CHARACTERIZATION OF NrsZ, A NOVEL SMALL REGULATORY NON-CODING RNA INVOLVED IN THE ADAPTATION OF PSEUDOMONAS AERUGINOSA PAO1

The opportunistic ubiquitous pathogen Pseudomonas aeruginosa strain PAOl is a versatile Gram-negative bacterium that has the extraordinary capacity to colonize a wide diversity of ecological niches and to cause severe and persistent infections in humans. To ensure an optimal coordination of the genes involved in nutrient utilization, this bacterium uses the NtrB/C and/or the CbrA/B two-component systems, to sense nutrients availability and to regulate in consequence the expression of genes involved in their uptake and catabolism. NtrB/C is specialized in nitrogen utilization, while the CbrA/B system is involved in both carbon and nitrogen utilization and both systems activate their target genes expression in concert with the alternative sigma factor RpoN. Moreover, the NtrB/C and CbrA/B two- component systems regulate the secondary metabolism of the bacterium, such as the production of virulence factors. In addition to the fine-tuning transcriptional regulation, P. aeruginosa can rapidly modulate its metabolism using small non-coding regulatory RNAs (sRNAs), which regulate gene expression at the post-transcriptional level by diverse and sophisticated mechanisms and contribute to the fast physiological adaptability of this bacterium. In our search for novel RpoN-dependent sRNAs modulating the nutritional adaptation of P. aeruginosa PAOl, we discovered NrsZ (Nitrogen regulated sRNA), a novel RpoN-dependent sRNA that is induced under nitrogen starvation by the NtrB/C two-component system. NrsZ has a unique architecture, formed of three similar stem-loop structures (SL I, II and II) separated by variant spacer sequences. Moreover, this sRNA is processed in short individual stem-loop molecules, by internal cleavage involving the endoribonuclease RNAse E. Concerning NrsZ functions in P. aeruginosa PAOl, this sRNA was shown to trigger the swarming motility and the rhamnolipid biosurfactants production. This regulation is due to the NrsZ-mediated activation of rhlA expression, a gene encoding for an enzyme essential for swarming motility and rhamnolipids production. Interestingly, the SL I structure of NrsZ ensures its regulatory function on rhlA expression, suggesting that the similar SLs are the functional units of this modular sRNA. However, the regulatory mechanism of action of NrsZ on rhlA expression activation remains unclear and is currently being investigated. Additionally, the NrsZ regulatory network was investigated by a transcriptome analysis, suggesting that numerous genes involved in both primary and secondary metabolism are regulated by this sRNA. To emphasize the importance of NrsZ, we investigated its conservation in other Pseudomonas species and demonstrated that NrsZ is conserved and expressed under nitrogen limitation in Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48 and Pseudomonas syringae pv. tomato DC3000, strains having different ecological features, suggesting an important role of NrsZ in the adaptation of Pseudomonads to nitrogen starvation. Interestingly the architecture of the different NrsZ homologs is similarly composed by SL structures and variant spacer sequences. However, the number of SL repetitions is not identical, and one to six SLs were predicted on the different NrsZ homologs. Moreover, NrsZ is processed in short molecules in all the strains, similarly to what was previously observed in P. aeruginosa PAOl, and the heterologous expression of the NrsZ homologs restored rhlA expression, swarming motility and rhamnolipids production in the P. aeruginosa NrsZ mutant. In many aspects, NrsZ is an atypical sRNA in the bacterial panorama. To our knowledge, NrsZ is the first described sRNA induced by the NtrB/C. Moreover, its unique modular architecture and its processing in similar short SL molecules suggest that NrsZ belongs to a novel family of bacterial sRNAs. -- L'agent pathogène opportuniste et ubiquitaire Pseudomonas aeruginosa souche PAOl est une bactérie Gram négative versatile ayant l'extraordinaire capacité de coloniser différentes niches écologiques et de causer des infections sévères et