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Melo S.A.,University of Houston | Melo S.A.,Beth Israel Deaconess Medical Center | Sugimoto H.,University of Houston | Sugimoto H.,Beth Israel Deaconess Medical Center | And 14 more authors.
Cancer Cell | Year: 2014

Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. © 2014 Elsevier Inc.


Guantes R.,Autonomous University of Madrid | Rastrojo A.,Centro Biologia Molecular Severo Ochoa | Neves R.,Weatherall Institute of Molecular Medicine | Neves R.,Center for Neuroscience and Cell Biology | And 4 more authors.
Genome Research | Year: 2015

Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype. © 2015 Guantes et al.


Melo C.A.,Netherlands Cancer Institute | Melo C.A.,Center for Neuroscience and Cell Biology | Drost J.,Netherlands Cancer Institute | Wijchers P.J.,University Utrecht | And 11 more authors.
Molecular Cell | Year: 2013

Binding within or nearby target genes involved in cell proliferation and survival enables the p53 tumor suppressor gene to regulate their transcription and cell-cycle progression. Using genome-wide chromatin-binding profiles, we describe binding of p53 also to regions located distantly from any known p53 target gene. Interestingly, many of these regions possess conserved p53-binding sites and all known hallmarks of enhancer regions. We demonstrate that these p53-bound enhancer regions (p53BERs) indeed contain enhancer activity and interact intrachromosomally with multiple neighboring genes to convey long-distance p53-dependent transcription regulation. Furthermore, p53BERs produce, in a p53-dependent manner, enhancer RNAs (eRNAs) that are required for efficient transcriptional enhancement of interacting target genes and induction of a p53-dependent cell-cycle arrest. Thus, our results ascribe transcription enhancement activity to p53 with the capacity to regulate multiple genes from a single genomic binding site. Moreover, eRNA production from p53BERs is required for efficient p53 transcription enhancement. © 2013 Elsevier Inc.


Martins T.,University of Coimbra | Baptista S.,University of Coimbra | Goncalves J.,University of Coimbra | Leal E.,Center for Neuroscience and Cell Biology | And 8 more authors.
Brain Research | Year: 2011

Methamphetamine (METH) is a powerful stimulant drug of abuse that has steadily gained popularity worldwide. It is known that METH is highly neurotoxic and causes irreversible damage of brain cells leading to neurological and psychiatric abnormalities. Recent studies suggested that METH-induced neurotoxicity might also result from its ability to compromise blood-brain barrier (BBB) function. Due to the crucial role of BBB in the maintenance of brain homeostasis and protection against toxic molecules and pathogenic organisms, its dysfunction could have severe consequences. In this study, we investigated the effect of an acute high dose of METH (30 mg/kg) on BBB permeability after different time points and in different brain regions. For that, young adult mice were sacrificed 1 h, 24 h or 72 h post-METH administration. METH increased BBB permeability, but this effect was detected only at 24 h after administration, being therefore a transitory effect. Interestingly, we also found that the hippocampus was the most susceptible brain region to METH, comparing to frontal cortex and striatum. Moreover, in an attempt to identify the key players in METH-induced BBB dysfunction we further investigated potential alterations in tight junction (TJ) proteins and matrix metalloproteinase-9 (MMP-9). METH was able to decrease the protein levels of zonula occludens (ZO)-1, claudin-5 and occludin in the hippocampus 24 h post-injection, and increased the activity and immunoreactivity of MMP-9. The pre-treatment with BB-94 (30 mg/kg), a matrix metalloproteinase inhibitor, prevented the METH-induced increase in MMP-9 immunoreactivity in the hippocampus. Overall, the present data demonstrate that METH transiently increases the BBB permeability in the hippocampus, which can be explained by alterations on TJ proteins and MMP-9. © 2011 Elsevier B.V. All rights reserved.


