Genome-wide transposon insertion scanning of environmental survival functions in the polycyclic aromatic hydrocarbon degrading bacterium Sphingomonas wittichii RW1.

Sphingomonas wittichii RW1 is a dibenzofuran and dibenzodioxin-degrading bacterium with potentially interesting properties for bioaugmentation of contaminated sites. In order to understand the capacity of the microorganism to survive in the environment we used a genome-wide transposon scanning approach. RW1 transposon libraries were generated with around 22 000 independent insertions. Libraries were grown for an average of 50 generations (five successive passages in batch liquid medium) with salicylate as sole carbon and energy source in presence or absence of salt stress at -1.5 MPa. Alternatively, libraries were grown in sand with salicylate, at 50% water holding capacity, for 4 and 10 days (equivalent to 7 generations). Library DNA was recovered from the different growth conditions and scanned by ultrahigh throughput sequencing for the positions and numbers of inserted transposed kanamycin resistance gene. No transposon reads were recovered in 579 genes (10% of all annotated genes in the RW1 genome) in any of the libraries, suggesting those to be essential for survival under the used conditions. Libraries recovered from sand differed strongly from those incubated in liquid batch medium. In particular, important functions for survival of cells in sand at the short term concerned nutrient scavenging, energy metabolism and motility. In contrast to this, fatty acid metabolism and oxidative stress response were essential for longer term survival of cells in sand. Comparison to transcriptome data suggested important functions in sand for flagellar movement, pili synthesis, trehalose and polysaccharide synthesis and putative cell surface antigen proteins. Interestingly, a variety of genes were also identified, interruption of which cause significant increase in fitness during growth on salicylate. One of these was an Lrp family transcription regulator and mutants in this gene covered more than 90% of the total library after 50 generations of growth on salicylate. Our results demonstrate the power of genome-wide transposon scanning approaches for analysis of complex traits.

Polymorphic mutations associated with the emergence of the multinucleoside/tide resistance mutations 69 insertion and Q151M.

BACKGROUND: We hypothesized that polymorphic mutations exist that are associated with the emergence of the multinucleoside resistance mutations (MNR), 69 insertion and Q151M. METHODS: The Swiss HIV Cohort Study was screened, and the frequencies of polymorphic mutations in HIV-1 (subtype B) were compared between patients detected with the 69 insertion (n = 17), Q151M (n = 29), ≥2 thymidine analogue mutations (TAM) 1 (n = 400) or ≥2 TAM 2 (n = 249). Logistic regressions adjusted for the antiretroviral treatment history were performed to analyze the association of the polymorphic mutations with MNR. RESULTS: The 69 insertion and TAM 1 were strongly associated and occurred in 94.1% (16 of 17) together. The 69 insertion seemed to emerge as a consequence of the TAM 1 pathway (median years until detection: 6.8 compared with 4.4 for ≥2 TAM 1, P Wilcoxon = 0.009). Frequencies of 8 polymorphic mutations (K43E, V60I, S68G, S162C, T165I, I202V, R211K, F214L) were significantly different between groups. Logistic regression showed that F214L and V60I were associated with the emergence of Q151M/TAM 2 opposed to 69 insertion/TAM 1. S68G, T165I, and I202V were associated with Q151M instead of TAM 2. CONCLUSIONS: Besides antiretroviral therapy, polymorphic mutations may contribute to the emergence of specific MNR mutations.

L'insertion sociale plurielle des femmes cadres supérieurs en Suisse : contribution à l'étude du bien-être subjectif au quotidien : approche qualitative intégrative

