The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template.

The interaction of Escherichia coli RNA polymerase with supercoiled DNA was visualized by cryo-electron microscopy of vitrified samples and by classical electron microscopy methods. We observed that when E. coli RNA polymerase binds to a promoter on supercoiled DNA, this promoter becomes located at an apical loop of the interwound DNA molecule. During transcription RNA polymerase shifts the apical loop along the DNA, always remaining at the top of the moving loop. This relationship between RNA polymerase and the supercoiled template precludes circling of the RNA polymerase around the DNA and prevents the growing RNA transcript from becoming entangled with the template DNA.

An exquisite cross-control mechanism among endothelial cell fate regulators directs the plasticity and heterogeneity of lymphatic endothelial cells.

Arteriovenous-lymphatic endothelial cell fates are specified by the master regulators, namely, Notch, COUP-TFII, and Prox1. Whereas Notch is expressed in the arteries and COUP-TFII in the veins, the lymphatics express all 3 cell fate regulators. Previous studies show that lymphatic endothelial cell (LEC) fate is highly plastic and reversible, raising a new concept that all 3 endothelial cell fates may co-reside in LECs and a subtle alteration can result in a reprogramming of LEC fate. We provide a molecular basis verifying this concept by identifying a cross-control mechanism among these cell fate regulators. We found that Notch signal down-regulates Prox1 and COUP-TFII through Hey1 and Hey2 and that activated Notch receptor suppresses the lymphatic phenotypes and induces the arterial cell fate. On the contrary, Prox1 and COUP-TFII attenuate vascular endothelial growth factor signaling, known to induce Notch, by repressing vascular endothelial growth factor receptor-2 and neuropilin-1. We show that previously reported podoplanin-based LEC heterogeneity is associated with differential expression of Notch1 in human cutaneous lymphatics. We propose that the expression of the 3 cell fate regulators is controlled by an exquisite feedback mechanism working in LECs and that LEC fate is a consequence of the Prox1-directed lymphatic equilibrium among the cell fate regulators.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

Prox1 interacts with Atoh1 and Gfi1, and regulates cellular differentiation in the inner ear sensory epithelia.

Inner ear hair cells and supporting cells arise from common precursors and, in mammals, do not show phenotypic conversion. Here, we studied the role of the homeodomain transcription factor Prox1 in the inner ear sensory epithelia. Adenoviral-mediated Prox1 transduction into hair cells in explant cultures led to strong repression of Atoh1 and Gfi1, two transcription factors critical for hair cell differentiation and survival. Luciferase assays showed that Prox1 can repress transcriptional activity of Gfi1 independently of Atoh1. Prox1 transduction into cochlear outer hair cells resulted in degeneration of these cells, consistent with the known phenotype of Gfi1-deficient mice. These results together with the widespread expression of endogenous Prox1 within the population of inner ear supporting cells point to the role for Prox1 in antagonizing the hair cell phenotype in these non-sensory cells. Further, in vivo analyses of hair cells from Gfi1-deficient mice suggest that the cyclin-dependent kinase inhibitor p57(Kip2) mediates the differentiation- and survival-promoting functions of Gfi1. These data reveal novel gene interactions and show that these interactions regulate cellular differentiation within the inner ear sensory epithelia. The data point to the tight regulation of phenotypic characteristics of hair cells and supporting cells.

Role of ATH5 in the specification of retinal ganglion cells and expression and cell compartmentalization of EFEMP1, a protein associated with Malattia Leventinese

