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Resume Introduction La consommation de tabac chez les sujets infectes par le VIH constitue un probleme essentiel, compte tenu des consequences plus severes et plus frequentes du tabagisme dans cette population. Peu d’etudes observationnelles relatives au sevrage tabagique ont ete conduites a ce jour dans cette population. Objectif L’objectif de l’etude etait d’evaluer l’efficacite de l’aide au sevrage tabagique chez 39 fumeurs qui avaient declare etre infectes par le VIH et qui ont consulte une premiere fois en tabacologie a l’hopital europeen Georges-Pompidou entre le 1er janvier 2011 et le 31 decembre 2012. Methodes Une etude retrospective a ete menee a partir des dossiers standardises de consultation de tabacologie (CDT) completes au cours de la periode 2011–2012. Les consultations de suivi ont ete etudiees et le sevrage tabagique a ete valide a chaque visite par la mesure du monoxyde de carbone expire ≤ 5 ppm. Les patients perdus de vue ont ete consideres comme fumeurs. L’abstinence maintenue validee a ete consignee au terme de 3 mois de suivi, puis 9 mois/1 an apres la premiere consultation. Resultats Parmi les 39 patients, 76,9 % etaient des hommes. Les consultants etaient de gros fumeurs (22,7 cigarettes/jour en moyenne), fortement dependants a la nicotine (33 % avaient un score ≥ 7 au test de Fagerstrom). En ce qui concerne les co-addictions, 41 % des consultants consommaient du cannabis dont un quart chaque jour, 10 % avaient un mesusage d’alcool et 13 % prenaient un traitement substitutif opiace. Sur le plan psychopathologique, un tiers de ces fumeurs declarait avoir un antecedent d’episode depressif avec a la premiere consultation, une symptomatologie anxieuse et/ou depressive (1/3 avait un score A-HAD ≥ 11 et 20 % un score D-HAD ≥ 11). Ces fumeurs ont tous recu un traitement pharmacologique d’aide au sevrage tabagique dans le cadre d’un suivi medical structure comportant des techniques comportementales : pour 85 % d’entre eux, des substituts nicotiniques sous forme de patchs et/ou formes orales et pour 15 %, de la varenicline®. Au terme de 3 mois, le taux d’abstinence maintenue validee par la mesure du monoxyde de carbone expire etait de 20,5 % et au terme de 9 mois/1 an, il etait de 13 %. Chez les fumeurs recus deux fois et plus, ce taux etait de 27,6 % a trois mois et 17,2 % au terme de 9 mois/un an. Discussion et conclusion Les resultats de cette etude locale de suivi suggerent que le sevrage est possible en pratique et qu’un suivi prolonge pourrait favoriser l’arret du tabac si les fumeurs continuent a adherer a la prise en charge. Ils soulignent egalement la necessite de prendre en compte les co-addictions frequentes dans cette population ainsi que les troubles anxieux et depressifs associes.

A selection of individuals from the biotech ecosystem give their views on the challenges facing the sector over the coming years.

Nat. Biotechnol. 34, 276–283 (2016); published online 10 March 2016; corrected after print 31 March 2016 In the version of this article initially published, Stanley Crooke's name was misspelled as “Crook” in the author list. The error has been corrected in the HTML and PDF versions of the article.

The methylation of 5'CpG 3' dinucleotides within genes creates potential targets for protein complexes that bind to methylated DNA sequences and to histone deacetylases (MBD-HDAC). This can lead to transcriptional repression by modification of chromatin. To test the importance of this repression in vivo and to determine when during development these epigenetic controls are placed on genes, two novel genes have been engineered by directed mutagenesis of the CpG-rich LacZ gene that are depleted of ( LagZ ) or completely lacking ( LagoZ ) CpG sequences. We report that the expression (transcriptional and translational) of the three genes is indistinguishable in transient assays in cleaving mouse embryos. Therefore, the complete absence of CpG sequences within three kilobases of coding sequence is compatible with its maintenance in the nucleus and with its expression. These molecules can now be used to study the ontogenesis of the CpG-dependent repressive system in intact organisms.

Retroviral vectors contain viral cis-acting elements to achieve the packaging, reverse transcription, integration, and expression of the retroviral genomic nucleic acid sequence. However, these elements are not useful in the integrated provirus and can be the cause of problems. We have developed a vector which eliminates the majority of these viral elements. This vector, a long terminal repeat (LTR) enhancer-deleted vectors, exploits the Cre-lox recombination system of the P1 bacteriophage. The Cre-lox system is neutral for eukaryotic cells. The 32-nucleotide loxP site is inserted within the U3 of the 3' LTR along with with the gene to be transduced (in place of the viral enhancers). Following the LTR-mediated loxP duplication, the LTRs can be recombined by the Cre enzyme. The structure of the resulting provirus in the host genome corresponds to a single LTR (deleted of the viral enhancers) carrying a single copy of the gene to be transduced. If the Cre expression unit is furnished after the integration of a loxP-containing virus, the efficiency of the recombination is not absolute. If the Cre expression unit is inserted between the two LTRs, only single LTR proviral structures are found following infection by the retroviral vector.

