Novel insulated gamma and lentis retroviral vectors towards safer genetic modification of stem cells

In otherwise successful gene therapy trials insertional mutagenesis has resulted in leukemia. The identification of new short synthetic genetic insulator elements (GIE) which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts with e.g. b-globin HS4, have met with poor efficacy and genetic instability. We have investigated potential improvement with two new candidate synthetic GIEs in SIN-gamma and lentiviral vectors. With each constructs two internal promoters have been tested: either the strong Fr- MuLV-U3 or the housekeeping hPGK.We could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro and lentivectors. In target cells a dramatic shift of expression is observed with an homogenous profile the level of which strictly depends on the promoter strength. These data remain stable in both HeLa cells over three months and cord blood HSCs for two months, irrespective of the multiplicity of infection (MOI). In comparison, control native and SIN vectors expression levels show heterogeneous, depend on the MOI and prove unstable. We have undertaken genotoxicity assessment in comparing integration patterns ingenuity in human target cells sampled over three months using high-throughput pyro-sequencing. Data will be presented. Further genotoxicity assessment will include in vivo studies. We have established insulated vectors which harbour both boundary and enhancer-blocking effect and show stable in prolonged in vitro culture conditions. Work performed with support of EC-DG research FP6-NoE, CLINIGENE: LSHB-CT-2006-018933

Qualités psychométriques de la version française de la TAS-20 et prévalence de l'alexithymie chez 264 adolescents tout-venant [The 20-item Toronto alexithymia scale: structural validity, internal consistency and prevalence of alexithymia in a Swiss adolescent sample]

L'objectif de cette étude est d'examiner la structure factorielle et la consistance interne de la TAS-20 sur un échantillon d'adolescents (n = 264), ainsi que de décrire la distribution des caractéristiques alexithymiques dans cet échantillon. La structure à trois facteurs de la TAS-20 a été confirmée par notre analyse factorielle confirmatoire. La consistance interne, mesurée à l'aide d'alpha de Cronbach, est acceptable pour le premier facteur (difficulté à identifier les sentiments (DIF)), bonne pour le second (difficulté à verbaliser les sentiments (DDF)), mais en revanche, faible pour le troisième facteur (pensées orientées vers l'extérieur (EOT)). Les résultats d'une Anova mettent en évidence une tendance linéaire indiquant que plus l'âge augmente plus le niveau d'alexithymie (score total TAS-20), la difficulté à identifier les sentiments et les pensées orientées vers l'extérieur diminuent. En ce qui concerne la prévalence de l'alexithymie, on remarque en effet que 38,5 % des adolescents de moins de 16 ans sont considérés comme alexithymiques, contre 30,1 % des 16-17 ans et 22 % des plus de 17 ans. Notre étude indique donc que la TAS-20 est un instrument adéquat pour évaluer l'alexithymie à l'adolescence, tout en suggérant quelques précautions étant donné l'aspect développemental de cette période.

Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency.

Female residents experiencing medical errors in general internal medicine: a qualitative study.

BACKGROUND: Doctors, especially doctors-in-training such as residents, make errors. They have to face the consequences even though today's approach to errors emphasizes systemic factors. Doctors' individual characteristics play a role in how medical errors are experienced and dealt with. The role of gender has previously been examined in a few quantitative studies that have yielded conflicting results. In the present study, we sought to qualitatively explore the experience of female residents with respect to medical errors. In particular, we explored the coping mechanisms displayed after an error. This study took place in the internal medicine department of a Swiss university hospital. METHODS: Within a phenomenological framework, semi-structured interviews were conducted with eight female residents in general internal medicine. All interviews were audiotaped, fully transcribed, and thereafter analyzed. RESULTS: Seven main themes emerged from the interviews: (1) A perception that there is an insufficient culture of safety and error; (2) The perceived main causes of errors, which included fatigue, work overload, inadequate level of competences in relation to assigned tasks, and dysfunctional communication; (3) Negative feelings in response to errors, which included different forms of psychological distress; (4) Variable attitudes of the hierarchy toward residents involved in an error; (5) Talking about the error, as the core coping mechanism; (6) Defensive and constructive attitudes toward one's own errors; and (7) Gender-specific experiences in relation to errors. Such experiences consisted in (a) perceptions that male residents were more confident and therefore less affected by errors than their female counterparts and (b) perceptions that sexist attitudes among male supervisors can occur and worsen an already painful experience. CONCLUSIONS: This study offers an in-depth account of how female residents specifically experience and cope with medical errors. Our interviews with female residents convey the sense that gender possibly influences the experience with errors, including the kind of coping mechanisms displayed. However, we acknowledge that the lack of a direct comparison between female and male participants represents a limitation while aiming to explore the role of gender.

