A dual model of normal vs isogenic Nrf2-depleted murine epithelial cells to explore oxidative stress involvement.

Cancer-derived cell lines are useful tools for studying cellular metabolism and xenobiotic toxicity, but they are not suitable for modeling the biological effects of food contaminants or natural biomolecules on healthy colonic epithelial cells in a normal genetic context. The toxicological properties of such compounds may rely on their oxidative properties. Therefore, it appears to be necessary to develop a dual-cell model in a normal genetic context that allows to define the importance of oxidative stress in the observed toxicity. Given that the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be the master regulator of antioxidant defenses, our aim was to develop a cellular model comparing normal and Nrf2-depleted isogenic cells to qualify oxidative stress-related toxicity. We generated these cells by using the CRISPR/Cas9 technique. Whole-genome sequencing enabled us to confirm that our cell lines were free of cancer-related mutations. We used 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product closely related to oxidative stress, as a model molecule. Here we report significant differences between the two cell lines in glutathione levels, gene regulation, and cell viability after HNE treatment. The results support the ability of our dual-cell model to study the role of oxidative stress in xenobiotic toxicity.

Asterics: a simple tool for the ExploRation and Integration of omiCS data.

BACKGROUND: The rapid development of omics acquisition techniques has induced the production of a large volume of heterogeneous and multi-level omics datasets, which require specific and sometimes complex analyses to obtain relevant biological information. Here, we present ASTERICS (version 2.5), a publicly available web interface for the analyses of omics datasets. RESULTS: ASTERICS is designed to make both standard and complex exploratory and integration analysis workflows easily available to biologists and to provide high quality interactive plots. Special care has been taken to provide a comprehensive documentation of the implemented analyses and to guide users toward sound analysis choices regarding some specific omics data. Data and analyses are organized in a comprehensive graphical workflow within ASTERICS workspace to facilitate the understanding of successive data editions and analyses leading to a given result. CONCLUSION: ASTERICS provides an easy to use platform for omics data exploration and integration. The modular organization of its open source code makes it easy to incorporate new workflows and analyses by external contributors. ASTERICS is available at https://asterics.miat.inrae.fr and can also be deployed using provided docker images.

The Histone Chaperone Network Is Highly Conserved in Physarum polycephalum.

The nucleosome is composed of histones and DNA. Prior to their deposition on chromatin, histones are shielded by specialized and diverse proteins known as histone chaperones. They escort histones during their entire cellular life and ensure their proper incorporation in chromatin. Physarum polycephalum is a Mycetozoan, a clade located at the crown of the eukaryotic tree. We previously found that histones, which are highly conserved between plants and animals, are also highly conserved in Physarum. However, histone chaperones differ significantly between animal and plant kingdoms, and this thus probed us to further study the conservation of histone chaperones in Physarum and their evolution relative to animal and plants. Most of the known histone chaperones and their functional domains are conserved as well as key residues required for histone and chaperone interactions. Physarum is divergent from yeast, plants and animals, but PpHIRA, PpCABIN1 and PpSPT6 are similar in structure to plant orthologues. PpFACT is closely related to the yeast complex, and the Physarum genome encodes the animal-specific APFL chaperone. Furthermore, we performed RNA sequencing to monitor chaperone expression during the cell cycle and uncovered two distinct patterns during S-phase. In summary, our study demonstrates the conserved role of histone chaperones in handling histones in an early-branching eukaryote.

Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Study of the colonic epithelial-mesenchymal dialogue through establishment of two activated or not mesenchymal cell lines: Activated and resting ones differentially modulate colonocytes in co-culture.

Continuous and rapid renewal of the colonic epithelium is crucial to resist the plethora of luminal deleterious agents. Subepithelial fibroblasts contribute to this turnover by regulating epithelial proliferation and differentiation. However, when intestinal homeostasis is disturbed, fibroblasts can acquire an activated phenotype and play a major role in the progression of intestinal pathologies. To evaluate the involvement of fibroblasts in the regulation of colonocytes under homeostatic or pathological conditions, we established resting and activated conditionally immortalized fibroblast cell lines (nF and mF) from mouse colonic mucosa. We then studied the epithelial-mesenchymal interactions between activated or resting fibroblasts and the normal mouse colonocytes (Co) using a co-culture model. Both fibroblastic cell lines were characterized by RT-qPCR, western blot and immunofluorescence assay. Our results showed that nF and mF cells were positive for fibroblastic markers such as vimentin and collagen 1, and negative for cytokeratin 18 and E-cadherin, attesting to their fibroblastic type. They also expressed proteins characteristic of the epithelial stem cell niche such as Grem1, CD90 or Wnt5a. Only rare nF fibroblasts were positive for α-SMA, whereas all mF fibroblasts strongly expressed this marker, supporting that mF cells were activated fibroblasts/myofibroblasts. In coculture, nF fibroblasts and Co cells strongly interacted via paracrine exchanges resulting in BMP4 production in nF fibroblasts, activation of BMP signaling in Co colonocytes, and decreased growth of colonocytes. Activated-type mF fibroblasts did not exert the same effects on Co cells, allowing colonocytes free to proliferate. In conclusion, these two colonic fibroblast lines, associated with Co cells in coculture, should allow to better understand the role of mesenchymal cells in the preservation of homeostasis and the development of intestinal pathologies.