persistantes chez l'être humain. Afin d'assurer une coordination optimale des gènes impliqués dans l'utilisation de différents nutriments, cette bactérie se sert de systèmes à deux composants tel que NtrB/C et CbrA/B afin de détecter la disponibilité des ressources nutritives, puis de réguler en conséquence l'expression des gènes impliqués dans leur importation et leur catabolisme. Le système NtrB/C régule l'utilisation des sources d'azote alors que le système CbrA/B est impliqué à la fois dans l'utilisation des sources de carbone et d'azote. Ces deux systèmes activent l'expression de leurs gènes-cibles de concert avec le facteur sigma alternatif RpoN. En outre, NtrB/C et CbrA/B régulent aussi le métabolisme secondaire, contrôlant notamment la production d'importants facteurs de virulence. En plus de toutes ces régulations génétiques fines ayant lieu au niveau transcriptionnel, P. aeruginosa est aussi capable de moduler son métabolisme en se servant de petits ARNs régulateurs non-codants (ARNncs), qui régulent l'expression génétique à un niveau post- transcriptionnel par divers mécanismes sophistiqués et contribuent à rendre particulièrement rapide l'adaptation physiologique de cette bactérie. Au cours de nos recherches sur de nouveaux ARNncs dépendant du facteur sigma RpoN et impliqués dans l'adaptation nutritionnelle de P. aeruginosa PAOl, nous avons découvert NrsZ (Nitrogen regulated sRNA), un ARNnc induit par la cascade NtrB/C-RpoN en condition de carence en azote. NrsZ a une architecture unique, composée de trois structures en tige- boucle (TB I, II et III) hautement similaires et séparées par des « espaceurs » ayant des séquences variables. De plus, cet ARNnc est clivé en petits fragments correspondant au trois molécules en tige-boucle, par un processus de clivage interne impliquant l'endoribonucléase RNase E. Concernant les fonctions de NrsZ chez P. aeruginosa PAOl, cet ARNnc est capable d'induire la motilité de type « swarming » et la production de biosurfactants, nommés rhamnolipides. Cette régulation est due à l'activation par NrsZ de l'expression de rhlA, un gène essentiel pour la motilité de type swarming et pour la production de rhamnolipides. Étonnamment, la structure TB I est capable d'assurer à elle seule la fonction régulatrice de NrsZ sur l'expression de rhlA, suggérant que ces molécules TBs sont les unités fonctionnelles de cet ARNnc modulaire. Cependant, le mécanisme moléculaire par lequel NrsZ active l'expression de rhlA demeure à ce jour incertain et est actuellement à l'étude. En plus, le réseau de régulations médiées par NrsZ a été étudié par une analyse de transcriptome qui a indiqué que de nombreux gènes impliqués dans le métabolisme primaire ou secondaire seraient régulés par NrsZ. Pour accentuer l'importance de NrsZ, nous avons étudié sa conservation dans d'autres espèces de Pseudomonas. Ainsi, nous avons démontré que NrsZ est conservé et exprimé en situation de carence d'azote par les souches Pseudomonas protegens Pf-5, Pseudomonas putida KT2442, Pseudomonas entomophila L48, Pseudomonas syringae pv. tomato DC3000, quatre espèces ayant des caractéristiques écologiques très différentes, suggérant que NrsZ joue un rôle important dans l'adaptation du genre Pseudomonas envers la carence en azote. Chez toutes les souches étudiées, les différents homologues de NrsZ présentent une architecture similaire faite de TBs conservées et d'espaceurs. Cependant, le nombre de TBs n'est pas identique et peut varier de une à six copies selon la souche. Les différentes versions de NrsZ sont clivées en petites molécules dans ces quatre souches, comme il a été observé chez P. aeruginosa PAOl. De plus, l'expression hétérologue des différentes variantes de NrsZ est capable de restaurer l'expression de rhlA, la motilité swarming et la production de rhamnolipides dans une souche de P. aeruginosa dont nrsZ a été inactivé. Par bien des aspects, NrsZ est un ARNnc atypique dans le monde bactérien. À notre connaissance, NrsZ est le premier ARNnc décrit comme étant régulé par le système NtrB/C. De plus, son unique architecture modulaire et son clivage en petites molécules similaires suggèrent que NrsZ appartient à une nouvelle famille d'ARNncs bactériens.

