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- Functional analysis of two novel TBX5 variants present in individuals with Holt-Oram syndrome with different clinical manifestationsPublication . Varela, Debora; Varela, Tatiana; Conceição, Natércia; Ferreira, Angela; Marques, Nuno; Silva, Ana Paula; Azevedo, Pedro; Pereira, Salome; Camacho, Ana; de Jesus, Ilidio; Cancela, M. LeonorHolt-Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5-a gene encoding a transcription factor important for heart and skeletal development-are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.
- Transcriptional regulation of human T-box 5 gene (TBX5) by bone- and cardiac-related transcription factorsPublication . Varela, Débora; Conceição, Natércia; Cancela, M. LeonorT-box 5 (TBX5) protein belongs to the T-box family whose members play a crucial role in cell-type specification, morphogenesis and organogenesis. TBX5 is a transcription factor important for cardiac development and upper limbs formation and its haploinsufficiency causes Holt-Oram syndrome (HOS). An increase in TBX5 dosage also leads to HOS, suggesting that TBX5 is a dose-sensitive transcription factor that needs to be tightly regulated but the molecular mechanisms involved remain unclear. In this work we report the cloning and functional analysis of human TBX5 promoter region 1 (upstream of exon 1) and promoter region 2 (upstream of exon 2), that probably regulate the transcription of the different transcript variants. In silico analysis showed several binding sites for cardiac and skeletal related transcription factors (TFs) and their functionality was assessed using promoter-luciferase constructions and TF-expressing vectors. MEF2A (Myocyte enhancer factor 2 A) was shown to positively regulate both TBX5 promoters, while EGR1 (early growth response 1) repressed both promoters. SOX9 (SRY (sex determining region Y)-box 9) repressed only the activity of promoter region 2. Interestingly, YY1 (Yin and yang 1) repressed promoter region 1 (that regulates the expression of variant 1 and 3), but activated promoter region 2 (that regulates the expression of variant 4). In conclusion, this work provides novel insights toward the better understanding of TBX5 transcriptional regulation by cardiac- and skeletal-related TFs.
- Out-of-hospital cardiac arrest in the Algarve region of Portugal: a retrospective registry trial with outcome dataPublication . Carvalho, Nuno Mourão; Martins, Cláudia; Cartaxo, Vera; Marreiros, Ana; Justo, Emília; Raposo, Carlos; Binnie, AlexandraBackground and importanceOut-of-hospital cardiac arrest is a leading cause of death in Europe. An understanding of region-specific factors is essential for informing strategies to improve survival. DesignThis retrospective observational study included all out-of-hospital cardiac arrest patients attended by the Emergency Medical Service of the Algarve in 2019. Outcome data were derived from hospital records. Main resultsIn 2019, there were 850 out-of-hospital cardiac arrests treated with cardiopulmonary resuscitation in the Algarve, representing a population incidence of 189/100 000. Return of spontaneous circulation occurred in 83 patients (9.8%), of whom 17 (2.0%) had survival to hospital discharge and 15 (1.8%) had survival with good neurologic outcome. Among patients in the Utstein comparator group, survival to hospital discharge was 21.4%. Predictors of return of spontaneous circulation were age, witnessed arrest, initial shockable rhythm, time of year, time to cardiopulmonary resuscitation, and time to advanced life support. Predictors of survival to hospital discharge were age, initial shockable rhythm, time to rhythm analysis, and time to advanced life support. Predictors of survival with good neurologic outcome were age, initial shockable rhythm, and time to return of spontaneous circulation. ConclusionsThe incidence of out-of-hospital cardiac arrest with cardiopulmonary resuscitation in the Algarve was higher than in other jurisdictions while return of spontaneous circulation, survival to hospital discharge, and survival with good neurologic outcome were comparatively low. An aging population, a geographically diverse region, and a low incidence of bystander cardiopulmonary resuscitation may have contributed to these outcomes. These results confirm the importance of early cardiopulmonary resuscitation, early rhythm assessment, and early advanced life support, all of which are potentially modifiable through public education, broadening of the defibrillator network and increased availability of advanced life support teams.
