La transition fruit immature/fruit mature: analyse globale des profils transcriptomiques

RNASeq allowed us to measure the mRNA expression levels in tomato fruits in the presence of external signals (hormone treatment). Analysis of this data identified a number of genes actively responding to treatment, thus shedding light on the process of the fruit ripening. Our RNASeq experiment revealed hundreds of new transcripts (genes and RNA) in tomato which were previously un-annotated. We identified a need and started a development of robust statistical methods for DE genes identificatio

Comparison of normalization methods for differential gene expression analysis in RNA-Seq experiments: A matter of relative size of studied transcriptomes

In recent years, RNA-Seq technologies became a powerful tool for transcriptome studies. However, computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. In particular, it is known that the choice of a normalization procedure leads to a great variability in results of differential gene expression analysis. The present study compares the most widespread normalization procedures and proposes a novel one aiming at removing an inherent bias of studied transcriptomes related to their relative size. Comparisons of the normalization procedures are performed on real and simulated data sets. Real RNA-Seq data sets analyses, performed with all the different normalization methods, show that only 50% of significantly differentially expressed genes are common. This result highlights the influence of the normalization step on the differential expression analysis. Real and simulated data sets analyses give similar results showing 3 different groups of procedures having the same behavior. The group including the novel method named “Median Ratio Normalization” (MR N) gives the lower number of false discoveries. Within this group the MR N method is less sensitive to the modification of parameters related to the relative size of transcriptomes such as the number of down- and upregulated genes and the gene expression levels. The newly proposed MR N method efficiently deals with intrinsic bias resulting from relative size of studied transcriptomes. Validation with real and simulated data sets confirmed that MR N is more consistent and robust than existing methods

Synergetic effect of recoverin and calmodulin on regulation of rhodopsin kinase.

Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca(2+)/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca(2+)-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca(2+)-sensing protein. The synergetic inhibition of RK by both Ca(2+)-sensors occurs over a broader range of Ca(2+)-concentration than by recoverin alone, indicating increased Ca(2+)-sensitivity of RK regulation in the presence of both Ca(2+)-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

<p>Abstract</p> <p>Background</p> <p>The phylum <it>Verrucomicrobia </it>is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the <it>Verrucomicrobia</it>, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.</p> <p>Results</p> <p>We report the complete genome sequence of strain V4, the first one from a representative of the <it>Verrucomicrobia</it>. Isolate V4, initially named "<it>Methylokorus infernorum</it>" (and recently renamed <it>Methylacidiphilum infernorum</it>) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of <it>M. infernorum </it>was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C₁-utilization pathways compared to other known methylotrophs. The <it>M. infernorum </it>genome does not encode tubulin, which was previously discovered in bacteria of the genus <it>Prosthecobacter</it>, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping <it>Planctomycetes</it>, <it>Verrucomicrobia </it>and <it>Chlamydiae </it>into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the <it>M. infernorum </it>lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of <it>M. infernorum </it>shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.</p> <p>Conclusion</p> <p>The results of genome analysis of <it>M. infernorum </it>support the monophyly of the PVC superphylum. <it>M. infernorum </it>possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways <it>via </it>horizontal gene transfer, in particular, from <it>Proteobacteria</it>.</p> <p>Reviewers</p> <p>This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.</p

Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

The excessive light illumination of mammalian retina is known to induce oxidative stress and photoreceptor cell death linked to progression of age-related macular degeneration. The photochemical damage of photoreceptors is suggested to occur via two apoptotic pathways that involve either excessive rhodopsin activation or constitutive phototransduction, depending on the light intensity. Both pathways are dramatically activated in the absence of rhodopsin desensitization by GRK1. Previously, we have shown that moderate illumination (halogen lamp, 1,500 lx, 1–5 h) of mammalian eyes provokes disulfide dimerization of recoverin, a calcium-dependent regulator of GRK1. Here, we demonstrate under in vivo conditions that both moderate long-term (metal halide lamp, 2,500 lx, 14 h, rat model) and intense short-term (halogen lamp, 30,000 lx for 3 h, rabbit model) illumination of the mammalian retina are accompanied by accumulation of disulfide dimer of recoverin. Furthermore, in the second case we reveal alternatively oxidized derivatives of the protein, apparently including its monomer with sulfinic group. Histological data indicate that thiol oxidation of recoverin precedes apoptosis of photoreceptors. Both disulfide dimer and oxidized monomer (or oxidation mimicking C39D mutant) of recoverin exhibit lowered α-helical content and thermal stability of their apo-forms, as well as increased Ca2+ affinity. Meanwhile, the oxidized monomer and C39D mutant of recoverin demonstrate impaired ability to bind photoreceptor membranes and regulate GRK1, whereas disulfide dimer exhibits notably improved membrane binding and GRK1 inhibition in absence of Ca2+. The latter effect is expected to slow down rhodopsin desensitization in the light, thereby favoring support of the light-induced oxidative stress, ultimately leading to photoreceptor apoptosis. Overall, the intensity and duration of illumination of the retina affect thiol oxidation of recoverin likely contributing to propagation of the oxidative stress and photoreceptor damage

