HOPPSIGEN: a database of human and mouse processed pseudogenes

Processed pseudogenes result from reverse transcribed mRNAs. In general, because processed pseudogenes lack promoters, they are no longer functional from the moment they are inserted into the genome. Subsequently, they freely accumulate substitutions, insertions and deletions. Moreover, the ancestral structure of processed pseudogenes could be easily inferred using the sequence of their functional homologous genes. Owing to these characteristics, processed pseudogenes represent good neutral markers for studying genome evolution. Recently, there is an increasing interest for these markers, particularly to help gene prediction in the field of genome annotation, functional genomics and genome evolution analysis (patterns of substitution). For these reasons, we have developed a method to annotate processed pseudogenes in complete genomes. To make them useful to different fields of research, we stored them in a nucleic acid database after having annotated them. In this work, we screened both mouse and human complete genomes from ENSEMBL to find processed pseudogenes generated from functional genes with introns. We used a conservative method to detect processed pseudogenes in order to minimize the rate of false positive sequences. Within processed pseudogenes, some are still having a conserved open reading frame and some have overlapping gene locations. We designated as retroelements all reverse transcribed sequences and more strictly, we designated as processed pseudogenes, all retroelements not falling in the two former categories (having a conserved open reading or overlapping gene locations). We annotated 5823 retroelements (5206 processed pseudogenes) in the human genome and 3934 (3428 processed pseudogenes) in the mouse genome. Compared to previous estimations, the total number of processed pseudogenes was underestimated but the aim of this procedure was to generate a high-quality dataset. To facilitate the use of processed pseudogenes in studying genome structure and evolution, DNA sequences from processed pseudogenes, and their functional reverse transcribed homologs, are now stored in a nucleic acid database, HOPPSIGEN. HOPPSIGEN can be browsed on the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server (http://pbil.univ-lyon1.fr/) or fully downloaded for local installation

Transcriptional Coregulators: Fine-Tuning Metabolism

Metabolic homeostasis requires that cellular energy levels are adapted to environmental cues. This adaptation is largely regulated at the transcriptional level, through the interaction between transcription factors, coregulators, and the basal transcriptional machinery. Coregulators, which function as both metabolic sensors and transcriptional effectors, are ideally positioned to synchronize metabolic pathways to environmental stimuli. The balance between inhibitory actions of corepressors and stimulatory effects of coactivators enables the fine-tuning of metabolic processes. This tight regulation opens therapeutic opportunities to manage metabolic dysfunction by directing the activity of cofactors toward specific transcription factors, pathways, or cells/tissues, thereby restoring whole-body metabolic homeostasis

Identitag, a relational database for SAGE tag identification and interspecies comparison of SAGE libraries

BACKGROUND: Serial Analysis of Gene Expression (SAGE) is a method of large-scale gene expression analysis that has the potential to generate the full list of mRNAs present within a cell population at a given time and their frequency. An essential step in SAGE library analysis is the unambiguous assignment of each 14 bp tag to the transcript from which it was derived. This process, called tag-to-gene mapping, represents a step that has to be improved in the analysis of SAGE libraries. Indeed, the existing web sites providing correspondence between tags and transcripts do not concern all species for which numerous EST and cDNA have already been sequenced. RESULTS: This is the reason why we designed and implemented a freely available tool called Identitag for tag identification that can be used in any species for which transcript sequences are available. Identitag is based on a relational database structure in order to allow rapid and easy storage and updating of data and, most importantly, in order to be able to precisely define identification parameters. This structure can be seen like three interconnected modules : the first one stores virtual tags extracted from a given list of transcript sequences, the second stores experimental tags observed in SAGE experiments, and the third allows the annotation of the transcript sequences used for virtual tag extraction. It therefore connects an observed tag to a virtual tag and to the sequence it comes from, and then to its functional annotation when available. Databases made from different species can be connected according to orthology relationship thus allowing the comparison of SAGE libraries between species. We successfully used Identitag to identify tags from our chicken SAGE libraries and for chicken to human SAGE tags interspecies comparison. Identitag sources are freely available on web site. CONCLUSIONS: Identitag is a flexible and powerful tool for tag identification in any single species and for interspecies comparison of SAGE libraries. It opens the way to comparative transcriptomic analysis, an emerging branch of biology

Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models

Background: While many biological studies can be performed on cell-based systems, the investigation of molecular pathways related to complex human dysfunctions - e.g. neurodegenerative diseases - often requires long-term studies in animal models. The nematode Caenorhabditis elegans represents one of the best model organisms for many of these tests and, therefore, versatile and automated systems for accurate time-resolved analyses on C. elegans are becoming highly desirable tools in the field. Results: We describe a new multi-functional platform for C. elegans analytical research, enabling automated worm isolation and culture, reversible worm immobilization and long-term high-resolution imaging, and this under active control of the main culture parameters, including temperature. We employ our platform for in vivo observation of biomolecules and automated analysis of protein aggregation in a C. elegans model for amyotrophic lateral sclerosis (ALS). Our device allows monitoring the growth rate and development of each worm, at single animal resolution, within a matrix of microfluidic chambers. We demonstrate the progression of individual protein aggregates, i.e. mutated human superoxide dismutase 1 - Yellow Fluorescent Protein (SOD1-YFP) fusion proteins in the body wall muscles, for each worm and over several days. Moreover, by combining reversible worm immobilization and on-chip high-resolution imaging, our method allows precisely localizing the expression of biomolecules within the worms' tissues, as well as monitoring the evolution of single aggregates over consecutive days at the sub-cellular level. We also show the suitability of our system for protein aggregation monitoring in a C. elegans Huntington disease (HD) model, and demonstrate the system's ability to study long-term doxycycline treatment-linked modification of protein aggregation profiles in the ALS model. Conclusion: Our microfluidic-based method allows analyzing in vivo the long-term dynamics of protein aggregation phenomena in C. elegans at unprecedented resolution. Pharmacological screenings on neurodegenerative disease C. elegans models may strongly benefit from this method in the near future, because of its full automation and high-throughput potential

