Biology subjects

Vieira, A. S.

Publications and source records attributed to Vieira, A. S..

4 recordsLinked to original sources

bioRxiv2023

An optimized protocol for the creation of constructs expressing sodium channel subunits to be used in functional studies of genetic epilepsies

BackgroundWe aim to develop a method for cloning highly unstable plasmids encoding sodium channel subunits expressed in the brain. MethodNEB Stable competent cells (NSCC) were transformed with the native plasmid. After purification, plasmids with the expected profile of enzymatic digestion were sequenced. Finally, mutant linear plasmids (MLP) created by site-directed mutagenesis were used to transform NSCC. ResultsWe found that choosing a suitable host for transformation and subcloning, the controlled temperature during cell growth, and a small volume of Luria Bertani (LB) medium allowed the generation of plasmids without undesirable mutations or structural alterations. Comparison with existing methodAt present, there is little information on a detailed method for cloning plasmids carrying the SCN1A gene. Using the method proposed here, we observed increased cell growth and precision in the sequence of the obtained plasmids. ConclusionWe have developed an optimized strategy for replicating unstable plasmids using NSCC with an appropriate volume of culture medium and an adequate temperature in the cloning and subcloning phases. We have also demonstrated how to create the desired plasmid constructs by using site-directed mutagenesis. We expect that our protocol will assist investigators interested in functional studies of sodium channels expressed in the brain.

molecular biology↗

bioRxiv2023

Identifying cellular markers of focal cortical dysplasia type II with cell-type deconvolution and single-cell signatures

Focal cortical dysplasia (FCD) is a brain malformation that causes medically refractory epilepsy. FCD is classified into three categories based on structural and cellular abnormalities, with FCD type II being the most common and characterized by disrupted organization of the cortex and abnormal neuronal development. In this study, we employed cell-type deconvolution and single-cell signatures to analyze bulk RNA-seq from multiple transcriptomic studies, aiming to characterize the cellular composition of brain lesions in patients with FCD IIa and IIb subtypes. Our deconvolution analyses revealed specific cellular changes in FCD IIb, including neuronal loss and an increase in reactive astrocytes (astrogliosis) when compared to FCD IIa. Astrogliosis in FCD IIb was further supported by a gene signature analysis and histologically confirmed by glia fibrilla acidic protein (GAP) immunostaining. Overall, our findings demonstrate that FCD II subtypes exhibit differential neuronal and glial compositions, with astrogliosis emerging as a hallmark of FCD IIb. These observations, validated in independent patient cohorts and confirmed using immunohistochemistry, offer novel insights into the involvement of glial cells in FCD type II pathophysiology and may contribute to the development of targeted therapies for this condition.

neuroscience↗

bioRxiv2023

The nuclear receptor Nr2f6 represses skeletal muscle oxidative metabolism and force production.

ObjectiveThe maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. However, the mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate, for the first time, the effects of Nr2f6 modulation on muscle biology in vivo and in vitro. MethodsGlobal RNA-seq was performed in Nr2f6-knockdown C2C12 myocytes (N=4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and overexpression C2C12 cell lines (N=3-6). Nr2f6 content was evaluated in in vitro and in vivo lipid overload models (N=3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N=4), ex vivo contractility experiments (N=5), and histological analysis (N=7). The conservation of Nr2f6 depletion effects was confirmed in primary human and mouse skeletal muscle cells. ResultsNr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 overexpression significantly increased the expression of myosin heavy chain genes (2-3-fold). Nr2f6 content in skeletal muscle decreased by 40% in lipid-overloaded myotubes and by 50% in mice fed a high-fat diet. Depletion of Nr2f6 increased myocyte lipid oxidative capacity by 75% and protected against lipid-induced cell death. This protection was associated with direct repression of uncoupling protein 3 (20%) and PGC-1 (30%) promoter activity following Nr2f6 overexpression. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofiber content (18%), accompanied by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an immune response molecular signature and a decrease in genes involved in oxidative metabolism and muscle contractility. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation. ConclusionIn summary, our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These findings have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.

biochemistry↗

bioRxiv2019

Physical Activity Induces Nucleus Accumbens Genes Expression Changes Preventing Chronic Pain Susceptibility Promoted by High-Fat Diet and Sedentary Behavior in Mice

High-fat diet (HFD)-induced obesity was reported to increase pain behavior independent of obesity status in rats, whereas weight loss interventions such as voluntary physical activity (PA) for adults with overweight or obesity was reported to promote pain reduction in humans with chronic pain (CP). However, is unknown whether an HFD and sedentary (SED) behavior is underlying to CP susceptibility and whether voluntary PA can prevent it. Moreover, differential gene expression in the nucleus accumbens (NAc) is considered to play a crucial role in CP susceptibility. The present study used an adapted model of the inflammatory prostaglandin E2 (PGE)-induced persistent hyperalgesia (PH-ST) protocol for mice, an HFD, and a voluntary PA paradigm to test these hypotheses. In addition, we performed a transcriptome in the NAc and a gene ontology enrichment tools to investigate the differential gene expression and identify the biological processes associated with CP susceptibility tested here. Our results demonstrated that HFD and sedentary behavior promoted CP susceptibility, which in turn was prevented by voluntary PA, even when the animals were fed an HFD. Transcriptome in the NAc found 2,204 differential expression genes related CP susceptibility promoted by HFD and sedentary behavior and prevented by voluntary PA. The gene ontology enrichment revealed 41 biological processes implicated in CP susceptibility. Analyzing collectively those biological processes, our results suggested that genes related to metabolic and mitochondria stress were up-regulated in the CP susceptibility group, whereas genes related to neuroplasticity and axonogenesis were up-regulated in the CP prevented group. These findings provide pieces of evidence that an HFD and sedentary behavior promoted gene expression changes in the NAc related to neurodegeneration and those changes were also underlying to CP susceptibility. Additionally, our findings confirmed other findings supporting the crucial role of voluntary PA to prevent CP susceptibility and add novel insights of differential gene expression in the NAc related to neuroplasticity.

neuroscience↗