The objective of this cohort study of a consecutive sample of infants exposed during pregnancy to anticonvulsant drugs was to determine if the mother received late pregnancy prophylaxis with vitamin K, and if any infants had signs of hemorrhagic disease. The medical records of 204 neonates exposed to anticonvulsant drugs in utero and 77 unexposed control neonates were retrospectively reviewed. No hemorrhagic disease was observed, and the incidence of bleeding tendencies was not higher in infants exposed to these drugs compared with control infants, despite no prenatal vitamin K supplementation in all but 1 epileptic woman.

Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.

prp6 and prp9 thermosensitive (ts) mutants are affected in pre-mRNA splicing and transport from the nucleus to the cytoplasm. PRP6 and PRP9 wild-type alleles have been sequenced. DNA sequence analysis reveals homologies in the 5' and 3' non-coding regions, suggesting a common regulation of gene expression. PRP6 and PRP9 genes encode a 899 amino acid and a 530 amino acid protein, respectively. The PRP6 protein has repeated motifs that evoke helix-loop-helix structures. Both PRP6 and PRP9 proteins have cysteine/histidine motifs loosely related to those found in zinc finger proteins. The substitution of some, but not all, of these residues by directed mutagenesis has a critical effect on the protein function. Homology searches reveal that two other proteins known to be involved in the nuclear splicing pathway--the yeast PRP11 and the human U1C proteins--contain similar sequences. The five cysteine/histidine motifs found in these four proteins display amino acid similarities in addition to the cysteine and histidine residues, indicating that they participate in biological structures or functions related to the splicing process. In addition, PRP6 and PRP9 exhibit leucine repeat motifs which may be implicated in protein interactions. The prp6 and prp9 ts mutations have been mapped and sequenced.

Les vecteurs retroviraux contiennent des elements viraux agissant en cis pour obtenir l'expression, l'encapsidation, la transcription inverse et l'integration de la sequence d'acide nucleique du genome retroviral. Ces elements ne sont toutefois pas utiles dans le provirus integre et peuvent etre la cause de nombreux problemes. L'invention concerne un vecteur retroviral qui elimine la majorite de ces elements viraux. Ce vecteur exploite entre autres le systeme de recombinaison Cre- lox du bacteriophage P1. Le site loxP a 32 nucleotides est insere a l'interieur de la sequence U3 de la 3'LTR avec le gene a introduire dans la cellule. Suite a la duplication de loxP par l'intermediaire de la LTR, les LTR peuvent etre recombinees par l'enzyme Cre. La structure du provirus qui en resulte dans le genome hote correspond a une seule LTR portant une copie unique du gene a introduire dans la cellule. Si l'unite d'expression de Cre est inseree entre les deux LTR, on ne trouve que des structures provirales a LTR unique a la suite d'une infection avec le vecteur retroviral.

The ability to introduce specific alterations of endogenous genes into the germ line of mice via targeted mutagenesis in embryonic stem (ES) cells has represented a major breakthrough in mouse genetics. Gene inactivation has been widely used to examine the effects of loss of function in various biological processes such as development, cellular biology, and physiology. This has already permitted the accumulation of new insights into gene function and also the creation of mouse models of human genetic diseases. Introduction of subtle mutations at specific locations of the mammalian genome is also useful to refine genetic analysis and to produce models of genetic diseases which do not necessarily result from null mutations. Several strategies have been developed, each aimed at generating subtle mutations in a given gene (6). One common limitation to all current gene-targeting procedures is the low frequency of correct targeting. This becomes a serious problem especially with use of two successive rounds of targeting, a method common to several strategies used for the generation of mutated genes devoid of foreign selection sequences. Therefore, attempts have been made to increase the efficiency of gene targeting by several means, such as increasing the size of the region of homologies with the target locus, using isogenic genomic DNA, or improving the selection procedures (6). In this report, we present an alternative approach to overcome these limitations which relies on the observation that double-strand ends of broken chromosomes are highly recombinogenic (reviewed in reference 5). Double-strand breaks (DSB) are frequently associated with DNA alteration events in eukaryotes (4, 31); during meiosis in Saccharomyces cerevisiae, for example, transient DSB are induced at a number of positions known to be hot spots for recombination (23). It has recently been shown that a unique DSB can specifically be induced in the yeast (12), plant (24), and mammalian (8, 22, 26, 28) genomes by using the yeast I-SceI meganuclease. The I-SceI protein is an endonuclease responsible for intron homing in yeast mitochondria, a process that apparently proceeds by DSB repair (18); I-SceI endonuclease can induce recombination in yeast nuclei (12). In mammalian cells, the yeast meganuclease I-SceI has been shown to efficiently induce a DSB in a chromosomal target containing an I-SceI recognition sequence. This allows DNA break repair with high frequency by recombination with a donor molecule homologous to the regions flanking the break (7, 8, 22, 25, 26, 28). We reasoned that the introduction of a DSB in an endogenous gene could increase targeting frequency at this natural locus through stimulation of the cellular recombination machinery. The gene encoding villin, a major component of the actin cytoskeleton of intestine and kidney cells (13), was chosen to develop this gene targeting strategy. We found that induction of a DSB in the target gene by using the meganuclease I-SceI resulted in greatly enhanced homologous replacement by the incoming DNA, even when the length of genomic DNA homology is reduced.