Video-assisted thoracoscopic resection of a small pulmonary nodule after computed tomography-guided localization with a hook-wire system. Experience in 45 consecutive patients.

BACKGROUND: This study is a single-institution validation of video-assisted thoracoscopic (VATS) resection of a small solitary pulmonary nodule (SPN) previously localized by a CT-guided hook-wire system in a consecutive series of 45 patients. METHODS: The records of all patients undergoing VATS resection for SPN preoperatively localized by CT-guided a hook-wire system from January 2002 to December 2004 were assessed with respect to failure to localize the lesion by the hook-wire system, conversion thoracotomy rate, duration of operation, postoperative complications, and histology of SPN. RESULTS: Forty-five patients underwent 49 VATS resections, with simultaneous bilateral SPN resection performed in 4. Preoperative CT-guided hook-wire localization failed in two patients (4%). Conversion thoracotomy was necessary in two patients (4%) because it was not possible to resect the lesion by a VATS approach. The average operative time was 50 min. Postoperative complications occurred in 3 patients (6%), one hemothorax and two pneumonia. The mean hospital stay was 5 days (range: 2-18 days). Histological assessment revealed inflammatory disease in 17 patients (38%), metastasis in 17 (38%), non-small-cell lung cancer (NSCLC) in 4 (9%), lymphoma in 3 (6%), interstitial fibrosis in 2 (4%), histiocytoma in one (2%), and hamartoma in one (2%). CONCLUSIONS: Histological analysis of resected SPN revealed unexpected malignant disease in more than 50% of the patients indicating that histological clarification of SPN seems warranted. Video-assisted thoracoscopic resection of SPN previously localized by a CT-guided hook-wire system is related to a low conversion thoracotomy rate, a short operation time, and few postoperative complications, and it is well suited for the clarification of SPN.

The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis.

A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described.

La fortuna di Luciano nel Rinascimento : il volgarizzamento del manoscritto Vaticano Chigiano L.VI.215 : edizione critica dei volgarizzamenti delle "Storie vere"