ABSTRACT : The development of the retina is a very complex process, occurring through the progressive restriction of cell fates, from pluripotent cell populations to complex tissues and organs. In all vertebrate species analyzed so far, retinal differentiation starts with the generation of retinal ganglion cells (RGC)s. One of the documented key essential events in the specification of RGCs is the expression of ATHS, an atonal homolog encoding a bHLH transcription factor. Despite the putative role of master regulator of RGC differentiation, the mechanism of integrating its functions into a coherent program underlying the production of this subclass of retinal neurons has not yet been elucidated. By using chromatin immunoprecipitation combined with microarray (ChIP-on-chip) we have screened for ATH5 direct targets in the developing chick retina at two consecutive periods: E3.5 (stage HH22) and E6 (stage HH30), covering the stages of progenitor proliferation, neuroepithelium patterning, RGC specification, cell cycle exit and early neuronal differentiation. In parallel, complementary analysis with Affymetrix expression microarrays was conducted. We compared RGCs versus retina to see if the targets correspond to genes preferentially expressed in RGCs. We also precociously overexpressed ATH5 in the retina of individual embryo, and contralateral retina vas used as a control. Our integrated approach allowed us to establish a compendium of ATH5-targets and enabled us to position ATH5 in the transcription network underlying neurogenesis in the retina. Malattia Leventinese (ML) is an autosomal, dominant retinal dystrophy characterized by extracellular, amorphous deposits known as drusen, between the retinal pigment epithelium (RPE) and Bruch's membrane. On the genetic level, it has been associated with a single missense mutation (R345W) in a widely expressed gene with unknown function called EFEMP1. We determined expression patterns of the EFEMP1 gene in normal and ML human retinas. Our data shown that the upregulation of EFEMP1 is not specific to ML eye, except for the region of the ciliary body. We also analyzed the cell compartmentalization of different versions of the protein (both wild type and mutant). Our studies indicate that both abnormal expression of the EFEMP1 gene and mutation and accumulation of EFEMP 1 protein (inside or outside the cells) might contribute to the ML pathology. Résumé : 1er partie : L'ontogenèse de la rétine est un processus complexe au cours duquel des cellules progénitrices sont engagée, par vagues successives, dans des lignées où elles vont d'abord être déterminées puis vont se différencier pour finalement construire un tissu rétinien composé de cinq classes de neurones (les photorécepteurs, les cellules horizontales, bipolaires, amacrines et ganglionnaires) et d'une seule de cellules gliales (les cellules de Muller). Chez tous les vertébrés, la neurogenèse rétinienne est d'abord marquée par la production des cellules ganglionnaires (RGCs). La production de cette classe de neurone est liée à l'expression du gène ATH5 qui est un homologue du gène atonal chez la Drosophile et qui code pour un facteur de transcription de la famille des protéines basic Helix-Loop-Helix (bHLH). Malgré le rôle central que joue ATH5 dans la production des RGCs, le mécanisme qui intègre la fonction de cette protéine dans le programme de détermination neuronale et ceci en relation avec le développement de la rétine n'est pas encore élucidé. Grâce à une technologie qui permet de combiner la sélection de fragments de chromatine liant ATH5 et la recherche de séquences grâce à des puces d'ADN non-codants (ChIP-on-chip), nous avons recherché des cibles potentielles de la protéine ATH5 dans la rétine en développement. Nous avons conduit cette recherche à deux stades de développement de manière à englober la phase de prolifération cellulaire, la détermination des RGCs, la sortie du cycle cellulaire ainsi que les premières étapes de la différentiation de ces neurones. Des expériences complémentaires nous ont permis de définir les patrons d'expression des gènes sélectionnés ainsi que l'activité promotrice des éléments de régulation identifiés lors de notre criblage. Ces approches expérimentales diverses et complémentaires nous ont permis de répertorier des gènes cibles de la protéine ATH5 et d'établir ainsi des liens fonctionnels entre des voies métaboliques dont nous ne soupçonnions pas jusqu'alors qu'elles puissent être associées à la production d'une classe de neurones centraux. 2ème partie : Malattia Leventinese (ML) est une maladie génétique qui engendre une dystrophie de la rétine. Elle se caractérise par l'accumulation de dépôt amorphe entre l'épithélium pigmentaire et la membrane de Bruch et connu sous le nom de drusen. Cette maladie est liée à une simple mutation non-sens (R345W) dans un gène dénommé EFEMP1 qui est exprimé dans de nombreux tissus mais dont la fonction reste mal définie. Une étude détaillée de l'expression de ce gène dans des rétines humaines a révélé une expression à un niveau élevé du gène EFEMP1 dans divers tissus de l'oeil ML mais également dans des yeux contrôles. Alors que l'accumulation d'ARN messager EFEMP1 dans les cellules de l'épithélium pigmentaire n'est pas spécifique à ML, l'expression de ce gène dans le corps cilié n'a été observée que dans l'oeil ML. Nous avons également comparé la sécrétion de la protéine sauvage avec celle porteuse de la mutation. En résumé, notre étude révèle que le niveau élevé d'expression du gène EFEMP1 ainsi que l'accumulation de la protéine dans certains compartiments cellulaires pourraient contribuer au développement de pathologies rétiniennes liées à ML.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Résistance aux diurétiques de l'anse en clinique [Resistance to loop diuretics in clinical practice]

Loop diuretics belong to the most common medications used in ambulatory and hospitalized patients, especially in situation of hypervolemia and chronic renal failure. Prolonged used of these agents in particular medical conditions can lead to attenuation of their diuretic effect, commonly known as "resistance" to diuretics. This article intends to review the main mechanisms of resistance to loop diuretic and the ways to counteract them in clinical practice.

