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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.

There is a particular need among HIV-infected patients to stop smoking because of the risk of smoking-related complications and the high prevalence of cigarette smoking among them. Only a few studies have focused on this population in real-world settings.Investigate the effectiveness of a smoking cessation support for HIV-infected patients at the Georges Pompidou University hospital (HEGP) smoking cessation service during the 2011-2012 period.A retrospective study of smoking cessation medical records was performed for 39 smokers who had visited for the first time the HEGP smoking cessation service during the 2011-2012 period and declared to be infected by the HIV on their smoking cessation self-questionnaire. The study has described smokers' characteristics and follow-up to measure the abstinence rate, validated by the patient declaration, the registration of the number of days without cigarettes between each visit and a measure of expired carbon monoxide ≤ 5ppm at each visit. We examined smokers lost to follow-up and they have been considered as smokers. Maintained abstinence rates at 3 month-follow-up and at 9 months/one year were registered.The 39 HIV-infected smokers registered in the study were mainly male (30/39), were heavy smokers with a consumption mean of nearly 23 cigarettes per day. One third presented high nicotine dependence with a Fagerström test ≥ 7. A depression history was reported among one third of them. Symptoms of anxiety and depression were declared by 20% and 33% respectively among them. Thirteen percent of them received opioid replacement therapies, 41% were cannabis users (one out of four were daily users) and 10 % declared alcohol abuse. 85% of patients received nicotine replacement therapy (patch and/or oral forms) and 15% vareniclineWith a severe smoking profile, frequent co-addictions and anxiodepressive symptoms, our results suggest that behavioral techniques combined with nicotine replacement therapy or varenicline

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.

Erratum to: “I-SceI meganuclease mediates highly efficient transgenesis in fish” Mechanisms of Development, Volume 120, Issue 2, February 2003, Pages 267; The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

The present invention concerns a method for in vivo generation of a linear polynucleotide with 5′ and 3′ free ends from a vector having no free end, said linear polynucleotide being integrated into the host cell genome. The vector having no free end according to the present invention comprise the polynucleotide to be linearized or excised flanked by a cleavage site, said cleavage site being preferably not found in the host cell genome. The present invention also relates to the resulting cells and their uses, for example for production of proteins or other genes, biomolecules, biomaterials, transgenic plants, vaccines, transgenic animals or for treatment or prophylaxis of a condition or disorder in an individual., La présente invention concerne une méthode de génération in vivo d'un polynucléotide linéaire présentant des extrémités libres 5' et 3' d'un vecteur dépourvu d'extrémité libre, ledit polynucléotide linéaire étant intégré dans le génome de la cellule hôte. Selon la présente invention, ledit vecteur dépourvu d'extrémité libre comprend le polynucléotide à linéariser ou à exciser flanqué d'un site de clivage, ledit site de clivage étant de préférence absent dans le génome de la cellule hôte. La présente invention concerne également des cellules ainsi obtenues et leurs utilisations, par exemple pour la production de protéines ou d'autres gènes, de biomolécules, de biomatériaux, de plantes transgéniques, de vaccins, d'animaux transgéniques ou pour le traitement ou la prophylaxie d'un état pathologique ou d'un trouble chez un individu.

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 chromatic. 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.

Methylation of genomic DNA at CpG sequences has a repressive effect on gene expression in vertebrates. The level of methylation of the genome varies widely during development in mammals. The DNA of early germ cells and blastocysts is largely hypomethylated but that of implantation embryos is hypermethylated. To test whether these variations might affect gene expression, we have studied the pattern of expression of a CpG-rich and of a CpG-poor LacZ transgene driven by a strong promotor of an ubiquitous gene. We find that the changes of the expression of the CpG-rich LacZ gene directly correlate with variations of methylation and that lowering the level of CpG in the reporter gene decreases dramatically its susceptibility to these variations. Therefore, in association with fluctuations in genome methylation a CpG-dependent system of repression must control positively and negatively gene expression during development and gametogenesis. We also find that the repression of the CpG-rich LacZ reporter can be abolished in 2-cell embryo by inhibitors of histone deacetylases suggesting that, at least at this stage, this CpG-dependent repression acts through histone deacetylation. In addition, we show that the β-globin locus control region and other genomic elements completely reverse the repression established at implantation in embryonic cells. The complex patterns of expression of the CpG-rich and poor LacZ reporter genes during gametogenesis and development-including a sex-dependent expression in the zygotic nucleus before the morula stage-are described. The possible implication of the constraints imposed on gene expression by DNA methylation revealed by this study are discussed.

