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.[Supplemental material is available for this article.] Cellular heterogeneity can result from noise generated during gene expression and plays an essential role in fundamental processes such as development, cell differentiation, and cancer (Raj and van Oudenaarden 2008;Eldar and Elowitz 2010;Balázsi et al. 2011). Gene expression noise may originate from stochasticity in the biochemical reactions at an individual gene (intrinsic noise) or from fluctuations in cellular components inducing a global effect (extrinsic noise) (Elowitz et al. 2002;Maheshri and O'Shea 2007). Extrinsic noise is often a dominant source of variation both in prokaryotes (Taniguchi et al. 2010) and eukaryotes (Raser and O'Shea 2004;Newman et al. 2006). Despite this, the origins of extrinsic fluctuations are mostly unknown, although random protein partitioning from cell growth and division (Rosenfeld et al. 2005;Volfson et al. 2006), upstream transcription factors (Volfson et al. 2006), or cell cycle stage (Zopf et al. 2013) have been shown to contribute to variability in protein levels. A common constraint across eukaryotic gene expression is its high energy cost (with ∼75% of the ATP cellular energy budget invested into mRNA and protein polymerization) (Forster et al. 2003;Wagner 2005;Lane and Martin 2010), where every step, from chromatin remodeling to transcription elongation, assembly of splicing factors, and translation, depends on energy (Fig. 1A). Since most of the energy required in normal cells is supplied by mitochondrial oxidative phosphorylation (Vander Heiden et al. 2009), variability in the number and/or functionality of mitochondria is a natural source of variability in ATP content...

Biophysical cues influence many aspects of cell behavior. Stiffness of the extracellular matrix is probed by cells and transduced into biochemical signals through mechanotransduction protein networks, strongly influencing stem cell behavior. Cellular stemness is intimately related with mechanical properties of the cell, like intracellular contractility and stiffness, which in turn are influenced by the microenvironment. Pluripotency is associated with soft and low-contractility cells. Hence, we postulated that soft cell culture substrates, presumably inducing low cellular contractility and stiffness, increase the reprogramming efficiency of mesenchymal stem/stromal cells (MSCs) into induced pluripotent stem cells (iPSCs). We demonstrate that soft substrates (1.5 or 15 kPa polydimethylsiloxane – PDMS) caused modulation of several cellular features of MSCs into a phenotype closer to pluripotent stem cells (PSCs). MSCs cultured on soft substrates presented more relaxed nuclei, lower maturation of focal adhesions and F-actin assembling, more euchromatic and less heterochromatic nuclear DNA regions, and increased expression of pluripotency-related genes. These changes correlate with the reprogramming of MSCs, with a positive impact on the kinetics, robustness of colony formation and reprogramming efficiency. Additionally, substrate stiffness influences several phenotypic features of iPS cells and colonies, and data indicates that soft substrates favor full iPSC reprogramming.

Polynorbornenes of different molecular weights were synthesised by addition polymerisation, using a nickel based catalyst. This polymerisation route leads to amorphous polymers that were shown to display unique properties: high glass transition temperature, rigid random coil conformation, and dense packing in the amorphous state. Gas permeation membranes were prepared from these addition type nonsubstituted polynorbornenes. A study of their gas transport properties was performed and compared with both substituted addition polynorbornenes and ring opening metathesis polymerised polynorbornenes. The observed behaviour, in terms of permeability and selectivity, is in accordance with the low free volume and the dense packing confirmed by positron annihilation spectroscopy and WAXD in these polymer structures. ᭧

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 cell's 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.

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Chromatin structure is a major regulator of transcription and gene expression. Herein we explore the use of osmotic modulation to modify the chromatin structure and reprogram gene expression. In this study we use the extracellular osmotic pressure as a chromatin structure and transcriptional modulator. Hyposmotic modulation promotes chromatin loosening and induces changes in RNA polymerase II (Pol II) activity. The chromatin decondensation opens space for higher amounts of DNA engaged RNA Pol II. Hyposmotic modulation constitutes an alternative route to manipulate cell fate decisions. This technology was tested in model protocols of induced pluripotency and transdifferentiation in cells growing in suspension and adherent to substrates, CD34+ umbilical-cord-blood (UCB), fibroblasts and B-cells. The efficiency and kinetics of these cell fate modulation processes were improved by transient hyposmotic modulation of the cell environment.

Reverse micelles are studied in sodium AOT/water/isooctane mixtures as functions of AOT concentration (C AOT ), water to AOT mole ratio (w 0 ) and temperature (T), from 294 to 333 K, using positron annihilation lifetime spectroscopy (LS). By four-component analysis of the spectra, it is possible to extract the LS parameters (intensities, I i , and lifetimes, τ i ) of the triplet positronium (o-Ps) present in the aqueous (I 3 , τ 3 ) and organic (I 4 , τ 4 ) phases. The latter lifetime is constant and corresponds with the value measured in pure isooctane, while τ 3 is remarkably lower than the value for pure water. This difference is attributed to the out-diffusion of o-Ps from the water cores to isooctane. The relevant rigorous diffusion equations imply two fitting parameters, the radius of the water aggregates (r 0 ) and a transmission factor (h). Fixation of r 0 ) 3.6 nm for C AOT ) 0.1 M, w 0 ) 20, and T ) 294 K, as known from previous work, allows the quantitative derivation of the r 0 values for all other conditions. The water spheres appear to present some permeability to o-Ps, with a transmission factor h ) 0.12 nm -1 . The sphere radius increases smoothly with C AOT and w 0 and, more importantly, with T. The changes with w 0 give r 0 ) 0.181w 0 and 0.186w 0 nm at 294 and 298 K, respectively, and are in excellent agreement with previous proposals. The sum of the intensities, I tot ) I 3 + I 4 , is much lower than the o-Ps intensity in pure isooctane. In particular, at C AOT > 0.04 M, I tot appears very close to the value found for pure water. The possibility of a strong inhibition of Ps formation due to the micelles, as proposed in previous work, is ruled out because of the negligible electron or positron scavenging ability of alkyl sulfonates. It is thus concluded that Ps formation occurs primarily in the aqueous part of the micelles, the water aggregates representing efficient traps for the positrons while they are slowing down in the solutions. IntroductionPositronium (Ps), the bound state of a positron (e + ) with an electron (e -), has been used increasingly in recent decades as an efficient probe of the physical and chemical properties of matter. 1 The most commonly used positron annihilation technique (PAT) is lifetime spectroscopy (LS), which allows us to obtain both the lifetimes (τ i ) and relative abundances (or intensities, I i ) of the various positron species. Normally, in order of increasing lifetimes, these are singlet Ps (p-Ps, i ) 1), free positron (i ) 2), and triplet Ps (o-Ps, i ) 3). Because of its distinct long lifetime, the latter appears to be the most useful probe. The o-Ps decay occurs with the emission of two γ-rays through the pick-off process, i.e., the annihilation of the positron with one of the (bound) electrons of the surrounding medium. Information is obtained through two distinct, independent processes, Ps formation and annihilation, both of which depend on various characteristics of the systems under investigation. In liquids, Ps formation may occur on a very short time ...

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