A gene-rich, transcriptionally active environment and the pre-deposition of repressive marks are predictive of susceptibility to KRAB/KAP1-mediated silencing.

AbstractBACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.

Regulation of adaptive behaviour during fasting by hypothalamic Foxa2.

The lateral hypothalamic area is considered the classic 'feeding centre', regulating food intake, arousal and motivated behaviour through the actions of orexin and melanin-concentrating hormone (MCH). These neuropeptides are inhibited in response to feeding-related signals and are released during fasting. However, the molecular mechanisms that regulate and integrate these signals remain poorly understood. Here we show that the forkhead box transcription factor Foxa2, a downstream target of insulin signalling, regulates the expression of orexin and MCH. During fasting, Foxa2 binds to MCH and orexin promoters and stimulates their expression. In fed and in hyperinsulinemic obese mice, insulin signalling leads to nuclear exclusion of Foxa2 and reduced expression of MCH and orexin. Constitutive activation of Foxa2 in the brain (Nes-Cre/+;Foxa2T156A(flox/flox) genotype) results in increased neuronal MCH and orexin expression and increased food consumption, metabolism and insulin sensitivity. Spontaneous physical activity of these animals in the fed state is significantly increased and is similar to that in fasted mice. Conditional activation of Foxa2 through the T156A mutation expression in the brain of obese mice also resulted in improved glucose homeostasis, decreased fat and increased lean body mass. Our results demonstrate that Foxa2 can act as a metabolic sensor in neurons of the lateral hypothalamic area to integrate metabolic signals, adaptive behaviour and physiological responses.

Origen de la multicel·lularitat : una aproximació genòmica i funcional

En el present estudi s'analitza l'origen i evolució de 2 molècules claus pera entendre la multicel·lularitat dels animals: les molècules d'adhesió integrines i els factors de transcripció T-box. S’utilitzen els genomes recentment publicats de protists unicel•lulars parents propers dels animals. S’analitza l’origen i evolució d’aquests gens mitjançant anàlisi filogènic, determinació de motius funcionals i també tècniques de biologia molecular. A més, es documenta un cas de transferència gènica horitzontal des d'un eucariota cap a un procariota, fenomen poc habitual. Les principals conclusions són que tant l’adhesoma d'integrina com els gens T-box tenen un origen molt anterior als animals, en un context unicel•lular, i que després foren cooptats pel llinatge multicel•lular dels animals.

Eina gràfica per la creació d'autòmats per a la representació del llenguatge natural

Aquest projecte tracta la implementació d’una eina gràfica multiplataforma de creació i edició de gramàtiques electròniques per representar el Llenguatge Natural. És una eina per lingüistes i projectes com Spanish FrameNet Project amb la quan poden representar fàcilment transductors en un format més visual, les transicions es representen en forma de “caixes”, i guardar els resultats. S’han implementat varies opcions per crear una eina còmode i personalitzable per l’usuari amb funcionalitats enfocades a les seves necessitats com importar/exportar autòmats des d’una Expressió Regular. Es tracta l’implementació de tots els components que s’han necessitat per crear la GUI així com la seva funcionalitat.

Habitat, breeding performance, diet and individual age in Swiss barn owls (Tyto alba)

Intensification of farming over the past 50 years has homogenised the landscape structure and contributed to the decline of bird populations in Europe. To better target the conservation of the Barn Owl Tyto alba, we assessed the influence of the landscape structure on breeding performance in western Switzerland. The analyses considered a 23-year data set of breeding parameters collected in an area dominated by intensive agriculture. Using a Geographic Information System approach, landscape characteristics were described around 194 nest sites. Our analyses showed that nest-box occupancy, laying date, clutch and brood size, egg volume and probability of producing a second annual clutch were not significantly associated with any of the eight principal landscape variables (agricultural land, woodland, urban area, hedgerows, cereals, sugar beet, maize and meadow). Nevertheless, the probability that a breeding pair occupied a nest-box decreased the more roads there were surrounding the nest-box. The absence of strong associations between habitat features and breeding parameters suggests that prey availability may be relatively similar between the different breeding sites. In our study area Barn Owls can always find suitable foraging habitats around most nest-boxes.