- One-minute and green synthesis of magnetic iron oxide nanoparticles assisted by design of experiments and high energy ultrasound: Application to biosensing and immunoprecipitationPublication . Pérez-Beltrán, Christian Hazael; Jose Garcia-Guzman, Juan; Ferreira, Bibiana; Estevez-Hernandez, Osvaldo; Lopez-Iglesias, David; Cubillana-Aguilera, Laura; Link, Wolfgang; Stanica, N.; Rosa Da Costa, Ana; Maria Palacios-Santander, JoseThe present study is focused on the ultrafast and green synthesis, via the co-precipitation method, of magnetic nanoparticles (MNPs) based on iron oxides using design of experiments (DOE) and high energy sonochemical approach, considering two main factors: amplitude (energy) of the ultrasound probe and sonication time. The combination of these techniques allowed the development of a novel one-minute green synthesis, which drastically reduced the amount of consumed energy, solvents, reagents, time and produced residues. This green sonochemical synthesis permitted to obtain mean particle sizes of 11 ? 2 nm under the optimized conditions of amplitude = 40% (2826 J) and time = 1 min. Their composition, structure, size, morphology and magnetic properties were assessed through X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning and transmission electron microscopy (SEM & TEM), and vibrating sample magnetometry (VSM). The characterization results indicate the proper formation of MNPs, and the correct functionalization of MNPs with different coating agents. The functionalized MNPs were used as: i) biosensor, which could detect mercury in water in the range of 0.030?0.060 ppm, and ii) support onto which polyclonal antibodies were anchored and successfully bound to an osteosarcoma cell line expressing the target protein (TRIB2-GFP), as part of an immunoprecipitation assay.
- Musculoskeletal complications associated with pathological iron toxicity and its molecular mechanismsPublication . Simão, Márcio; Cancela, M. LeonorIron is fundamental for several biological functions, but when in excess can lead to the development of toxic events. Some tissues and cells are more susceptible than others, but systemic iron levels can be controlled by treating patients with iron-chelating molecules and phlebotomy. An early diagnostic can be decisive to limit the progression of musculoskeletal complications like osteoarthritis and osteoporosis because of iron toxicity. In iron-related osteoarthritis, aggravation can be associated to a few events that can contribute to joints articular cartilage exposure to high iron concentrations, which can promote articular degeneration with very little chance of tissue regeneration. In contrast, bone metabolism is much more dynamic than cartilage, but progressive iron accumulation and ageing can be decisive factors for bone health. The iron overload associated with hereditary diseases like hemochromatosis, hemophilias, thalassemias and other hereditary anaemias increase the negative impact of iron toxicity in joints and bone, as well as in life quality, even when iron levels can be controlled. The molecular mechanisms by which iron can compromise cartilage and bone have been illusive and only in the last 20 years studies have started to shed some light into the molecular mechanisms associated with iron toxicity. Ferroptosis and the regulation of intracellular iron levels is instrumental in the balance between detoxification and induced cell death. In addition, these complications are accompanied with multiple susceptibility factors that can aggravate iron toxicity and should be identified. Therefore, understanding tissues microenvironment and cell communication is fundamental to contextualize iron toxicity.
- Decavanadate and metformin-decavanadate effects in human melanoma cellsPublication . de Sousa-Coelho, Ana Luísa; Aureliano, Manuel; Fraqueza, Gil; Serrão, Gisela; Gonçalves, João; Sánchez-Lombardo, Irma; Link, Wolfgang; Ferreira, BibianaDecavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive-non-competitive inhibition with an IC50 value about 6 times higher (87 mu M) than the previously described for decavanadate (15 mu M). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/ threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.
- Isolated vocal cord paresis as a presentation of acute ischemic strokePublication . Valente, Diana; Gil, Inês; Jacinto, Filipe; Freitas, Luís; Nzwalo, Hipólito; Felix, CatarinaStroke presenting with predominant laryngeal dysfunction manifestations is often part of a clinical constellation of neurological signs associated with acute medullar lesion [1]. Isolated laryngeal symptoms as a stroke presentation are extremely rare due to the intimal anatomical relation of swallowing-related structures with the ascending/descending tracts in the lateral medulla [2]. Herein we present a case with isolated vocal cord paresis as the sole manifestation of acute ischemic stroke.