Moreira V.M.,University of Coimbra | Salvador J.A.R.,University of Coimbra | Salvador J.A.R.,Center for Neuroscience and Cell Biology | Simoes S.,Center for Neuroscience and Cell Biology | And 3 more authors.
European Journal of Medicinal Chemistry | Year: 2013

A series of novel oleanane-type pentacyclic triterpenoids bearing a boronate ester moiety at C3 have been synthesized by palladium-catalyzed cross-coupling of bis(pinacolato)diboron with vinyl triflates, in the presence of base, and these compounds were fully characterized by 1D and 2D NMR techniques. Evaluation of their antiproliferative effects on a panel of hematological-based and solid tumor cell lines identified three active oleanolic vinyl boronates that inhibited the growth of leukemia (Jurkat, K562), Burkitt's lymphoma (Jijoye), cervix (Hela), colon (SW480), and ovary (SKOV-3) cancer cells without concomitant inhibition of non-tumoral human fibroblasts. Their mechanisms of action were investigated on the leukemia Jurkat cell line. The results show that the incorporation of boron in the oleanolic acid core combined with the presence of amide bonds afforded compounds with desirable biological effects such as apoptosis induction and inhibition of proteasomal activity on tumor cells, which makes them potential templates for further development in the anticancer drug setting. © 2013 Elsevier Masson SAS. All rights reserved.


Melo C.A.,Netherlands Cancer Institute | Melo C.A.,Center for Neuroscience and Cell Biology | Leveille N.,Netherlands Cancer Institute | Agami R.,Netherlands Cancer Institute | Agami R.,Rotterdam University
Cell Research | Year: 2013

Recently, various studies shed light on the functional significance of enhancer RNAs. Two recent studies published in Nature by Li et al. and Lam et al. highlight the importance of these newly characterized RNA molecules and their key role in controlling transcriptional programs. © 2013 IBCB, SIBS, CAS.


Melo C.A.,Netherlands Cancer Institute | Melo C.A.,Center for Neuroscience and Cell Biology | Melo S.A.,University of Houston | Melo S.A.,University of Porto
Methods in Molecular Biology | Year: 2014

RNA-induced silencing complex is the cytoplasmic effector machine of the microRNA (miRNA) pathway and contains a single-stranded miRNA guiding it to its target mRNAs. The biogenesis of mature miRNAs is a regulatory process achieved by complex machinery composed of few protein components, among which the ribonuclease III Dicer plays a central role. Dicer is essential for miRNA maturation and catalyzes one of the rate-limiting steps of miRNA production. In this chapter, we describe a protocol to study the catalytic activity of Dicer in cell extracts by measuring their ability to process precursor RNA (pre-miRNAs) into functional mature miRNAs. Impairment of the miRNA biogenesis machinery due to loss-of-function mutations in effectors of the pathway such as Dicer has been demonstrated before. Dicing assay can be used in cancer to assess Dicer enzymatic activity compared with healthy controls. Therefore, this experimental approach is likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases. © Springer Science+Business Media New York 2014.


Zuzarte M.,University of Coimbra | Goncalves M.J.,University of Coimbra | Cavaleiro C.,University of Coimbra | Cruz M.T.,Center for Neuroscience and Cell Biology | And 5 more authors.
Industrial Crops and Products | Year: 2013