Electrode location and clinical outcome in hippocampal electrical stimulation for mesial temporal lobe epilepsy.

PURPOSE: To study the clinical outcome in hippocampal deep brain stimulation (DBS) for the treatment of patients with refractory mesial temporal lobe epilepsy (MTLE) according to the electrode location. METHODS: Eight MTLE patients implanted in the hippocampus and stimulated with high-frequency DBS were included in this study. Five underwent invasive recordings with depth electrodes to localize ictal onset zone prior to chronic DBS. Position of the active contacts of the electrode was calculated on postoperative imaging. The distances to the ictal onset zone were measured as well as atlas-based hippocampus structures impacted by stimulation were identified. Both were correlated with seizure frequency reduction. RESULTS: The distances between active electrode location and estimated ictal onset zone were 11±4.3 or 9.1±2.3mm for patients with a >50% or <50% reduction in seizure frequency. In patients (N=6) showing a >50% seizure frequency reduction, 100% had the active contacts located <3mm from the subiculum (p<0.05). The 2 non-responders patients were stimulated on contacts located >3mm to the subiculum. CONCLUSION: Decrease of epileptogenic activity induced by hippocampal DBS in refractory MTLE: (1) seems not directly associated with the vicinity of active electrode to the ictal focus determined by invasive recordings; (2) might be obtained through the neuromodulation of the subiculum.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

The vigilance components of begging and sibling competition

The conflict between siblings over how parental resources are divided has promoted the evolution of specific behaviour to outcompete each other. Young animals look out for parents' arrival in order to start begging as quickly as possible, since a rapid begging reaction increases the likelihood of being fed before nestmates. If the young can physically intercept the parents, selection might be operating on the offspring ability to monitor parent arrival (vigilance towards parents) and any sudden modifications in siblings' behaviour (vigilance towards siblings). To investigate the adaptive value of nestling vigilance in the context of family interactions, we recorded which direction barn owl Tyto alba siblings were facing in 89 two-chick broods before the first parental feeding visit of the night. Nestlings were more vigilant towards nest entrance than expected by chance suggesting that vigilance towards parents is an important component of sibling competition. When positioned near the nest-box entrance where parents predictably deliver food, the younger individual (i.e. junior) looked more towards the entrance than its older sibling. Thus, when the likelihood of obtaining a food item is relatively high, juniors are more vigilant than seniors to detect the incoming parent. When positioned at the back of the nest, the senior looked relatively more frequently towards its sibling than the junior did in the same situation. This suggests that when the likelihood of obtaining a food item is relatively low, seniors are more vigilant than juniors to observe their sibling. Because vigilance was not related to hunger level and prey obtaining, we propose the hypothesis that vigilance towards parents and siblings only indirectly influences the outcome of sibling competition.

Evaluation of two minimally invasive techniques for electroencephalogram recording in wild or freely behaving animals.

Insight into the function of sleep may be gained by studying animals in the ecological context in which sleep evolved. Until recently, technological constraints prevented electroencephalogram (EEG) studies of animals sleeping in the wild. However, the recent development of a small recorder (Neurologger 2) that animals can carry on their head permitted the first recordings of sleep in nature. To facilitate sleep studies in the field and to improve the welfare of experimental animals, herein, we test the feasibility of using minimally invasive surface and subcutaneous electrodes to record the EEG in barn owls. The EEG and behaviour of four adult owls in captivity and of four chicks in a nest box in the field were recorded. We scored a 24-h period for each adult bird for wakefulness, slow-wave sleep (SWS), and rapid-eye movement (REM) sleep using 4 s epochs. Although the quality and stability of the EEG signals recorded via subcutaneous electrodes were higher when compared to surface electrodes, the owls' state was readily identifiable using either electrode type. On average, the four adult owls spent 13.28 h awake, 9.64 h in SWS, and 1.05 h in REM sleep. We demonstrate that minimally invasive methods can be used to measure EEG-defined wakefulness, SWS, and REM sleep in owls and probably other animals.