Résumé II lavoro verte sui volgarizzamenti quattro-cinquecenteschi di Luciano di Samosata, importante capitolo nella fortuna dell'autore greco, che diede l'avvio a quel vasto fenomeno chiamato "lucianesimo", esteso in Europa fino al XIX sec. In particolare fornisco l'edizione critica e commentata delle Storie vere volgarizzate, contenute nella prima, assai ampia (41 opuscoli), e per molto tempo unica, silloge lucianea in volgare, che ho datato a poco prima del 1480. Essa ci è giunta tramite un unico manoscritto, il Vaticano Chigiano L.VI.215, confezionato a Ferrara per Ercole I d'Este, nonché in almeno otto edizioni veneziane apparse fra il 1525 e il 1551. La princeps, da cui dipendono in vario modo tutte le edizioni successive, è pubblicata da Niccolò Zoppino. I1 ms. e le prime due edizioni (1525; 1527 Bindoni e Pasini) tacciono il nome del traduttore, che compare solo nell'edizione del 1529 (Zoppino): Niccolò Leoniceno (1428-1524), medico umanista e valente grecista, attivo a Ferrara dal 1464 al 1524, studioso e traduttore di Ippocrate e Galeno, editore di Aristotele e volgarizzatore di storici per Ercole d'Este. L'edizione ha richiesto uno studio preliminare sulle numerose traduzioni in latino e in volgare di Luciano, per valutare meglio le modalità della sua fortuna umanistica. Confrontando ms. e stampe, per le Storie vere si hanno due volgarizzamenti totalmente diversi, fin dal titolo: La vera historia nel ms., Le vere narrazioni nelle cinquecentine. Ma per l'ultimo quarto di testo, ms. e stampe in sostanza coincidono. La collazione ha coinvolto anche il testo greco (con gli apparati delle edizioni critiche) e la versione latina dell'umanista umbro Lilio Tifernate (1417/18-1486) risalente al 1439-43 ca., intitolata De veris narrationibus, di cui si hanno almeno tre redazioni d'autore; una quarta è invece dovuta probabilmente a Benedetto Bordon, che la inserì nella sua silloge latina di Luciano del 1494. Ho cosa stabilito che il volgarizzamento del ms. Chigiano, La vera historia, è stato eseguito direttamente dal greco, fatto eccezionale nel panorama delle traduzioni umanistiche, mentre quello a stampa, Le vere narrazioni, deriva dalla redazione Bordon del De veris narrationibus. La diversità dei titoli dipende dalle varianti dei codici greci utilizzati dai traduttori: il Vat. gr. 1323, o una sua copia, è utilizzato sia dal volgarizzatore del Chigiano, sia da Bordon, indipendentemente l'uno dall'altro; il Marc. gr. 434, o una sua copia, dal Tifernate. Il titolo latino mantenuto da Bordon risale al Tifernate. Per quanto riguarda l'attribuzione dei due volgarizzamenti, come già per altri due testi della silloge da me studiati (Lucio 01 Asino e Timone), anche per La vera historia del Chigiano è accettabile il nome di Niccolò Leoniceno, poiché: 1) essa è tradotta direttamente dal greco, correttamente e con buona resa in volgare, 2) Paolo Giovio -che conobbe di persona il Leoniceno -, negli Elogia veris clarorum virorum imaginibus apposita ricorda che i volgarizzamenti di Luciano e di Dione eseguiti dal Leoniceno piacquero molto ad Ercole d'Este, 3) nessuno nella prima metà del sec. XVI rivendica, per sé o per un suo maestro, il volgarizzamento di Luciano. Le vere narrazioni a stampa, tradotte dal latino del Bordon, dopo il 1494 e prima del 1525, per la parte che diverge dalla Vera historia rimangono invece anonime. Dato che si tratta di due volgarizzamenti distinti, ho allestito l'edizione a fronte dei due testi fin dove essi divergono, seguendo per l'uno il ms., per l'altro la princeps; per la parte finale, in cui confluiscono, mi baso invece sul manoscritto e relego in apparato le varianti più vistose della princeps (non è emerso un chiaro rapporto di dipendenza fra i due testimoni). Oltre all'apparato critico con le lezioni rifiutate, fornisco un commento con la giustificazione delle scelte e il confronto con i corrispondenti passi greci e latini.

ICD-11 for quality and safety: overview of the who quality and safety topic advisory group.

This paper outlines the approach that the WHO's Family of International Classifications (WHO-FIC) network is undertaking to create ICD-11. We also outline the more focused work of the Quality and Safety Topic Advisory Group, whose activities include the following: (i) cataloguing existing ICD-9 and ICD-10 quality and safety indicators; (ii) reviewing ICD morbidity coding rules for main condition, diagnosis timing, numbers of diagnosis fields and diagnosis clustering; (iii) substantial restructuring of the health-care related injury concepts coded in the ICD-10 chapters 19/20, (iv) mapping of ICD-11 quality and safety concepts to the information model of the WHO's International Classification for Patient Safety and the AHRQ Common Formats; (v) the review of vertical chapter content in all chapters of the ICD-11 beta version and (vi) downstream field testing of ICD-11 prior to its official 2015 release. The transition from ICD-10 to ICD-11 promises to produce an enhanced classification that will have better potential to capture important concepts relevant to measuring health system safety and quality-an important use case for the classification.