Expression of pSTAT5 predicts FLT3 internal tandem duplications in acute myeloid leukemia.

Mutations of the Fms-like tyrosine kinase 3 (FLT3) can be detected in a significant number of acute myeloid leukemias (AML). Seventy-five cases of acute myeloid leukemia were evaluated for FLT3-internal tandem duplications (ITD) by polymerase chain reaction. Paraffin-embedded formalin-fixed trephine biopsies of these cases were evaluated for expression of phosphorylated signal transducer and activator of transcription 1 (pSTAT1), pSTAT3, and pSTAT5. Specific expression of pSTAT5 was proven in leukemic blasts in situ by double staining with a blast-specific marker. Expression of pSTAT5 in > or =1% of blasts was highly predictive of FLT3-ITD. Neither expression of pSTAT1 nor pSTAT3 were associated with FLT3 mutations. Altogether we conclude that pSTAT5 expression can precisely be assessed by immunohistochemistry in routinely processed bone marrow trephines, STAT5 is highly likely the preferred second messenger of FLT3-mediated signaling in AML, and expression of pSTAT5 is predictive of FLT3-ITD.

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Summary : Internal ribosome entry sites (IRES) are used by viruses as a strategy to bypass inhibition of cap-dependent translation that commonly results from viral infection. IRES are also used in eukaryotic cells to control mRNA translation under conditions of cellular stress (apoptosis, heat shock) or during the G2 phase of the cell cycle when general protein synthesis is inhibited. Variation in cellular expression levels has been shown to be inherited. Expression is controlled, among others, by transcriptional factors and by the efficiency of cap-mediated translation and ribosome activity. We aimed at identifying genomic determinants of variability in IRES-mediated translation of two representative IRES [Encephalomyocarditis virus (EMCV) and X-linked Inhibitor-of-Apoptosis (XIAP) IRES]. We used bicistronic lentiviral constructions expressing two fluorescent reporter transgenes. Lentiviruses were used to transduce seven different laboratory cell lines and B lymphoblastoid cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209); representing an in vitro approach to family structure allowing genome scan analyses. The relative expression of the two markers was assessed by FACS. IRES efficiency varies according to cellular background, but also varies, for a same cell type, among individuals. The control of IRES activity presents an inherited component (h2) of 0.47 and 0.36 for EMCV and XIAP IRES, respectively. A genome scan identified a suggestive Quantitative Trait Loci (LOD 2.35) involved in the control of XIAP IRES activity. Résumé : Les sites internes d'entrée des ribosomes (IRES = internal ribosome entry sites) sont utilisés par les virus comme une stratégie afin d'outrepasser l'inhibition de traduction qui résulte communément d'une infection virale. Les IRES sont également utilisés par les cellules eucaryotes pour contrôler la traduction de l'ARN messager dans des conditions de stress cellulaire (apoptose, choc thermique) ou durant la phase G2 du cycle cellulaire, situations durant lesquelles la synthèse générale des protéines est inhibée. La variation des niveaux d'expression cellulaire de transcription est un caractère héréditaire. L'expression des gènes est contrôlée entre autre par les facteurs de transcription et par l'efficacité de la traduction initiée par la coiffe ainsi que par l'activité des ribosomes. Durant cette étude nous avons eu pour but d'identifier les déterminants génomiques responsables de la variabilité de la traduction contrôlée par l'IRES. Ceci a été effectué en étudiant deux IRES représentatifs : l'IRES du virus de l'encéphalomyocardite (EMCV) et l'IRES de l'inhibiteur de l'apoptose XIAP (X-linked Inhibitor-of-Apoptosis). Nous avons utilisés des lentivirus délivrant un transgène bicistronique codant pour deux gènes rapporteurs fluorescents. Ces lentivirus ont été utilisés pour transduire sept différentes lignées cellulaires de laboratoire et des lignées cellulaires lymphoblastoïdes B du Centre d'Etude du Polymorphisme Humain (CEPH; 15 pedigrees; n=209) qui représentent une approche in vitro de la structure familiale et qui permettent des analyses par balayage du génome. L'expression relative des deux marqueurs fluorescents a été analysée par FACS. Nos résultats montrent que l'efficacité des IRES varie en fonction du type de cellules. Il varie aussi, pour le même type de cellules, selon les individus. Le contrôle de l'activité de l'IRES est un caractère héritable (héritabilité h2) de 0.47 et 0.36 pour les IRES de EMCV et XIAP respectivement. Le balayage du génome a permis l'identification d'un locus à effets quantitatifs [QTL Quantitative Trait Loci (LOD 2.35)] impliqué dans le contôle de l'activité de l'IRES de XIAP.

