Coulomb dissociation of O-16 into He-4 and C-12

We measured the Coulomb dissociation of O-16 into He-4 and C-12 within the FAIR Phase-0 program at GSI Helmholtzzentrum fur Schwerionenforschung Darmstadt, Germany. From this we will extract the photon dissociation cross section O-16(alpha,gamma)C-12, which is the time reversed reaction to C-12(alpha,gamma)O-16. With this indirect method, we aim to improve on the accuracy of the experimental data at lower energies than measured so far. The expected low cross section for the Coulomb dissociation reaction and close magnetic rigidity of beam and fragments demand a high precision measurement. Hence, new detector systems were built and radical changes to the (RB)-B-3 setup were necessary to cope with the high-intensity O-16 beam. All tracking detectors were designed to let the unreacted O-16 ions pass, while detecting the C-12 and He-4

Association of Forced Vital Capacity with the Developmental Gene <i>NCOR2</i>

Background Forced Vital Capacity (FVC) is an important predictor of all-cause mortality in the absence of chronic respiratory conditions. Epidemiological evidence highlights the role of early life factors on adult FVC, pointing to environmental exposures and genes affecting lung development as risk factors for low FVC later in life. Although highly heritable, a small number of genes have been found associated with FVC, and we aimed at identifying further genetic variants by focusing on lung development genes. Methods Per-allele effects of 24,728 SNPs in 403 genes involved in lung development were tested in 7,749 adults from three studies (NFBC1966, ECRHS, EGEA). The most significant SNP for the top 25 genes was followed-up in 46,103 adults (CHARGE and SpiroMeta consortia) and 5,062 chi

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Effets de la Tosyl Phénylalaline Chlorométhyl Cétone (TPCK) sur l'activation de la NADPH oxydase de neutrophiles humains

La NADPH oxydase se situe dans la membrane des cellules phagocytaires. Elle est constituée de deux protéines membranaires, formant le cytochrome b558, et de quatre protéines cytosoliques. Lors de l'activation, les facteurs cytosoliques sont phosphorylées, ils transloquent et s'associent au cytochrome qui produit alors l'anion superoxyde. L'activation fait intervenir des voies de signalisation complexes. Le Tosyl-Phénylalanine-Chlorométhyl-Cétone (TPCK), inhibiteur de protéases, inhibe la NADPH oxydase. Cependant, aucune protéase ne semble impliquée dans la production d'anion superoxyde et le TPCK inhibe d'autres protéines. Au laboratoire, des tests cinétiques ont montré que l'incubation des neutrophiles avec le TPCK, avant ou après activation par le phorbol myristate acétate (PMA), inhibe l'activation de la NADPH oxydase en modifiant une protéine cytosolique de 15 kDa. Par immunoblot, il est montré ici que le TPCK inhibe et réverse la translocation de p47phox et p67phox. Par contre, il n'a aucun effet sur la translocation de Rac 2. Le TPCK peut aussi empêcher l'association à la membrane de p67phox et Rac 2, présentes dans les membranes de cellules non activées. Le marquage des cellules au [32P], montre que le TPCK inhibe et réverse la phosphorylation de p47phox. La purification partielle de la cible, marquée avec du TPCK tritié, n'a pas permis de l'identifier. L'ensemble des résultats montre que le TPCK touche un élément de la signalisation cellulaire et active une phosphatase qui pourrait être impliquée dans la l'activation/désactivation de la NADPH oxydase et dans l'organisation du cytosquelette. L'identification de la cible permettra de mieux expliquer les effets observés. A long terme, cela pourra peut-être permettre de réguler l'activité de cette enzyme localement grâce à la synthèse d'inhibiteurs plus spécifiques que le TPCK. Ces inhibiteurs pourront alors être utilisés dans un but thérapeutique dans les maladies inflammatoires impliquant la NADPH oxydase.NADPH oxidase is located in phagocytic cells membrane. It is made of two membrane proteins, bath forming the cytochrome b558, and four cytosolic proteins. After activation, cytosolic factors are phosphorylated, they transolocate to the membrane and associate to the cytochrome, which produces superoxide anion. NADPH oxidase activation requires complex signaling pathways. Tosyl Phenylalanine Chloromethyl Ketone (TPCK), a protease inhibitor, is known to inhibit this enzyme activity. However, no protease implicated in superoxide production bas ever been identified. Furthermore, it is now known that TPCK also inhibits the activity of other proteins. This laboratory bas shown that incubation of neutrophils with this inhibitor, before and after stimulation with Phorbol Myristate Acetate (PMA), inhibits activation of NADPH oxidase by modifying a cytosolic protein of 15 kDa. Using immunoblots, it is shown here that p47phox and p67phox translocation is inhibited and reversed by TPCK treatment. The TPCK bas no effect on Rac 2 translocation. It is also observed that TPCK prevents p67phox and Rac 2 membrane association in non-activated cells. Using [32P] labeling and immuno-precipitation, it is shown that p47phox phosphorylation is also inhibited and reversed by TPCK. A partial purification of the TPCK target, using tritiated TPCK, didn't allow the identification of this protein. Taken together, these results show that TPCK target is an important element in cellular signaling. Incubation of neutrophils with TPCK certainly activates a phosphatase, which could be involved in NADPH oxidase activation/deactivation and cytoskeleton organization. The target identification is an important step to explain TPCK effects. Furthermore, this will give the opportunity to synthesize specific NADPH oxidase inhibitors to locally control superoxide anion production in a therapeutic goal.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Etudes mécanistiques de flavodéshydrogénases d'a-hydroxyacides