Per-antenna rate and power control for MIMO layered architectures in the low- and high-power regimes

In a MIMO layered architecture, several codewordsare transmitted from a multiplicity of antennas. Although thespectral efficiency is maximized if the rates of these codewordsare separately controlled, the feedback rate within the linkadaptation loop is reduced if they are constrained to be identical.This poses a direct tradeoff between performance andfeedback overhead. This paper provides analytical expressionsthat quantify the difference in spectral efficiency between bothapproaches for arbitrary numbers of antennas. Specifically, thecharacterization takes place in the realm of the low- and highpowerregimes via expansions that are shown to have a widerange of validity.In addition, the possibility of adjusting the transmit powerof each codeword individually is considered as an alternative tothe separate control of their rates. Power allocation, however,turns out to be inferior to rate control within the context of thisproblem.

Characterization of the ligand-binding site of the serotonin 5-HT3 receptor: the role of glutamate residues 97, 224, AND 235.

Ligand-gated ion channels of the Cys loop family are receptors for small amine-containing neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiology, radioligand binding, fluorescence measurements, and immunochemistry. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model.

A large deletion in the adRP gene PRPF31: evidence that haploinsufficiency is the cause of disease.

PURPOSE: To report a large deletion that encompasses more than 90% of PRPF31 gene and two other neighboring genes in their entirety in an adRP pedigree that appears to show only the typical clinical features of retinitis pigmentosa. METHODS: To identify PRPF31 mutation in a dominant RP family (ADRP2) previously linked to the RP11 locus, the 14 exons of PRPF31 were screened for mutations by direct sequencing. To investigate the possibility of a large deletion, microsatellite markers near PRPF31 gene were analyzed by non-denaturing PAGE. RESULTS: Initial screening of PRPF31 gene in the ADRP2 family did not reveal an obvious mutation. A large deletion was however suspected due to lack of heterozygosity for nearly all PRPF31 intragenic single nucleotide polymorphysm (SNPs). In order to estimate the size of the deletion, SNPs and microsatellite markers spanning and flanking PRPF31 were analyzed in the entire ADRP2 family. Haplotype analysis with the above markers suggested a deletion of approximately 30 kb that included the putative promoter region of a novel gene OSCAR, the entire genomic content of genes NDUFA3, TFPT and more than 90% of PRPF31 gene. Sequence analysis of the region flanking the potential deletion showed a high presence of Alu elements implicating Alu mediated recombination as the mechanism responsible for this event. CONCLUSIONS: This mutation provides evidence that haploinsufficiency rather than aberrant function of mutated proteins is the cause of disease in these adRP patients with mutations in PRPF31 gene.

Improving social measurements: the experience of the ESS first rounds in Spain

With the two aims of monitoring social change and improving social measurement, the European Social Survey is now closing its third round. This paper shows how the accumulated experience of the two first rounds has been used to validate the questionnaire, better adapt the sampling design to the country characteristics and efficiently commit fieldwork in Spain. For example, the dynamic character of the population nowadays makes necessary to estimated design effects at each round from the data of the previous round. The paper also demonstrates how, starting with a response rate of 52% at first round, a 66% response rate is achieved at the third round thanks to an extensive quality control conducted by the polling agency and the ESS national team and based on a detailed analysis of the non-response cases and the incidences reported by the interviewed in the contact form.

The SLC2 family of facilitated hexose and polyol transporters.

The SLC2 family of glucose and polyol transporters comprises 13 members, the glucose transporters (GLUT) 1-12 and the H(+)- myo-inositol cotransporter (HMIT). These proteins all contain 12 transmembrane domains with both the amino and carboxy-terminal ends located on the cytoplasmic side of the plasma membrane and a N-linked oligosaccharide side-chain located either on the first or fifth extracellular loop. Based on sequence comparison, the GLUT isoforms can be grouped into three classes: class I comprises GLUT1-4; class II, GLUT6, 8, 10, and 12 and class III, GLUT5, 7, 9, 11 and HMIT. Despite their sequence similarity and the presence of class-specific signature sequences, these transporters carry various hexoses and HMIT is a H(+)/ myo-inositol co-transporter. Furthermore, the substrate transported by some isoforms has not yet been identified. Tissue- and cell-specific expression of the well-characterized GLUT isoforms underlies their specific role in the control of whole-body glucose homeostasis. Numerous studies with transgenic or knockout mice indeed support an important role for these transporters in the control of glucose utilization, glucose storage and glucose sensing. Much remains to be learned about the transport functions of the recently discovered isoforms (GLUT6-13 and HMIT) and their physiological role in the metabolism of glucose, myo-inositol and perhaps other substrates.

Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation.

