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The majority of gene expression studies focus on the search for genes whose mean expression is different between two or more populations of samples in the so-called "differential expression analysis" approach. However, a difference in variance in gene expression may also be biologically and physiologically relevant. In the classical statistical model used to analyze RNA-sequencing (RNA-seq) data, the dispersion, which defines the variance, is only considered as a parameter to be estimated prior to identifying a difference in mean expression between conditions of interest. Here, we propose to evaluate four recently published methods, which detect differences in both the mean and dispersion in RNA-seq data. We thoroughly investigated the performance of these methods on simulated datasets and characterized parameter settings to reliably detect genes with a differential expression dispersion. We applied these methods to The Cancer Genome Atlas datasets. Interestingly, among the genes with an increased expression dispersion in tumors and without a change in mean expression, we identified some key cellular functions, most of which were related to catabolism and were overrepresented in most of the analyzed cancers. In particular, our results highlight autophagy, whose role in cancerogenesis is context-dependent, illustrating the potential of the differential dispersion approach to gain new insights into biological processes and to discover new biomarkers. Author summary: Gene expression is the process by which genetic information is translated into functional molecules. Transcription is the first step of this process, consisting of synthesizing messenger RNAs. During recent decades, genome-wide transcriptional profiling technologies have made it possible to assess the expression levels of thousands of genes in parallel in a variety of biological contexts. In statistical analyses, the expression of a gene is estimated by counting sequencing reads over a set of samples and is defined by two dimensions: mean and variance. The overwhelming majority of gene expression studies focus on identifying genes whose mean expression significantly changes when comparing samples of different conditions of interest to gain knowledge of biological processes. In this classical approach, the variance is usually considered only as a noise parameter to be estimated before assessing the mean expression. However, finely estimating the variance of expression may be biologically relevant since a modification of this parameter may reflect a change in gene expression regulation. Here, we propose to evaluate the performance of statistical methods that identify such differentially variant genes. We highlighted the potential of this approach by analyzing cancer datasets, thus identifying key cellular functions in tumor progression. [ABSTRACT FROM AUTHOR]

We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion. The microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress. [ABSTRACT FROM AUTHOR]

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines. [ABSTRACT FROM AUTHOR]

The p63 protein plays a key role in regulating human keratinocyte proliferation and differentiation. Although some p63-regulating microRNAs (miRNAs) have been identified in the control of epidermal homeostasis, little is known about miRNAs acting downstream of p63. In this paper, we characterized multiple p63-regulated miRNAs (miR-17, miR-20b, miR-30a, miR-106a, miR-143 and miR-455-3p) and elucidated their roles in the onset of keratinocyte differentiation. We identified RB, p21 and multiple MAPKs as targets of these p63-controlled miRNAs. Upon inhibition of most of these miRNAs, we observed defects in commitment to differentiation that could be reversed by siRNA-mediated silencing of their targets. Furthermore, knockdown of MAPK8 and MAPK9 efficiently restored expression of the early differentiation markers keratin 1 and keratin 10 in p63-silenced primary human keratinocytes. These results highlight new mechanistic roles of multiple miRNAs, particularly the miR-17 family (miR-17, miR-20b and miR-106a), as regulatory intermediates for coordinating p63 with MAPK signaling in the commitment of human mature keratinocytes to early differentiation. [ABSTRACT FROM AUTHOR]

Background: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. Methodology/Principal Findings: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value,0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test. In situ hybridization of pre-mir-302a, pre-let-7b and mir-365, using specific labelled locked nucleic acids and confocal microscopy, demonstrated that these miRNA were localized in mitochondria of human myoblasts. Total RNA was extracted from enriched mitochondria isolated by an immunomagnetic method from a culture of human myotubes. The detection of 742 human miRNA (miRBase) were monitored by RT-qPCR at three increasing mtRNA inputs. Forty six miRNA were significantly expressed (2nd derivative method Cp.35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value,0.05). Conclusions/Significance: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria. [ABSTRACT FROM AUTHOR]

Epigenomes commonly refer to the sequence of presence/absence of specific epigenetic marks along eukaryotic chromatin. Complete histone-borne epigenomes have now been described at single-nucleosome resolution from various organisms, tissues, developmental stages, or diseases, yet their intra-species natural variation has never been investigated. We describe here that the epigenomic sequence of histone H3 acetylation at Lysine 14 (H3K14ac) differs greatly between two unrelated strains of the yeast Saccharomyces cerevisiae. Using single-nucleosome chromatin immunoprecipitation and mapping, we interrogated 58,694 nucleosomes and found that 5,442 of them differed in their level of H3K14 acetylation, at a false discovery rate (FDR) of 0.0001. These Single Nucleosome Epi-Polymorphisms (SNEPs) were enriched at regulatory sites and conserved non-coding DNA sequences. Surprisingly, higher acetylation in one strain did not imply higher expression of the relevant gene. However, SNEPs were enriched in genes of high transcriptional variability and one SNEP was associated with the strength of gene activation upon stimulation. Our observations suggest a high level of inter-individual epigenomic variation in natural populations, with essential questions on the origin of this diversity and its relevance to gene x environment interactions. [ABSTRACT FROM AUTHOR]

p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin. [ABSTRACT FROM AUTHOR]

Background: ID2 is a member of a subclass of transcription regulators belonging to the general bHLH (basic-helix-loophelix) family of transcription factors. In normal cells, ID2 is responsible for regulating the balance between proliferation and differentiation. More recent studies have demonstrated that ID2 is involved in tumor progression in several cancer types such as prostate or breast. Methodology/Principal Findings: In this work, we investigated, for the first time, the relationship between the expression of ID2 in non-small cell lung cancer (NSCLC) patients and the clinicopathological features and prognosis of these patients. Immunohistochemistry was performed on tissue microarrays, which included 62 NSCLC tumors. In malignant tissues, ID2 expression has been detected in both the nuclear and cytoplasmic compartments, but we have demonstrated that only nuclear expression of ID2 is inversely correlated with the differentiation grade of the tumor (p = 0.007). Interestingly, among patients with poorly differentiated tumors, high nuclear expression of ID2 was an independent and unfavorable prognostic factor for survival (p = 0.036). Conclusions: These results suggest that ID2 could be involved in tumor dedifferentiation processes of NSCLC, and could be used as prognostic marker for patients with poorly differentiated tumors. [ABSTRACT FROM AUTHOR]