- The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficitsPublication . Koppenol, Rebekah; Conceição, André; Afonso, Inês T.; Afonso-Reis, Ricardo; Costa, Rafael G; Tomé, Sandra; Teixeira, Diogo; Pinto-da-Silva, Joana; Codêsso, José Miguel; Brito, David V.C.; Mendonça, Liliana; Marcelo, Adriana; Pereira de Almeida, Luís; Matos, Carlos A; Nóbrega, ClévioKoppenol et al. show that overexpression of G3BP1 in cell models of SCA2 and SCA3 leads to a reduction in ataxin-2 and ataxin-3 aggregation. G3BP1 lentiviral delivery reduces motor deficits and neuropathology in preclinical models, suggesting that G3BP1 may be a potential therapeutic target for polyQ disorders. Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.
- BARX2/FOXA1/HK2 axis promotes lung adenocarcinoma progression and energy metabolism reprogrammingPublication . Xie, Kai; Feng, Jian; Fan, Dingwei; Wang, Shi; Luo, Jing; Ren, Zhijian; Zheng, Chao; Diao, Yifei; De Mello, Ramon Andrade; Tavolari, Simona; Brandi, Giovanni; Roden, Anja C.; Ren, Binhui; Shen, Yi; Xu, LinBackground: Metabolic reprogramming is an emerging cancer feature that has recently drawn special attention since it promotes tumor cell growth and proliferation. However, the mechanism of the Warburg effect is still largely unknown. This research aimed to reveal the effects of BarH-like homeobox 2 (BARX2) in regulating tumor progression and glucose metabolism in lung adenocarcinoma (LUAD).Methods: Expression of BARX2 was measured by quantitative real-time polymerase chain reaction (qRTPCR) in LUAD cell line and tissues, and the tumor-promoting function of BARX2 in LUAD cells was detected in vitro and in vivo xenograft models. The metabolic effects of BARX2 were examined by detecting glucose uptake, the production levels of lactate and pyruvate, and the extracellular acidification rate (ECAR). Chromatin immunoprecipitation (ChIP) assay and luciferase reporter gene assay were used to identify the underlying molecular mechanism of BARX2 regulation of HK2. Further studies showed that transcription factor FOXA1 directly interacts with BARX2 and promotes the transcriptional activity of BARX2.Results: BARX2 was remarkably up-regulated in LUAD tissues and positively linked to advanced clinical stage and poor prognosis. In vitro and in vivo data indicated ectopic expression of BARX2 enhanced cell proliferation and tumorigenesis, whereas BARX2 knockdown suppressed these effects. Metabolic-related experiments showed BARX2 promoted the reprogramming of glucose metabolism. Mechanistically, the BARX2/FOXA1/HK2 axis promoted LUAD progression and energy metabolism reprogramming.Conclusions: In summary, our research first defined BARX2 as a tumor-promoting factor in LUAD andthat it may act as a novel prognostic biomarker and new therapeutic target for the disease.
- Post-transcriptional silencing of Bos taurus prion family genes and its impact on granulosa cell steroidogenesisPublication . Pimenta, Jorge M.B.G.A.; Pires, Virgínia M.R.; Nolasco, Sofia; Castelo-Branco, Pedro; Marques, Carla C.; Apolónio, Joana; Azevedo, Rita; Fernandes, Mónica T.; Lopes-da-Costa, Luís; Prates, José; Pereira, Rosa M.L.N.Prion proteins constitute a major public health concern, which has partly overshadowed their physiological roles in several scenarios. Indeed, these proteins were implicated in male fertility but their role in female fertility is relatively less explored. This study was designed to evaluate the role of SPRN and PRNP prion family genes in bovine follicular steroidogenesis pathways. Post-transcriptional SPRN and PRNP silencing with siRNAs was established in bovine granulosa cell (GC) in vitro culture, and gene expression and progesterone and estradiol concentrations were evaluated. SPRN knockdown, led to a down regulation of CYP11A1 mRNA levels (2.1-fold), and PRNP knockdown led to an upregulation of SPRN mRNA levels (2.3-fold). CYP19A1 expression and estradiol synthesis was not detected in any experimental group. Finally, SPRN knockdown led to a mild reduction in progesterone production in GCs and this was the only experimental group that did not exhibit an increment in progesterone levels after 48 h of culture. As a conclusion, it was possible to detect the expression of the SPRN gene in bovine GCs, a potential interaction between SPRN and PRNP regulation, and the impact of SPRN expression on CYP11A1 and progesterone levels. These findings bring new insights into the role of these genes in ovarian steroidogenesis and female reproductive physiology. (c) 2022 Elsevier Inc. All rights reserved.