Phylogenetic relationships and divergence times among dormice (Rodentia, Gliridae) based on three nuclear genes

We examined phylogenetic relationships among six species representing three subfamilies, Glirinae, Graphiurinae and Leithiinae with sequences from three nuclear protein-coding genes (apolipoprotein B, APOB; interphotoreceptor retinoid-binding protein, IRBP; recombination-activating gene 1, RAG1). Phylogenetic trees reconstructed from maximum-parsimony (MP), maximum-likelihood (ML) and Bayesian-inference (BI) analyses showed the monophyly of Glirinae (Glis and Glirulus) and Leithiinae (Dryomys, Eliomys and Muscardinus) with strong support, although the branch length maintaining this relationship was very short, implying rapid diversification among the three subfamilies. Divergence time estimates were calculated from ML (local clock model) and Bayesian-dating method using a calibration point of 25 Myr (million years) ago for the divergence between Glis and Glirulus, and 55 Myr ago for the split between lineages of Gliridae and Sciuridae on the basis of fossil records. The results showed that each lineage of Graphiuros, Glis, Glirulus and Muscardinus dates from the Late Oligocene to the Early Miocene period, which is mostly in agreement with fossil records. Taking into account that warm climate harbouring a glirid-favoured forest dominated from Europe to Asia during this period, it is considered that this warm environment triggered the prosperity of the glirid species through the rapid diversification. Glirulus japonicas is suggested to be a relict of this ancient diversification during the warm period.

Sex-chromosome turnovers: the hot-potato model.

Abstract Sex-determining systems often undergo high rates of turnover but for reasons that remain largely obscure. Two recent evolutionary models assign key roles, respectively, to sex-antagonistic (SA) mutations occurring on autosomes and to deleterious mutations accumulating on sex chromosomes. These two models capture essential but distinct key features of sex-chromosome evolution; accordingly, they make different predictions and present distinct limitations. Here we show that a combination of features from the two models has the potential to generate endless cycles of sex-chromosome transitions: SA alleles accruing on a chromosome after it has been co-opted for sex induce an arrest of recombination; the ensuing accumulation of deleterious mutations will soon make a new transition ineluctable. The dynamics generated by these interactions share several important features with empirical data, namely, (i) that patterns of heterogamety tend to be conserved during transitions and (ii) that autosomes are not recruited randomly, with some chromosome pairs more likely than others to be co-opted for sex.

Le principe de transparence dans le canton de Vaud : évaluation des chapitres I, III, IV et VI de la Loi du 24 septembre 2002 sur l'information

La Loi vaudoise sur l'information (LInfo), en vigueur depuis le 1er septembre 2003, contient les bases légales relatives à l'information communiquée par l'Etat aux médias et au public. A l'image de plus en plus de législations en la matière, elle consacre le principe de l'information sur demande, selon lequel tout individu a désormais le droit de consulter des documents officiels et d'obtenir de l'information de la part des autorités sans devoir motiver sa demande. La présente évaluation s'intéresse à comprendre comment ce principe de transparence est défini dans cette loi, quelle est son application par l'administration, dans quelle mesure il est utilisé par les citoyens et enfin quels sont ses effets six ans après son entrée en vigueur. Das waadtländische Informationsgesetz (LInfo), in Kraft seit dem 1. September 2003, liefert die rechtliche Grundlage betreffend Informationen die durch den Kanton an die Medien und die Öffentlichkeit kommuniziert werden. Als Grundlage dient dazu das Prinzip um Erhalt von Informationen, nach welchem jede Person das Recht hat offizielle Dokumente einzusehen und Informationen von den Behörden zu erhalten ohne die Anfrage begründen zu müssen. Der vorliegende Beitrag hinterfragt wie dieses Transparenzprinzip im Gesetz definiert ist und wie es durch die Verwaltung angewendet wird. Weiter wird untersucht in welchem Ausmass es durch die Bürger und Bürgerinnen angewendet wird und was das Gesetz sechs Jahre nach seinem Inkrafttreten bewirkt hat.

Internal arsenite bioassay calibration using multiple reporter cell lines

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error-prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli-based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or β-galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for β-galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l−1).