After primary growth, most dicotyledonous plants undergo secondary growth. Secondary growth involves an increase in the diameter of shoots and roots through formation of secondary vascular tissue. A hallmark of secondary growth initiation in shoots of dicotyledonous plants is the initiation of meristematic activity between primary vascular bundles, i.e. in the interfascicular regions. This results in establishment of a cylindrical meristem, namely the vascular cambium. Surprisingly, despite its major implications for plant growth and the accumulation of biomass, the molecular regulation of secondary growth is only poorly understood. Here, we combine histological, molecular and genetic approaches to characterize interfascicular cambium initiation in the Arabidopsis thaliana inflorescence shoot. Using genome-wide transcriptional profiling, we show that stress-related and touch-inducible genes are up-regulated in stem regions where secondary growth takes place. Furthermore, we show that the products of COI1, MYC2, JAZ7 and the touch-inducible gene JAZ10, which are components of the JA signalling pathway, are cambium regulators. The positive effect of JA application on cambium activity confirmed a stimulatory role of JA in secondary growth, and suggests that JA signalling triggers cell divisions in this particular context.

Improving social measurements: The experience of the ESS first rounds in Spain

With the two aims of monitoring social change and improving social measurement, the European Social Survey is now closing its third round. This paper shows how the accumulated experience of the two first rounds has been used to validate the questionnaire, better adapt the sampling design to the country characteristics and efficiently commit fieldwork in Spain. For example, the dynamic character of the population nowadays makes necessary to estimated design effects at each round from the data of the previous round. The paper also demonstrates how, starting with a response rate of 52% at first round, a 66% response rate is achieved at the third round thanks to an extensive quality control conducted by the polling agency and the ESS national team and based on a detailed analysis of the non-response cases and the incidences reported by the interviewed in the contact form.

The Anarak, Jandaq and Posht-e-Badam metamorphic complexes in central Iran: New geological data, relationships and tectonic implications

The Anarak, Jandaq and Posht-e-Badam metamorphic complexes occupy the NW part of the Central-East Iranian Microcontinent and are juxtaposed with the Great Kavir block and Sanandaj-Sirjan zone. Our recent findings redefine the origin of these complexes, so far attributed to the Precambrian-Early Paleozoic orogenic episodes, and now directly related to the tectonic evolution of the Paleo-Tethys Ocean. This tectonic evolution was initiated by Late Ordovician-Early Devonian rifting events and terminated in the Triassic by the Eocimmerian collision event due to the docking of the Cimmerian blocks with the Asiatic Turan block. The ``Variscan accretionary complex'' is a new name we proposed for the most widely distributed metamorphic rocks connected to the Anarak and Jandaq complexes. This accretionary complex exposed from SW of Jandaq to the Anarak and Kabudan areas is a thick and fine grain siliciclastic sequence accompanied by marginal-sea ophiolitic remnants, including gabbro-basalts with a supra-subduction-geochemical signature. New Ar-40/Ar-39 ages are obtained as 333-320 Ma for the metamorphism of this sequence under greenschist to amphibolite facies. Moreover, the limy intercalations in the volcano-sedimentary part of this complex in Godar-e-Siah yielded Upper Devonian-Tournaisian conodonts. The northeastern part of this complex in the Jandaq area was intruded by 215 +/- 15 Ma arc to collisional granite and pegmatites dated by ID-TIMS and its metamorphic rocks are characterized by Some Ar-40/Ar-39 radiometric ages of 163-156 Ma. The ``Variscan'' accretionary complex was northwardly accreted to the Airekan granitic terrane dated at 549 +/- 15 Ma. Later, from the Late Carboniferous to Triassic, huge amounts of oceanic material were accreted to its southern side and penetrated by several seamounts such as the Anarak and Kabudan. This new period of accretion is supported by the 280-230 Ma Ar-40/Ar-39 ages for the Anarak mild high-pressure metamorphic rocks and a 262 Ma U-Pb age for the trondhjemite-rhyolite association of that area. The Triassic Bayazeh flysch filled the foreland basin during the final closure of the Paleo-Tethys Ocean and was partly deposited and/or thrusted onto the Cimmerian Yazd block. The Paleo-Tethys magmatic arc products have been well-preserved in the Late Devonian-Carboniferous Godar-e-Siah intra-arc deposits and the Triassic Nakhlak fore-arc succession. On the passive margin of the Cimmerian block, in the Yazd region, the nearly continuous Upper Paleozoic platform-type deposition was totally interrupted during the Middle to Late Triassic. Local erosion, down to Lower Paleozoic levels, may be related to flexural bulge erosion. The platform was finally unconformably covered by Liassic continental molassic deposits of the Shemshak. One of the extensional periods related to Neo-Tethyan back-arc rifting in Late Cretaceous time finally separated parts of the Eocimmerian collisional domain from the Eurasian Turan domain. The opening and closing of this new ocean, characterized by the Nain and Sabzevar ophiolitic melanges, finally transported the Anarak-Jandaq composite terrane to Central Iran, accompanied by large scale rotation of the Central-East Iranian Microcontinent (CEIM). Due to many similarities between the Posht-e-Badam metamorphic complex and the Anarak-Jandaq composite terrane, the former could be part of the latter, if it was transported further south during Tertiary time. (C) 2007 Elsevier B.V. All rights reserved.