guerra, Rui; Pires, Rosa; Afonso, Andreia M.; Brazio, António; Silva, Leonardo; Lucas, Marcia Rosendo; Daniel, Mariana; Panagopoulos, ThomasAlgarve Citrus are non-climacteric Protected Geographical Indication (PGI) commodities. They are harvested with minimal levels of juice content (>35 %), soluble solids content (SSC) (>10 %) and maturation index (MI) (>8), as required by the respective PGI normative reference. These internal quality attributes (IQA) are usually determined in small samples of fruit collected from the orchards close to harvest. This study aimed to use geostatistics to help predict the optimal harvest date (OHD) of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, namely, 'Newhall', and 'Valencia Late', at two different edaphoclimatic conditions observed in the locations of Quarteira, at the coast, and Paderne, near a mountainous area. Two orchards of 0.5-0.7 ha per cultivar were chosen and a total of 25 trees were georeferenced within each orchard, comprising 100 sampling points/trees. Firmness, juice content, SSC and MI of fruit were determined through time. In general, the fruit grown in Quarteira showed higher SSC and MI and lower firmness values, ripening two months earlier than those grown in Paderne, although the full effect of the various edaphoclimatic factors on these results are not fully understood. However, geospatial modelling of ripening has shown a large variability within the orchards, with some IQA evolution patterns observed in some orchards and/or cultivars but not in the others. Specifically, 1) a negative correlation between the firmness and MI spatial patterns; 2) a variable decay rate of firmness, much faster in Paderne for 'Valencia Late'; 3) local minima in juice content, below 35 %, observed in restricted spatial areas and in specific time periods, and which were clearer in 'Newhall'. These local variations highlight the need for an optimized management based on geospatial modelling. For example, the variable decay rate of firmness must be taken into account during fruit harvest and postharvest handling. On the other side, the observation of localized plots with juice content below 35 % must be contextualized in the broader picture of the entire orchard which, in the present study, always had consistent temporal average level above 35 %. This study has provided evidence that fruit ripening variability should be considered in the site-specific orchard management of citrus to optimize their harvest date.
- Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductantsPublication . Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Leonardo Mendes-Silva; Melo, Eduardo; Harding, Heather P.; Ron, DavidProtein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1(CtoS) purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.
- Induced pluripotent stem cells, a giant leap for mankind therapeutic applicationsPublication . Bragança, José; Lopes, João A. P.; Silva, Leonardo; Santos, JoãoInduced pluripotent stem cells (iPSC) technology has propelled the field of stem cells biology, providing new cells to explore the molecular mechanisms of pluripotency, cancer biology and aging. A major advantage of human iPSC, compared to the pluripotent embryonic stem cells, is that they can be generated from virtually any embryonic or adult somatic cell type without destruction of human blastocysts. In addition, iPSC can be generated from somatic cells harvested from normal individuals or patients, and used as a cellular tool to unravel mechanisms of human development and to model diseases in a manner not possible before. Besides these fundamental aspects of human biology and physiology that are revealed using iPSC or iPSC-derived cells, these cells hold an immense potential for cell-based therapies, and for the discovery of new or personalized pharmacological treatments for many disorders. Here, we review some of the current challenges and concerns about iPSC technology. We introduce the potential held by iPSC for research and development of novel health-related applications. We briefly present the efforts made by the scientific and clinical communities to create the necessary guidelines and regulations to achieve the highest quality standards in the procedures for iPSC generation, characterization and long-term preservation. Finally, we present some of the audacious and pioneer clinical trials in progress with iPSC-derived cells.
- CITED2 and the modulation of the hypoxic response in cancerPublication . Fernandes, Mónica T; Calado, Sofia; Mendes-Silva, Leonardo; Bragança, JoséCITED2 (CBP/p300-interacting transactivator with Glu/Asp-rich C-terminal domain, 2) is a ubiquitously expressed protein exhibiting a high affinity for the CH1 domain of the transcriptional co-activators CBP/p300, for which it competes with hypoxia-inducible factors (HIFs). CITED2 is particularly efficient in the inhibition of HIF-1α-dependent transcription in different contexts, ranging from organ development and metabolic homeostasis to tissue regeneration and immunity, being also potentially involved in various other physiological processes. In addition, CITED2 plays an important role in inhibiting HIF in some diseases, including kidney and heart diseases and type 2-diabetes. In the particular case of cancer, CITED2 either functions by promoting or suppressing cancer development depending on the context and type of tumors. For instance, CITED2 overexpression promotes breast and prostate cancers, as well as acute myeloid leukemia, while its expression is downregulated to sustain colorectal cancer and hepatocellular carcinoma. In addition, the role of CITED2 in the maintenance of cancer stem cells reveals its potential as a target in non-small cell lung carcinoma and acute myeloid leukemia, for example. But besides the wide body of evidence linking both CITED2 and HIF signaling to carcinogenesis, little data is available regarding CITED2 role as a negative regulator of HIF-1α specifically in cancer. Therefore, comprehensive studies exploring further the interactions of these two important mediators in cancer-specific models are sorely needed and this can potentially lead to the development of novel targeted therapies.