RESUME« L'insertion sociale plurielle des femmes cadres supérieurs en Suisse. Contribution à l'étude du bien-être subjectif au quotidien. Approche intégrative qualitative. »Depuis une quarantaine d'années, nombreux sont les travaux qui étudient les relations entre les différents milieux de vie au quotidien et, plus particulièrement, l'impact de leurs articulations singulières sur la santé. Nous y identifions deux axes principaux : l'un aborde ce phénomène selon une perspective de « conflit travail-famille » en termes de « stress », l'autre se focalise davantage sur la promotion du « bien-être » au travers d'une approche d'« équilibre travail-vie ». Or, l'ensemble de ces recherches considère comme «pathogènes» les désajustements, les contradictions et les tensions vécus au quotidien. Selon cette tendance, le « bien-être » apparaît comme un état d'équilibre ultime indépendant du contexte de vie du sujet. Toutefois, peu de recherches portent sur la dimension située du bien-être dans son rapport à l'activité concrète au sein des milieux deNotre étude s'intéresse à cette question auprès des femmes cadres supérieurs, selon une perspective critique et développementale en psychologie de la santé (Lyons & Chamberlain, 2006 ; Santiago-Delefosse, 2002,2011 ; Malrieu, 1989 ; Vygotski, 1985). En effet, cette population constitue un terrain privilégié pour comprendre le sens donné à l'activité à partir des contraintes, responsabilités et demandes perçues dans des contextes parfois contradictoires, et pour analyser le rôle de ces derniers dans un bien-être subjectif.En cohérence avec notre positionnement théorique, nous avons mené des entretiens qualitatifs focalisés sur l'activité quotidienne auprès de 20 femmes, et ceci en deux temps (T1-T2) (40 entretiens). Les résultats issus des analyses du contenu des discours permettent de définir le vécu de l'articulation des milieux de vie chez nos participantes, selon trois axes à la fois interdépendants et autonomes. Chaque axe se définit par une série de supports spécifiques jouant un rôle structurant dans leur bien-être subjectif. Ainsi, le premier axe se caractérise par des supports de maîtrise subjective, ainsi que par l'appropriation de contraintes sociales et corporelles, selon un rythme de l'activité soutenu. Le deuxième s'accompagne de supports qui favorisent la prise de distance au travers du relâchement du rythme et du lâcher prise de la maîtrise, par la création d'espaces personnels et sociaux « pour soi ». Enfin, le troisième porte sur le positionnement de soi par rapport à autrui en termes de « personnalisation ».Construits en rapport à la corporéité et à autrui au sein de contextes spécifiques, ces différents supports prennent leur sens au sein d'un système de pratiques global, unique pour chaque femme. Selon cette conception critique, le bien-être subjectif chez les femmes cadres se définit comme le fruit d'un processus mouvant issu des tensions vécues entre les trois axes de l'articulation des milieux de vie. Il est par conséquent social, corporel et psychologique.Nos résultats ouvrent des perspectives de recherche et d'intervention, notamment en santé et travail. Ces ouvertures sont orientées vers une approche intégrative en psychologie de la santé, c'est-à-dire, de la prise en compte dans des questions de santé et de bien-être du processus de construction du sujet en relation à son insertion sociale plurielle.ABSTRACT« Plural social participation among women senior managers in Switzerland. Contribution to the study of subjective well-being in everyday life. An integrative and qualitativeapproach.»For the last forty years, a large body of literature has studied the relationships between different social realms in everyday life and, more particularly, the impact of their singular intertwinements with health. We identify two main trends : The first one focuses on this phenomenon through the « work-family conflict » perspective in terms of « stress » whilst the second one is more concerned by the promotion of « well-being » through a « work-life balance » approach. However, both of these trends consider disadjustments, contradictions and tensions in everyday life as « pathogenic ». According to this conception, « well- being » appears as an ultimate state of balance which is indépendant from the subject's life context. Nevertheless, few studies have examined the situated dimension of well-being in its link to concrete activity in social realms.Our research is concerned with this issue among women senior managers from a critical and developmental perspective in Health Psychology (Lyons & Chamberlain, 2006 ; Santiago-Delefosse, 2002, 2011 ; Malrieu, 1989 ; Vygotski, 1985). In fact, this population represents a favourable field so as to study : how the meaning of daily activity is constructed accross different and often conflictive social realms ; to understand the many ways in which this population deals with perceived constraints, responsibilities and requests, and to analyse the role of situated plural activity in subjective well-being.Consitent with our theoretical framework, we have designed a qualitative method. We have conducted two-time (T1-T2) interviews with 20 women by focusing on their daily activity (40 interviews). The Thematic Content Analysis reveals that four different social realms are articulated among our participants through three main axes, which are at the same time interdependent and autonomous. Each one of these axis is defined by a certain number of specific supports that play an important role in these women's subjective well- being. The first axis is concerned by several supports that signify a « feeling of control » along with the appropriation of social and body constraints by means of a rapid pace of activity. The second one regards the use of supports that contribute to « put things into perspective » by means of the slowing down of the pace of activity and through letting go of the feeling of control. This mechanism includes the creation of personal and social spaces of « one's own ». The third axis is defined by the positioning of the self in regard to others through a process of « personnalization ».Developed in specific contexts through the intertwinnements between the body and social others, supports belonging to these axes acquire significance and meaning on the basis of their relationship to a global system of activities of which they are part. However, this articulation is uniquely defined for each participant. According to this critical approach, subjective well-being among women senior managers emerges as a meaningful and changing process, situated in a plural social context. This is, it appears as the result of conflictual interactions defining the three different axis that we have identified. Subjective well-being is hence a social, embodied and psychological phenomena that is closely linked to the articulation of different social realms. Our findings open new research and practice perspectives, especially concerning health and work issues. These perspectives convey an integrative approach in Health Psychology by considering health and well-being by taking into account the process of construction of the subject in regard to his or her plural social participation.