Lavandula L. and Thymus L. comprise several relevant species for the food, cosmetic, perfumery and pharmaceutical industries. Considering the traditional medicinal use of L. stoechas and T. herba-barona and the lack of scientific studies on their biological activities, the present study was designed to elucidate the composition and antifungal activity of their essential oils against fungi responsible for human infections as well as the anti-inflammatory potential and their cytotoxicity on a macrophage cell line. Moreover, the antifungal activity against fungi responsible for food contamination is also reported.Flowering parts of the plants were submitted to hydrodistillation in a Clevenger-type apparatus and the oils were analysed by GC and GC-MS. The minimal inhibitory and minimal lethal concentrations of the oils against fungi strains were determined using a macrodilution broth method. For the anti-inflammatory activity, an in vitro model of lipopolysaccharide-stimulated macrophages was used and the inhibition of nitric oxide production quantified. Assessment of the oils cytotoxicity was performed using the MTT reduction assay.L. stoechas essential oil was rich in fenchone (37.0%) and camphor (27.3%) while T. herba-barona oil showed high amounts of two phenols, carvacrol (54.0%) and thymol (30.2%). The latter was the most active oil against the tested fungi but evidenced high cytotoxicity on macrophages. L. stoechas was active against dermatophyte strains and showed potential anti-inflammatory activity at concentrations without affecting cell viability.These results support the use of L. stoechas in the development of phytopharmaceuticals or food supplements/nutraceuticals for the management of dermatophytosis and/or inflammatory-related diseases. Regarding T. herba-barona, it can be used as a preservative in storage products, due to its ability to inhibit Aspergilllus growth. © 2012 Elsevier B.V.


Pinho A.L.,Retzius vag | Pinho A.L.,Center for Neuroscience and Cell Biology | Ullen F.,Retzius vag | Castelo-Branco M.,University of Coimbra | And 2 more authors.
Cerebral Cortex | Year: 2016

Neuroimaging studies of internally generated behaviors have shown seemingly paradoxical results regarding the dorsolateral prefrontal cortex (DLPFC), which has been found to activate, not activate or even deactivate relative to control conditions. On the one hand, the DLPFC has been argued to exert top-down control over generative thought by inhibiting habitual responses; on the other hand, a deactivation and concomitant decrease in monitoring and focused attention has been suggested to facilitate spontaneous associations and novel insights. Here, we demonstrate that prefrontal engagement in creative cognition depends dramatically on experimental conditions, that is, the goal of the task. We instructed professional pianists to perform improvisations on a piano keyboard during fMRI and play, eitherwith a certainemotional content (happy/fearful), or using certain keys (tonal/atonal pitch-sets).We found lower activity in primarily the right DLPFC, dorsal premotor cortex and inferior parietal cortex during emotional conditions compared with pitch-set conditions. Furthermore, the DLPFC was functionally connected to the defaultmode network during emotional conditions and to the premotor network during pitch-set conditions. The results thus support the notion of two broad cognitive strategies for creative problem solving, relying on extrospective and introspective neural circuits, respectively. © The Author 2016.


PubMed | Center for Neuroscience and Cell Biology, University of Beira Interior and University of Coimbra
Type: | Journal: Journal of controlled release : official journal of the Controlled Release Society | Year: 2016

Modulation of the subventricular zone (SVZ) neurogenic niche can enhance brain repair in several disorders including Parkinsons disease (PD). Herein, we used biocompatible and traceable polymeric nanoparticles (NPs) containing perfluoro-1,5-crown ether (PFCE) and coated with protamine sulfate to complex microRNA-124 (miR-124), a neuronal fate determinant. The ability of NPs to efficiently deliver miR-124 and prompt SVZ neurogenesis and brain repair in PD was evaluated. In vitro, miR-124 NPs were efficiently internalized by neural stem/progenitors cells and neuroblasts and promoted their neuronal commitment and maturation. The expression of Sox9 and Jagged1, two miR-124 targets and stemness-related genes, were also decreased upon miR-124 NP treatment. In vivo, the intracerebral administration of miR-124 NPs increased the number of migrating neuroblasts that reached the granule cell layer of the olfactory bulb, both in healthy and in a 6-hydroxydopamine (6-OHDA) mouse model for PD. MiR-124 NPs were also able to induce migration of neurons into the lesioned striatum of 6-OHDA-treated mice. Most importantly, miR-124 NPs proved to ameliorate motor symptoms of 6-OHDA mice, monitored by the apomorphine-induced rotation test. Altogether, we provide clear evidences to support the use of miR-124 NPs as a new therapeutic approach to boost endogenous brain repair mechanisms in a setting of neurodegeneration.

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