With the up to date published data, the risk of clomiphene exposure during pregnancy cannot be determined. After a review of animal experimentation results, we will discuss the human data. Most of the publications with human exposition concern treatments given before the beginning of pregnancy. The questions addressed to the Centre de Pharmacovigilance of Paris F. Widal will be detailed. From May 1983 to July 1995 39 requests are available with only 25 cases of known outcomes. For these 25 cases, five elective terminations and three spontaneous abortions are reported. Only one defect was observed among the 17 births (9 females, 8 males): unilateral fibula agenesis the imputability of which is difficult to establish. These data seems reassuring; however, the number of patients studied is small. Other studies on clomiphene-exposed pregnancies are necessary in order to investigate this risk.

In this article, we describe two novel methods to modify the genome of mammalian cells. The first method utilizes a new family of recombinant retroviruses (RRV) which leads to transduction in the mammalian genome of, not a complete recombinant retroviruses, but a single LTR (sLTR) that carries the foreign gene. Therefore, all cis viral regulatory elements of the virus, including those necessary for the control of transcription, reverse transcription and integration, are absent from the transduced material. In addition to being safer because of their inability to retrotranspose, the sLTR family of recombinant retrovirus (sLTR-RRV) overcome the problems associated with the presence of viral sequences that negatively control the expression of the transduced material in stem cells.

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.

International audience; The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.

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.

A prospective transversal study was undertaken by occupational health physicians on more than 4,000 individuals including almost 800 women working in the same company with the following aims: a) to compare the prevalence of a certain number of classically recognized cardiovascular risk factors in 2 types of populations, service versus manufacturing industries, in the Paris region; and b) to understand these populations better so as to develop educational and information campaigns specifically adapted to their needs. Several elements were recorded in a computer data bank during the physical examination: blood pressure, blood cholesterol, smoking history, blood triglycerides, blood sugar, the ratio of measured to theoretical weight and physical activity. For each of these parameters, a value above a determined threshold level was defined as a risk factor. For men working in service industries, the percentages of heavy smokers (24%), hypercholesterolemia (18%), and hyperglycemia (7%) were significantly higher than in the manufacturing sector (19.3, 15 and 2.4%, respectively). In manufacturing industries, the percentages of men with hypertension (15%), hypertriglyceridemia (10%), who were overweight (24%) or had a low high density lipoprotein level (10%) were significantly higher than in the service sector (3.1, 7.5, 20 or 8.2%, respectively). The percentage of women smoking more than 20 g of tobacco per day (25.5%) in the service sector was significantly higher than that in manufacturing industries (4.25%). In contrast, in the manufacturing sector, the percentages of hypertensive (6%) and obese (24%) women were significantly higher than those in the service industries (1.1 and 16%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; The expression of murine leukemia provirus in embryonal carcinoma (EC) cells is blocked by a mechanism still incompletely understood. The blockage is not overcome by deleting a large portion of the enhancer region (in U3) in recombinant retroviruses (M-MuLVneo delta Enh). This confirms the presence of negative elements outside the viral 82-bp repeats. However, a few sites in the genomes of EC cells permit M-MuLVneo delta Enh proviral expression. One such site, identified in PCC4, PCC3, and LT, was studied. The complete analysis of the mechanism of activation by Northern (RNA) blotting, cloning, and sequencing of partial cDNA copies of the viral transcript and of the site of integration establishes that viral transcripts are initiated from an upstream host-cell promoter and are spliced from a host donor to a cryptic viral acceptor at position 542 in the Moloney murine leukemia virus (M-MuLV) genome. In consequence, the mature transcripts are host cell-virus fusion transcripts from which M-MuLV sequences, including the cis-active negative elements of the 5' long terminal repeat-containing region, are absent. The provirus integrates apparently randomly into any of the three most proximal introns of the transcriptional unit. The host cell promoter contains a TATA box and 14 potential SpI binding sites included in a 1.0-kb GC-rich island. These elements promote gene expression of recombinant vectors in EC and differentiated cells. The mechanism described points to a mechanism by which retroviruses can be transcribed from upstream nonviral elements and can acquire host genes by 5' annexation of exons.

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.