SDR enzymes oxidize specific lipidic alkynylcarbinols into cytotoxic protein-reactive species.

Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.

Annual transcriptome of a key zooplankton species, the copepod Calanus finmarchicus.

The copepod Calanus finmarchicus (Crustacea, Copepoda) is a key zooplanktonic species with a crucial position in the North Atlantic food web and significant contributor to ocean carbon flux. Like many other high latitude animals, it has evolved a programmed arrested development called diapause to cope with long periods of limited food supply, while growth and reproduction are timed to take advantage of seasonal peaks in primary production. However, anthropogenic warming is inducing changes in the expected timing of phytoplankton blooms, suggesting phenological mismatches with negative consequences for the N. Atlantic ecosystem. While diapause mechanisms are mainly studied in terrestrial arthropods, specifically on laboratory model species, such as the fruit fly Drosophila, the molecular investigations of annual rhythms in wild marine species remain fragmentary. Here we performed a rigorous year-long monthly sampling campaign of C. finmarchicus in a Scottish Loch (UK; 56.45°N, 5.18°W) to generate an annual transcriptome. The mRNA of 36 samples (monthly triplicate of 25 individuals) have been deeply sequenced with an average depth of 137 ± 4 million reads (mean ± SE) per sample, aligned to the reference transcriptome, and filtered. We detail the quality assessment of the datasets and provide a high-quality resource for the investigation of wild annual transcriptomic rhythms (35,357 components) in a key diapausing zooplanktonic species.

Transcription-associated topoisomerase 2α (TOP2A) activity is a major effector of cytotoxicity induced by G-quadruplex ligands.

G-quadruplexes (G4) are non-canonical DNA structures found in the genome of most species including human. Small molecules stabilizing these structures, called G4 ligands, have been identified and, for some of them, shown to induce cytotoxic DNA double-strand breaks. Through the use of an unbiased genetic approach, we identify here topoisomerase 2α (TOP2A) as a major effector of cytotoxicity induced by two clastogenic G4 ligands, pyridostatin and CX-5461, the latter molecule currently undergoing phase I/II clinical trials in oncology. We show that both TOP2 activity and transcription account for DNA break production following G4 ligand treatments. In contrast, clastogenic activity of these G4 ligands is countered by topoisomerase 1 (TOP1), which limits co-transcriptional G4 formation, and by factors promoting transcriptional elongation. Altogether our results support that clastogenic G4 ligands act as DNA structure-driven TOP2 poisons at transcribed regions bearing G4 structures.

Widely rhythmic transcriptome in Calanus finmarchicus during the high Arctic summer solstice period.

Solar light/dark cycles and seasonal photoperiods underpin daily and annual rhythms of life on Earth. Yet, the Arctic is characterized by several months of permanent illumination ("midnight sun"). To determine the persistence of 24h rhythms during the midnight sun, we investigated transcriptomic dynamics in the copepod Calanus finmarchicus during the summer solstice period in the Arctic, with the lowest diel oscillation and the highest altitude of the sun's position. Here we reveal that in these extreme photic conditions, a widely rhythmic daily transcriptome exists, showing that very weak solar cues are sufficient to entrain organisms. Furthermore, at extremely high latitudes and under sea-ice, gene oscillations become re-organized to include <24h rhythms. Environmental synchronization may therefore be modulated to include non-photic signals (i.e. tidal cycles). The ability of zooplankton to be synchronized by extremely weak diel and potentially tidal cycles, may confer an adaptive temporal reorganization of biological processes at high latitudes.

Daily transcriptomes of the copepod Calanus finmarchicus during the summer solstice at high Arctic latitudes.