A microfluidic device for longitudinal studies of C.elegans neurodegenerative disease models

We describe a microfluidic device for automated culture and long-term high resolution imaging of Caenorhabditis elegans nematodes, which we specifically employed as model organisms for the analysis of neurodegenerative disease progression. In this platform, we integrated: (i) a microfluidic design tailored for the confinement of worms at the L2 larval stage in separate culture chambers by means of passive hydrodynamics; (ii) an optimized protocol for worm feeding and progeny removal at desired rate, allowing follow-up of the same worms over long-term studies; (iii) a technique for reversible C.elegans immobilization, based on the thermoreversible gelation of Pluronic F127 (PF127) inside the microfluidic chip; (iv) active control of the device temperature; (v) a compact device assembly, suitable for automated multi-dimensional imaging on any upright or inverted microscope

Tetracyclines Disturb Mitochondrial Function across Eukaryotic Models: A Call for Caution in Biomedical Research.

In recent years, tetracyclines, such as doxycycline, have become broadly used to control gene expression by virtue of the Tet-on/Tet-off systems. However, the wide range of direct effects of tetracycline use has not been fully appreciated. We show here that these antibiotics induce a mitonuclear protein imbalance through their effects on mitochondrial translation, an effect that likely reflects the evolutionary relationship between mitochondria and proteobacteria. Even at low concentrations, tetracyclines induce mitochondrial proteotoxic stress, leading to changes in nuclear gene expression and altered mitochondrial dynamics and function in commonly used cell types, as well as worms, flies, mice, and plants. Given that tetracyclines are so widely applied in research, scientists should be aware of their potentially confounding effects on experimental results. Furthermore, these results caution against extensive use of tetracyclines in livestock due to potential downstream impacts on the environment and human health

Label-free three-dimensional imaging of Caenorhabditis elegans with visible optical coherence microscopy

Fast, label-free, high-resolution, three-dimensional imaging platforms are crucial for high-throughput in vivo time-lapse studies of the anatomy of Caenorhabditis elegans, one of the most commonly used model organisms in biomedical research. Despite the needs, methods combining all these characteristics have been lacking. Here, we present label-free imaging of live Caenorhabditis elegans with three-dimensional sub-micrometer resolution using visible optical coherence microscopy (visOCM). visOCM is a versatile optical imaging method which we introduced recently for tomography of cell cultures and tissue samples. Our method is based on Fourier domain optical coherence tomography, an interferometric technique that provides three-dimensional images with high sensitivity, high acquisition rate and micrometer-scale resolution. By operating in the visible wavelength range and using a high NA objective, visOCM attains lateral and axial resolutions below 1 μm. Additionally, we use a Bessel illumination offering an extended depth of field of approximately 40 μm.We demonstrate that visOCM’s imaging properties allow rapid imaging of full sized living Caenorhabditis elegans down to the sub-cellular level. Our system opens the door to many applications such as the study of phenotypic changes related to developmental or ageing processes

An evolutionarily conserved role for the aryl hydrocarbon receptor in the regulation of movement

The BXD genetic reference population is a recombinant inbred panel descended from crosses between the C57BL/6 (B6) and DBA/2 (D2) strains of mice, which segregate for about 5 million sequence variants. Recently, some of these variants have been established with effects on general metabolic phenotypes such as glucose response and bone strength. Here we phenotype 43 BXD strains and observe they have large variation (∼5-fold) in their spontaneous activity during waking hours. QTL analyses indicate that ∼40% of this variance is attributable to a narrow locus containing the aryl hydrocarbon receptor (Ahr), a basic helix-loop-helix transcription factor with well-established roles in development and xenobiotic metabolism. Strains with the D2 allele of Ahr have reduced gene expression compared to those with the B6 allele, and have significantly higher spontaneous activity. This effect was also observed in B6 mice with a congenic D2 Ahr interval, and in B6 mice with a humanized AHR allele which, like the D2 allele, is expressed much less and has less enzymatic activity than the B6 allele. Ahr is highly conserved in invertebrates, and strikingly inhibition of its orthologs in D. melanogaster and C. elegans (spineless and ahr-1) leads to marked increases in basal activity. In mammals, Ahr has numerous ligands, but most are either non-selective (e.g. resveratrol) or highly toxic (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)). Thus, we chose to examine a major environmental influence-long term feeding with high fat diet (HFD)-to see if the effects of Ahr are dependent on major metabolic differences. Interestingly, while HFD robustly halved movement across all strains, the QTL position and effects of Ahr remained unchanged, indicating that the effects are independent. The highly consistent effects of Ahr on movement indicate that changes in its constitutive activity have a role on spontaneous movement and may influence human behavior