Quand négocier l'ouverture du terrain c'est déjà enquêter. Obtenir la passation de questionnaires aux congressistes de partis marocains

Négocier le lancement pour la première fois une enquête par questionnaires et à grande échelle, sur les membres de 8 partis politiques marocains en plein congrès national, est une voie d'accès ethnographique privilégiée pour comprendre les modalités de prise de décision au sein de ces organisations. Cet article se penche sur les circonstances qui favorisent ou entravent l'ouverture du terrain. L'insertion dans des cercles de confiance, la « scientificité » du projet, la création d'un précédent par le premier parti à autoriser l'enquête semblent être les clés du succès. Toutefois, un refus révèle que tolérer une telle recherche revient pour les dirigeants à accepter avec plus ou moins d'enthousiasme une production « démocratisée » et hors contrôle de l'image de leur parti.

Hereditary hepatic and systemic amyloidosis caused by a new deletion/insertion mutation in the apolipoprotein AI gene.

We report a Spanish family with autosomal-dominant non-neuropathic hereditary amyloidosis with a unique hepatic presentation and death from liver failure, usually by the sixth decade. The disease is caused by a previously unreported deletion/insertion mutation in exon 4 of the apolipoprotein AI (apoAI) gene encoding loss of residues 60-71 of normal mature apoAI and insertion at that position of two new residues, ValThr. Affected individuals are heterozygous for this mutation and have both normal apoAI and variant molecules bearing one extra positive charge, as predicted from the DNA sequence. The amyloid fibrils are composed exclusively of NH2-terminal fragments of the variant, ending mainly at positions corresponding to residues 83 and 92 in the mature wild-type sequence. Amyloid fibrils derived from the other three known amyloidogenic apoAI variants are also composed of similar NH2-terminal fragments. All known amyloidogenic apoAI variants carry one extra positive charge in this region, suggesting that it may be responsible for their enhanced amyloidogenicity. In addition to causing a new phenotype, this is the first deletion mutation to be described in association with hereditary amyloidosis and it significantly extends the value of the apoAI model for investigation of molecular mechanisms of amyloid fibrillogenesis.

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Ex-PRESS R-50 miniature glaucoma implant insertion under the conjunctiva combined with cataract extraction

Rapport de synthèse : Le glaucome à angle ouvert est une neuropathie optique chronique progressive pour laquelle de nombreux traitements tant médicaux que chirurgicaux ont été proposés. La prise en charge chirurgicale s'articule principalement autour de deux chirurgies filtrantes, la trabéculectomie et la sclérectomie profonde avec implant de collagène. Cependant, les complications postopératoires de ces deux interventions étant relativement fréquentes, la recherche s'est orientée vers des traitements alternatifs dont la mise en place de micro-drains. Ces implants de drainage diminuent la pression intraoculaire en créant un court-circuit du flux d'humeur aqueuse de la chambre antérieure vers l'espace sous-conjonctival avec formation d'une bulle de filtration. L'implant Ex-PRESS R-50 est un implant miniature (2.5 mm de long pour 400 µm de diamètre) en acier inoxydable et biocompatible. La présente étude s'est proposée d'étudier l'efficacité et la sécurité de l'implant miniature Ex-Press R-50 lors d'une opération combinée cataracte-glaucome. Trente-cinq yeux de 35 patients (âge moyen: 75 ans) ont été inclus dans l'étude. Tous les patients ont bénéficié d'une opération de la cataracte par phacoemulsification et mise en place d'un implant de chambre postérieure suivie de l'implantation du micro-drain. Les pressions intraoculaires préopératoires et postopératoires, la meilleure acuité visuelle corrigée, le nombre de médicaments anti-glaucomateux ainsi que le type et le nombre de complications ont été évalués mensuellement puis tous les 6 mois pendant 4 ans. Le succès total a été défini par une pression postopératoire finale inférieure à 18mmHg sans traitement médical associé, le succès partiel par une pression postopératoire finale inférieure à 18mmHg avec ou sans traitement médical associé.. Le suivi moyen a été de 36.9 mois avec une baisse de la pression intraoculaire significative d'environ 25%. Une augmentation de l'acuité visuelle a été observée après l'opération de la cataracte et le nombre de médicaments anti-glaucomateux a été réduit de 57%. Dix patients ont bénéficié d'un traitement supplémentaire de la bulle de filtration par injection d'anti-métabolite (mitomycine C). Nous avons observé 8 complications majeures (4 érosions conjonctivales et 4 obstructions de l'orifice interne du micro-drain), toutes suivies de l'ablation de l'implant et de la réalisation d'une chirurgie classique du glaucome. En se basant sur les courbes de Kaplan-Meier à 48 mois, le taux de succès total était de 32.7% et le succès partiel de 53.7%. Nous pouvons conclure suite à ce travail que l'implant miniature Ex-PRESS R-50 est associé à un nombre trop élevé de complications, même si les cas non compliqués ont bénéficié d'une baisse significative de la pression intraoculaire. La modification de l'architecture du micro-drain ainsi que de la technique chirurgicale devrait augmenter le taux de succès.