Promoter architecture of mouse olfactory receptor genes.

Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Promoter and transcription analysis of penicillin-binding protein genes in Streptococcus gordonii.

An optimally cross-linked peptidoglycan requires both transglycosylation and transpeptidation, provided by class A and class B penicillin-binding proteins (PBPs). Streptococcus gordonii possesses three class A PBPs (PBPs 1A, 1B, and 2A) and two class B PBPs (PBPs 2B and 2X) that are important for penicillin resistance. High-level resistance (MIC, > or =2 microg/ml) requires mutations in class B PBPs. However, although unmutated, class A PBPs are critical to facilitate resistance development (M. Haenni and P. Moreillon, Antimicrob. Agents Chemother. 50:4053-4061, 2006). Thus, their overexpression might be important to sustain the drug. Here, we determined the promoter regions of the S. gordonii PBPs and compared them to those of other streptococci. The extended -10 box was highly conserved and complied with a sigma(A)-type promoter consensus sequence. In contrast, the -35 box was poorly conserved, leaving the possibility of differential PBP regulation. Gene expression in a penicillin-susceptible parent (MIC, 0.008 microg/ml) and a high-level-resistant mutant (MIC, 2 microg/ml) was monitored using luciferase fusions. In the absence of penicillin, all PBPs were constitutively expressed, but their expression was globally increased (1.5 to 2 times) in the resistant mutant. In the presence of penicillin, class A PBPs were specifically overexpressed both in the parent (PBP 2A) and in the resistant mutant (PBPs 1A and 2A). By increasing transglycosylation, class A PBPs could promote peptidoglycan stability when transpeptidase is inhibited by penicillin. Since penicillin-related induction of class A PBPs occurred in both susceptible and resistant cells, such a mutation-independent facilitating mechanism could be operative at each step of resistance development.

Field trials of an improved cost-effective device for detecting peridomestic populations of Triatoma infestans (Hemiptera: Reduviidae) in rural Argentina

An improved device for detecting peridomestic Triatoma infestans consisting of one-liter recycled Tetra Brik milk boxes with a central structure was tested using a matched-pair study design in two rural areas in Argentina. In Olta (La Rioja), the boxes were installed beneath the thatched roofs and on the vertical wooden posts of each peridomestic structure. After a 5-month exposure, at least one of the recovered boxes detected 88% of the 24 T. infestans-positive sites, and 86% of the 7 negative sites by timed manual collections at baseline. In Amamá (Santiago del Estero), the boxes were paired with the best performing prototype tested before (shelter unit). After 3 months, some evidence of infestation was detected in 89% (boxes) and 79% (shelters) of 18-19 sites positive by timed collections, whereas 19% and 16% of 32 negative sites were positive, respectively. Neither device differed significantly in the qualitative or quantitative collection of every sign of infestation. The installation site did not modify significantly the boxes' sampling efficiency in both study areas. As the total cost of each box was half as expensive as each shelter unit, the boxes are thus the most cost-effective and easy-to-use tool for detecting peridomestic T. infestans currently available.

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni.

Activité professionnelle des utilisateurs d'implant cochléaire [Professional occupation after cochlear implantation]

This study verifies whether cochlear implants helps deaf adults to maintain or develop their professional occupations. Sixty-seven patients received a questionnaire concerning their professional activities before and after implantation. At the time of implantation 34 were professionally active. After the implantation 29 remained active, 4 of them reporting positive developments in their careers. Five patients became inactive. The previously inactive patients remained inactive. There was no difference in auditory performances between professionally active or inactive patients. Cochlear implants enable most implanted adults to maintain and even progress in their professions. However, deafness still represents an obstacle to social integration as inactive patients who searched for a job were rejected after the job interviews.