Assembling the Marine Metagenome, One Cell at a Time

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa

Software Trajectory Analysis: An Empirically Based Method for Automated Software Process Discovery

Ph.D. University of Hawaii at Manoa 2015.Includes bibliographical references.Recurrent behaviors are considered to be the basic building blocks of any human-driven goaloriented process, reflecting the development of efficient ways for dealing with common tasks based on past performance. Thus, the ability to discover recurrent behaviors is utterly important for a bottom-up systematic study, modeling, and improvement of human-driven processes. In the context of software development, whose ultimate goal is the delivery of software, the ability to recognize recurrent behaviors enables the understanding, formal description, and effective guidance of evolving software processes. While a number of approaches for recurrent behavior discovery and software process modeling and improvement have been previously proposed, they typically built upon online intrusive techniques, such as observations and interviewing, therefore expensive, suffering from biases, and unwelcome by software developers. In this exploratory study, I have developed and tested the idea of software process discovery via off-line analysis of software process artifacts. For this, I have prototyped and evaluated the Software Trajectory Analysis framework, which is built upon the definition of the “software trajectory” data type, that is a temporally ordered sequence of software artifact measurements, and a novel technique for temporal data classification, that enables the discovery and ranking of software trajectory characteristic patterns. By analogy with the notion of trajectory in Physics, which describes a projectile path in metric space, a software trajectory describes the software process and product progression in a space of chosen software metrics, whereas its recurrent structural patterns are related to the recurrent behaviors. The claim of this dissertation is that (1) it is possible to discover recurrent behaviors off-line via systematic study of software artifacts, (2) the Software Trajectory Analysis framework provides an effective off-line approach to recurrent software process-characteristic behaviors discovery. In addition to the extensive experimental evaluation of a proposed algorithm for time series characteristic pattern discovery, three empirical case studies were carried out to evaluate the claim: two using software artifacts from public software repositories and one using the public dump of a Q&A web site. The results suggest that Software Trajectory Analysis is capable of discovering software process-characteristic recurrent behaviors off-line, though their sensible interpretation is sometimes difficult

La transition fruit immature/fruit mature: analyse globale des profils transcriptomiques

National audienceRNASeq allowed us to measure the mRNA expression levels in tomato fruits in the presence of external signals (hormone treatment). Analysis of this data identified a number of genes actively responding to treatment, thus shedding light on the process of the fruit ripening. Our RNASeq experiment revealed hundreds of new transcripts (genes and RNA) in tomato which were previously un-annotated. We identified a need and started a development of robust statistical methods for DE genes identificatio

Finding discriminative and interpretable patterns in sequences of surgical activities

International audienceObjective Surgery is one of the riskiest and most important medical acts that is performed today. Understanding the ways in which surgeries are similar or different from each other is of major interest to understand and analyze surgical behaviors. This article addresses the issue of identifying discriminative patterns of surgical practice from recordings of surgeries. These recordings are sequences of low-level surgical activities representing the actions performed by surgeons during surgeries.Materials and method To discover patterns that are specific to a group of surgeries, we use the vector space model (VSM) which is originally an algebraic model for representing text documents. We split long sequences of surgical activities into subsequences of consecutive activities. We then compute the relative frequencies of these subsequences using the tf*idf framework and we use the Cosine similarity to classify the sequences. This process makes it possible to discover which patterns discriminate one set of surgeries recordings from another set.Results Experiments were performed on 40 neurosurgeries of anterior cervical discectomy (ACD). The results demonstrate that our method accurately identifies patterns that can discriminate between (1) locations where the surgery took place, (2) levels of expertise of surgeons (i.e., expert vs. intermediate) and even (3) individual surgeons who performed the intervention. We also show how the tf*idf weight vector can be used to both visualize the most interesting patterns and to highlight the parts of a given surgery that are the most interesting.Conclusions Identifying patterns that discriminate groups of surgeon is a very important step in improving the understanding of surgical processes. The proposed method finds discriminative and interpretable patterns in sequences of surgical activities. Our approach provides intuitive results, as it identifies automatically the set of patterns explaining the differences between the groups