Ricardo Pires das Neves

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

Background Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis. Methods and Findings In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis. Conclusions Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.

Association between active genes occurs at nuclear speckles and is modulated by chromatin environment

Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse α-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human α-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

Connecting Variability in Global Transcription Rate to Mitochondrial Variability

The authors demonstrate a connection between variability in the rate of transcription and differences in cellular mitochondrial content.

Nanoparticles for intracellular-targeted drug delivery

Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

Mitochondrial variability as a source of extrinsic cellular noise.

We present a study investigating the role of mitochondrial variability in generating noise in eukaryotic cells. Noise in cellular physiology plays an important role in many fundamental cellular processes, including transcription, translation, stem cell differentiation and response to medication, but the specific random influences that affect these processes have yet to be clearly elucidated. Here we present a mechanism by which variability in mitochondrial volume and functionality, along with cell cycle dynamics, is linked to variability in transcription rate and hence has a profound effect on downstream cellular processes. Our model mechanism is supported by an appreciable volume of recent experimental evidence, and we present the results of several new experiments with which our model is also consistent. We find that noise due to mitochondrial variability can sometimes dominate over other extrinsic noise sources (such as cell cycle asynchronicity) and can significantly affect large-scale observable properties such as cell cycle length and gene expression levels. We also explore two recent regulatory network-based models for stem cell differentiation, and find that extrinsic noise in transcription rate causes appreciable variability in the behaviour of these model systems. These results suggest that mitochondrial and transcriptional variability may be an important mechanism influencing a large variety of cellular processes and properties.

Global variability in gene expression and alternative splicing is modulated by mitochondrial content

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.

Towards the Maturation and Characterization of Smooth Muscle Cells Derived from Human Embryonic Stem Cells

In this study we demonstrate that CD34+ cells derived from human embryonic stem cells (hESCs) have higher smooth muscle cell (SMC) potential than CD34− cells. We report that from all inductive signals tested, retinoic acid (RA) and platelet derived growth factor (PDGFBB) are the most effective agents in guiding the differentiation of CD34+ cells into smooth muscle progenitor cells (SMPCs) characterized by the expression of SMC genes and proteins, secretion of SMC-related cytokines, contraction in response to depolarization agents and vasoactive peptides and expression of SMC-related genes in a 3D environment. These cells are also characterized by a low organization of the contractile proteins and the contractility response is mediated by Ca2+, which involves the activation of Rho A/Rho kinase- and Ca2+/calmodulin (CaM)/myosin light chain kinase (MLCK)-dependent pathways. We further show that SMPCs obtained from the differentiation of CD34+ cells with RA, but not with PDGFBB, can be maturated in medium supplemented with endothelin-1 showing at the end individualized contractile filaments. Overall the hESC-derived SMCs presented in this work might be an unlimited source of SMCs for tissue engineering and regenerative medicine.

Efficient Pro-survival/angiogenic miRNA Delivery by an MRI-Detectable Nanomaterial

Herein, we report the use of biodegradable nanoparticles (NPs) containing perfluoro-1,5-crown ether (PFCE), a fluorine-based compound (NP170-PFCE) with the capacity to track cells in vivo by magnetic ressonance imaging (MRI) and efficiently release miRNA. NP170-PFCE complexed with miRNAs accumulate whitin the cells endolysosomal compartment and interact with higher frequency with argonaute2 (Ago2) and GW182 proteins, which are involved in the biological action of miRNAs, than commercial complexes formed by commercial reagents and miRNA, which in turn accumulate in the cell cytoplasm. The release of miRNA132 (miR132) from the NPs increased 3-fold the survival of endothelial cells (ECs) transplanted in vivo and 3.5-fold the blood perfusion in ischemic limbs relatively to control.

A biophysical model for transcription factories.

SummaryTranscription factories are nuclear domains where gene transcription takes placealthough the molecular basis for their formation and maintenance are unknown. In thisstudy, we explored how the properties of chromatin as a polymer may contribute to thestructure of transcription factories. We found that transcriptional active chromatincontains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Singlefibre analysis showed that this modification spans the entire body of the gene.Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active andinactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in thechromatin fibre. The result of this change in flexibility is that chromatin couldbehave like a multi-block copolymer with repetitions of stiff-flexible(active-inactive chromatin) components. Copolymers with such structure self-organizethrough spontaneous phase separation into microdomains. Consistent with such modelH4K16ac chromatin form foci that associates with nascent transcripts. We propose thattranscription factories are the result of the spontaneous concentration of H4K16acchromatin that are in proximity, mainly in cis.

Mitochondrial levels determine variability in cell death by modulating apoptotic gene expression

Fractional killing is the main cause of tumour resistance to chemotherapy. This phenomenon is observed even in genetically identical cancer cells in homogeneous microenvironments. To understand this variable resistance, here we investigate the individual responses to TRAIL in a clonal population of HeLa cells using live-cell microscopy and computational modelling. We show that the cellular mitochondrial content determines the apoptotic fate and modulates the time to death, cells with higher mitochondrial content are more prone to die. We find that all apoptotic protein levels are modulated by the mitochondrial content. Modelling the apoptotic network, we demonstrate that these correlations, and especially the differential control of anti- and pro-apoptotic protein pairs, confer mitochondria a powerful discriminatory capacity of apoptotic fate. We find a similar correlation between the mitochondria and apoptotic proteins in colon cancer biopsies. Our results reveal a different role of mitochondria in apoptosis as the global regulator of apoptotic protein expression.It is unclear what causes variation in cell death in response to chemotherapy. Here, the authors show that cellular mitochondrial content modulates apoptotic protein levels, which in turn regulates response to agents such as TRAIL.