The different types of internal hernia after laparoscopic Roux-En-Y gastric by-bass for morbid obesity : MDCT features : P5

Purpose: 1. To provide an overview of the different types of internal hernia (IH) occurring after laparoscopic Roux-en-Y gastric bypass (LRYGBP) performed for morbid obesity. 2. To describe the correspondent MDCT features in relation with the underlying anatomical landmarks in order to differentiate their localisation and to direct the surgeon during following laparoscopic closure of mesenteric defects. Methods and materials: LRYGBP performed for morbid obesity is associated with less perioperative complications, shorter hospital stay and a more rapid recovery compared with the open surgical procedure. However, a relatively high incidence of IH is seen that may be due to the laparoscopic approach, but also caused by rapid weight loss with consecutive loosening of the mesenteric sutures. Results: After briefly reviewing the surgical procedure of LRYGBP (ante- versus retrocolic) we describe the exact anatomical landmarks of the different types of IH occurring at any time after operation: They are caused by surgical defects either at the level of the transverse colon mesentery, at the Petersen's space, which represents an opening between the mesocolon and jejunal mesentery, or at the enteroenterostomy site. Typical MDCT features of each IH type in axial and coronal plane as well as targeted vascular reconstructions are demonstrated. Conclusion: Exact knowledge about underlying pathophysiology and anatomical landmarks is essential for distinguishing the different types of IH occurring after LRYGBP on MDCT, since radiological features are difficult to recognize and may even overlap. The radiologist should be aware of the potential anatomic sites to ensure subsequent straightforward laparoscopic exploration.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.

Methylation of GpG Island promoters in uveal melanoma

ABSTRACT : Background: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. Aim: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. Methods: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. Results: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/ 23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. Conclusions: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma. RAPPORT DE SYNTHESE : L'inactivation de gènes par une hyperméthylation de leur promoteur apparaît être un événement épigénétique fréquent, se retrouvant dans de nombreuses tumeurs. Dans cette étude, nous avons investigué dans des mélanomes primaires de l'uvée l'état de méthylation du promoteur de gènes fréquemment impliqués dans le développent tumoral tels que p16, TIMP3, RASSFI, RARB, FHIT, hTERT et APC. La méthylation des promoteurs de gènes a été étudiée par methylationsensitive single-strand conformation analysis (MS-SSCA) et dot blot assay (MS-DBA) dans une série de 23 mélanomes primaires de l'uvée. Tous les échantillons tissulaires provenaient de matériel fixé dans le formol et conservé dans des blocs de parraffine. Nous avons identifié une fréquence relativement élevée (52%) pour la méthylation du promoteur de hTERT. En ce qui concerne le reste des gènes étudiés, nous avons retrouvé des fréquences de méthylation de promoteurs relativement basses avec 13% pour RASSF1, 13% pour RARB 13%, 9% pour TIMP3 et 4% pour p16. Nous n'avons pas retrouvé d'hyperméthylation des promoteurs des gènes APC et FRIT. La méthylation de hTERT apparaît être un événement important dans la biologie du mélanome de l'uvée. L'hyperméthylation des autres gènes évalués ne semble pas être cruciale dans le développent de cette tumeur. Comme la méthylation des promoteurs des gènes APC, RASSF1 et RARB a été fréquemment observée dans le mélanome de la peau, notre étude tend à démontrer que des mécanismes épigénétiques différents surviennent dans le développement respectif de ces tumeurs.