The zooplankter Calanus finmarchicus is a member of the so-called "Calanus Complex", a group of copepods that constitutes a key element of the Arctic polar marine ecosystem, providing a crucial link between primary production and higher trophic levels. Climate change induces the shift of C. finmarchicus to higher latitudes with currently unknown impacts on its endogenous timing. Here we generated a daily transcriptome of C. finmarchicus at two high Arctic stations, during the more extreme time of Midnight Sun, the summer solstice. While the southern station (74.5 °N) was sea ice-free, the northern one (82.5 °N) was sea ice-covered. The mRNAs of the 42 samples have been sequenced with an average of 126 ± 5 million reads (mean ± SE) per sample, and aligned to the reference transcriptome. We detail the quality assessment of the datasets and the complete annotation procedure, providing the possibility to investigate daily gene expression of this ecologically important species at high Arctic latitudes, and to compare gene expression according to latitude and sea ice-coverage.

Evidence for late Pleistocene origin of Astyanax mexicanus cavefish.

BACKGROUND: Cavefish populations belonging to the Mexican tetra species Astyanax mexicanus are outstanding models to study the tempo and mode of adaptation to a radical environmental change. They are currently assigned to two main groups, the so-called "old" and "new" lineages, which would have populated several caves independently and at different times. However, we do not have yet accurate estimations of the time frames of evolution of these populations. RESULTS: We reanalyzed the geographic distribution of mitochondrial and nuclear DNA polymorphisms and we found that these data do not support the existence of two cavefish lineages. Using IMa2, a program that allows dating population divergence in addition to demographic parameters, we found that microsatellite polymorphism strongly supports a very recent origin of cave populations (< 20,000 years). We identified a large number of single-nucleotide polymorphisms (SNPs) in transcript sequences of pools of embryos (Pool-seq) belonging to Pachón cave population and a surface population from Texas. Based on summary statistics that can be computed with this SNP data set together with simulations of evolution of SNP polymorphisms in two recently isolated populations, we looked for sets of demographic parameters that allow the computation of summary statistics with simulated populations that are similar to the ones with the sampled populations. In most simulations for which we could find a good fit between the summary statistics of observed and simulated data, the best fit occurred when the divergence between simulated populations was less than 30,000 years. CONCLUSIONS: Although it is often assumed that some cave populations have a very ancient origin, a recent origin of these populations is strongly supported by our analyses of independent sets of nuclear DNA polymorphism. Moreover, the observation of two divergent haplogroups of mitochondrial and nuclear genes with different geographic distributions support a recent admixture of two divergent surface populations, before the isolation of cave populations. If cave populations are indeed only several thousand years old, many phenotypic changes observed in cavefish would thus have mainly involved the fixation of genetic variants present in surface fish populations and within a very short period of time.

A comparative transcriptomic approach to understanding the formation of cork.

KEY MESSAGE: The transcriptome comparison of two oak species reveals possible candidates accounting for the exceptionally thick and pure cork oak phellem, such as those involved in secondary metabolism and phellogen activity. Cork oak, Quercus suber, differs from other Mediterranean oaks such as holm oak (Quercus ilex) by the thickness and organization of the external bark. While holm oak outer bark contains sequential periderms interspersed with dead secondary phloem (rhytidome), the cork oak outer bark only contains thick layers of phellem (cork rings) that accumulate until reaching a thickness that allows industrial uses. Here we compare the cork oak outer bark transcriptome with that of holm oak. Both transcriptomes present similitudes in their complexity, but whereas cork oak external bark is enriched with upregulated genes related to suberin, which is the main polymer responsible for the protective function of periderm, the upregulated categories of holm oak are enriched in abiotic stress and chromatin assembly. Concomitantly with the upregulation of suberin-related genes, there is also induction of regulatory and meristematic genes, whose predicted activities agree with the increased number of phellem layers found in the cork oak sample. Further transcript profiling among different cork oak tissues and conditions suggests that cork and wood share many regulatory mechanisms, probably reflecting similar ontogeny. Moreover, the analysis of transcripts accumulation during the cork growth season showed that most regulatory genes are upregulated early in the season when the cork cambium becomes active. Altogether our work provides the first transcriptome comparison between cork oak and holm oak outer bark, which unveils new regulatory candidate genes of phellem development.

Complete Genome Sequence and Annotation of the Staphylococcus aureus Strain HG001.

Staphylococcus aureus is an opportunistic Gram-positive pathogen responsible for a wide range of infections from minor skin abscesses to life-threatening diseases. Here, we report the draft genome assembly and current annotation of the HG001 strain, a derivative of the RN1 (NCT8325) strain with restored rbsU (a positive activator of SigB).

Whole blood transcriptome analysis reveals potential competition in metabolic pathways between negative energy balance and response to inflammatory challenge.