Pratiques d'utilisation et d'appropriation d'un dispositif d'aide à l'insertion : le cas du contrat emploi-solidarité

C'est à travers l'analyse d'une politique publique d'aide à l'emploi (le contrat emploi solidarité) que nous avons exploré les mécanismes d'insertion professionnelle d'hommes et de femmes privés d'emploi. La sur-représentation des femmes dans ce dispositif, comme dans ceux les plus éloignés de l'emploi, nous a amené à inscrire notre raisonnement dans une problématique de rapports sociaux de sexe. C'est-à-dire que nous faisons l'hypothèse que les pratiques des opérateurs de terrain s'appuyant sur les assignations de sexe, produisent un traitement différencié des politiques d'insertion entre les hommes et les femmes. Celui-ci se caractérise notamment par la non prise en compte des difficultés et des besoins spécifiques des femmes et par une absence de novation dans les formations qui leur sont proposées. Les logiques institutionnelles ainsi à l'oeuvre contribuent implicitement à un renforcement des inégalités entre les sexes et à une reproduction de la division sexuelle du travail déjà observée sur le marché de l'emploi

Hepatitis C virus RNA replication requires a conserved structural motif within the transmembrane domain of the NS5B RNA-dependent RNA polymerase.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. Here, we report that the TMD of the HCV RdRp can be functionally replaced by a newly identified analogous membrane anchor of the GB virus B (GBV-B) NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B membrane anchor replicated with reduced efficiency. Compensatory amino acid changes at defined positions within the TMD improved the replication efficiency of these chimeras. These observations highlight a conserved structural motif within the TMD of the HCV NS5B RdRp that is required for RNA replication.

Challenge of transition in the socio-professional insertion of youngsters with neurodisabilities.

Objective: To evaluate the activities of patients with neurodisabilities and assess their insertion problems in the professional world. Methods: It is based on medical records of 267 patients (224 with neurodevelopmental diseases and 43 with neuromuscular diseases), aged 16-25 years, followed in the transition clinic of young adults in the neurorehabilitation services of a tertiary center. Results: Nearly half of them (46.8%) were in a protected environment, 37.08% studied and only 3.4% worked. Their studies are much longer and they are less in university than Swiss people of same age. The competitiveness criteria are no mental retardation and to be completely independent. Finally, 29.2% reported work problems, the foremost being the lack of adaptation in the workplace. Conclusion: These results highlight the need to increase the integration of young adults with neuromotor disorders in the labor market.

A mechanism of carbapenem resistance due to a new insertion element (ISPa133) in Pseudomonas aeruginosa

This study explored the evolutionary mechanism by which the clinical isolate PA110514 yields the imipenemresistant derivative PA116136. Both isolates were examined by PFGE and SDS-PAGE, which led to the identification of a new insertion sequence, ISPa133. This element was shown to have distinct chromosomal locations in each of the original isolates that appeared to explain the differences in imipenem susceptibilty. In strain PA110514, ISPa133 is located 56 nucleotides upstream of the translational start codon, which has no effect on expression of the porin OprD. However, in strain PA116136 ISPa133 it is located in front of nucleotide 696 and, by interrupting the coding region, causes a loss of OprD expression, thus conferring imipenem resistance. In vitro experiments mimicking the natural conditions of selective pressure yielded imipenem-resistant strains in which ISPa133 similarly interrupted oprD. A mechanism is proposed whereby ISPa133 acts as a mobile switch, with its position in oprD depending on the degree of selective pressure exerted by imipenem

A mechanism of carbapenem resistance due to a new insertion element (ISPa133) in Pseudomonas aeruginosa

This study explored the evolutionary mechanism by which the clinical isolate PA110514 yields the imipenemresistant derivative PA116136. Both isolates were examined by PFGE and SDS-PAGE, which led to the identification of a new insertion sequence, ISPa133. This element was shown to have distinct chromosomal locations in each of the original isolates that appeared to explain the differences in imipenem susceptibilty. In strain PA110514, ISPa133 is located 56 nucleotides upstream of the translational start codon, which has no effect on expression of the porin OprD. However, in strain PA116136 ISPa133 it is located in front of nucleotide 696 and, by interrupting the coding region, causes a loss of OprD expression, thus conferring imipenem resistance. In vitro experiments mimicking the natural conditions of selective pressure yielded imipenem-resistant strains in which ISPa133 similarly interrupted oprD. A mechanism is proposed whereby ISPa133 acts as a mobile switch, with its position in oprD depending on the degree of selective pressure exerted by imipenem