The different types of internal hernia after laparoscopic Roux-En-Y gastric by-pass for morbid obesity: MDCT features [C-419]

Learning Objectives: 1. To provide an overview of the different types of internal hernia (IH) occurring after laparoscopic Roux‑en‑Y gastric bypass (LRYGBP) for morbid obesity. 2. To describe correspondent MDCT features in relation with the underlying anatomical landmarks in order to differentiate their localisation and to direct the surgeon during following laparoscopic closure of mesenteric defects. Background: LRYGBP for morbid obesity is associated with less perioperative complications, shorter hospital stay and a more rapid recovery compared with the open surgical procedure. However, a relatively high incidence of IH is seen that may be due to the laparoscopic approach, but also caused by rapid weight loss with consecutive loosening of the mesenteric sutures. Procedure Details: After briefly reviewing the surgical procedure of LRYGBP (ante‑ versus retrocolic), we describe the exact anatomical landmarks of the different types of IH occurring at any time after operation: They are caused by surgical defects at the level of the transverse colon mesentery, at the Petersen's space, which represents an opening between the mesocolon and jejunal mesentery, or at the entero‑enterostomy site. Typical MDCT features of each IH type in axial and coronal planes as well as targeted vascular reconstructions are demonstrated. Conclusion: Exact knowledge about underlying pathophysiology and anatomical landmarks is essential for distinguishing the different types of IH occurring after LRYGBP on MDCT, since radiological features are difficult to recognize and may even overlap. The radiologist should be aware of the potential anatomic sites to ensure subsequent straightforward laparoscopic exploration.

Characteristic bimodal profiles of RNA polymerase II at thousands of active mammalian promoters.

BACKGROUND: In mammals, ChIP-seq studies of RNA polymerase II (PolII) occupancy have been performed to reveal how recruitment, initiation and pausing of PolII may control transcription rates, but the focus is rarely on obtaining finely resolved profiles that can portray the progression of PolII through sequential promoter states. RESULTS: Here, we analyze PolII binding profiles from high-coverage ChIP-seq on promoters of actively transcribed genes in mouse and humans. We show that the enrichment of PolII near transcription start sites exhibits a stereotypical bimodal structure, with one peak near active transcription start sites and a second peak 110 base pairs downstream from the first. Using an empirical model that reliably quantifies the spatial PolII signal, gene by gene, we show that the first PolII peak allows for refined positioning of transcription start sites, which is corroborated by mRNA sequencing. This bimodal signature is found both in mouse and humans. Analysis of the pausing-related factors NELF and DSIF suggests that the downstream peak reflects widespread pausing at the +1 nucleosome barrier. Several features of the bimodal pattern are correlated with sequence features such as CpG content and TATA boxes, as well as the histone mark H3K4me3. CONCLUSIONS: We thus show how high coverage DNA sequencing experiments can reveal as-yet unnoticed bimodal spatial features of PolII accumulation that are frequent at individual mammalian genes and reminiscent of transcription initiation and pausing. The initiation-pausing hypothesis is corroborated by evidence from run-on sequencing and immunoprecipitation in other cell types and species.