Negative Energy Balance (NEB) is considered to increase susceptibility to mastitis. The objective of this study was to improve our understanding of the underlying mechanisms by comparing transcriptomic profiles following NEB and a concomitant mammary inflammation. Accordingly, we performed RNA-seq analysis of blood cells in energy-restricted ewes and control-diet ewes at four different time points before and after intra mammary challenge with phlogogenic ligands. Blood leucocytes responded to NEB by shutting down lipid-generating processes, including cholesterol and fatty acid synthesis, probably under transcriptional control of SREBF 1. Furthermore, fatty acid oxidation was activated and glucose oxidation and transport inhibited in response to energy restriction. Among the differentially expressed genes (DEGs) in response to energy restriction, 64 genes were also differential in response to the inflammatory challenge. Opposite response included the activation of cholesterol and fatty acid synthesis during the inflammatory challenge. Moreover, activation of glucose oxidation and transport coupled with the increase of plasma glucose concentration in response to the inflammatory stimuli suggested a preferential utilization of glucose as the energy source during this stress. Leucocyte metabolism therefore undergoes strong metabolic changes during an inflammatory challenge, which could be in competition with those induced by energy restriction.

Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula.

The legume-Rhizobium symbiosis leads to the formation of a new organ, the root nodule, involving coordinated and massive induction of specific genes. Several genes controlling DNA methylation are spatially regulated within the Medicago truncatula nodule, notably the demethylase gene, DEMETER (DME), which is mostly expressed in the differentiation zone. Here, we show that MtDME is essential for nodule development and regulates the expression of 1,425 genes, some of which are critical for plant and bacterial cell differentiation. Bisulphite sequencing coupled to genomic capture enabled the identification of 474 regions that are differentially methylated during nodule development, including nodule-specific cysteine-rich peptide genes. Decreasing DME expression by RNA interference led to hypermethylation and concomitant downregulation of 400 genes, most of them associated with nodule differentiation. Massive reprogramming of gene expression through DNA demethylation is a new epigenetic mechanism controlling a key stage of indeterminate nodule organogenesis during symbiotic interactions.

Sequencing and characterization of a multi-organ Arctic charr transcriptome: A toolbox for investigating polymorphism and seasonal life in a high Arctic fish.

The Arctic charr (Salvelinus alpinus L.) inhabits fresh water ecosystems of the high North. The species has developed a strong phenotypic plasticity and variability in life history characteristics which has made this species an attractive model for investigations on phenotype plasticity, morph formation and ecological speciation. Further, the extreme seasonal variations in environmental conditions (e.g. food availability) in the high North induce seasonal changes in phenotype, which require precise timing mechanisms and physiological preparations. Individual gating of life-history strategies (e.g. formation of resident and sea-migrating morphs) and transitions (e.g. maturation) depends on conditional traits (size/energy status) at specific assessment time windows, and complex neuroendocrine regulation, which so far is poorly understood. In the absence of a reference genome, and in order to facilitate the investigation of the complex biological mechanisms of this unique fish model, the present study reveals a reference transcriptome for the Arctic charr. Using Roche 454 GS FLX+, we targeted various organs being either at the crossroads of many key pathways (neuroendocrine, metabolic, behavioral), of different ontological origins or displaying complementary physiological functions. The assemblage yielded 34,690 contigs greater than 1000bp with an average length (1690bp) and annotation rate (52%) within the range, or even higher, than what has been previously obtained with other teleost de novo transcriptomes. We dramatically improve the publically available transcript data on this species that may indeed be useful for various disciplines, from basic research to applied aspects related to conservation issues and aquaculture.

Jflow: a workflow management system for web applications.

SUMMARY: Biologists produce large data sets and are in demand of rich and simple web portals in which they can upload and analyze their files. Providing such tools requires to mask the complexity induced by the needed High Performance Computing (HPC) environment. The connection between interface and computing infrastructure is usually specific to each portal. With Jflow, we introduce a Workflow Management System (WMS), composed of jQuery plug-ins which can easily be embedded in any web application and a Python library providing all requested features to setup, run and monitor workflows. AVAILABILITY AND IMPLEMENTATION: Jflow is available under the GNU General Public License (GPL) at http://bioinfo.genotoul.fr/jflow. The package is coming with full documentation, quick start and a running test portal. CONTACT: Jerome.Mariette@toulouse.inra.fr.

The oak gene expression atlas: insights into Fagaceae genome evolution and the discovery of genes regulated during bud dormancy release.

BACKGROUND: Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. RESULTS: The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. CONCLUSION: In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes.

A unigene set for European beech (Fagus sylvatica L.) and its use to decipher the molecular mechanisms involved in dormancy regulation.

Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in nonmodel species. We generated 47,357 Sanger ESTs and 2.2M Roche-454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21,057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28,079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model-based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.