La famille des L-2-hydroxyacide oxydases à FMN comprend à la fois des déshydrogénases-oxydases et des déshydrogénases-transférases d'électrons. Ses membres possèdent plus de 30% d'identité de séquence et un repliement très proche, comme l'indiquent les structures des glycolate oxydase, oxydase d'hydroxyacide à longue chaîne (HAO) et flavocytochrome b2 (FCB2). Les résidus catalytiques sont invariants à une exception près pour la HAO. Le substrat physiologique de celle-ci n'est toujours pas connu, cependant il a été récemment montré qu'elle serait responsable de oxydation du créatol, qui n'est pas un hydroxyacide, en méthylguanidine. Pour cette famille, nombre d'expériences sont en faveur d'un mécanisme par carbanion, mais de récents résultats ont conduit certains auteurs à pencher pour un mécanisme par transfert d'hydrure. Nous montrons dans ce travail que le 2-hydroxyphényl acétohydroxamate (HYPAH), un homologue du mandélate (un des meilleurs substrat connu) et du créatol, est oxydé par la HAO. Ce composé se fixe également au site actif du FCB2. Des études comparative de pH en fonction de l'activité pour le mandélate et l'HYPAH, qui se fixerait sous forme ionisée, nous ont renseigné sur les résidus du site actif impliqués dans la catalyse. Nous avons également montré que le benzohydroxamate était un inhibiteur compétitif de la HAO, nos résultats ne laissent pas envisager l'existence d'un intermédiaire éndiolate. Dans une seconde partie, nous avons analysé le comportement du trifluorolactate (F3Lac) et du trifluoropyruvate (F3Pyr) avec le FCB2 et la HAO. La dépolarisation engendrée par les atomes de fluor doit être suffisante pour empêcher le départ d'un hydrure. Nous avons trouvé que le F3Lac est substrat pour le FCB2 et la HAO. La faible activité mesurée pourrait être due au potentiels rédox du couple F3Lac/F3Pyr, nous avons donc testé le F3Pyr dans des conditions de transhydrogénation et nous avons trouvé que la vitesse de cette réaction était rapide dans ce sens.The FMN-dependent L-2-hydroxy acid-oxidizing enzymes family comprises both dehydogenases-oxidases and dehydrogenases-electron transferases. Members display over 30% sequence identity and a strongly conserved fold, as indicated by the structures of glycolate oxidase, its isozyme long-chain hydroxyacid acid oxidase (HAO), flavocytochrome b2 (FCB2) and mandelate dehydrogenase. The catalytic residues are invariant, with one exception in HAO. For this enzyme family, evidence exists in favor of a carbanion mechanism, but recent results have led some authors to consider the possibility of a hydride transfer mechanism. The physiological substrate of HAO is unknown, still it has been recently reported that it could convert creatol, which is not a 2-hydroxyacid, to methylguanidine. We show in this work that 2-hydroxyphenyl acetohydroxamate (HYPAH), a compound that bears similarity both to mandelate (one of the best substrate known) and to creatol, is turned over by HAO. The compound also binds to the active site of homologous FCB2. Comparative pH-rate studies for mandelate and its hydroxamate suggest that HYPAH may bind in its ionized form and gave us information about the catalysis-implicated residues. In addition to these studies, we have found that HAO is competitively inhibited by benzohydroxamate, our results do not support the existence of an enolic intermediate for HAO. We have analyzed, in a second part of this work, the behavior of trifluorolactate (F3Lac) and trifluropyruvate (F3Pyr) towards FCB2 and HAO. The electrostatic attraction exerted by the fluorine atoms must be strong enough to prevent departure of a hydride ion. We find that F3Lac is a substrate both for FCB2 and HAO. The decreased efficiencies measured could be due to a higher redox potential of the F3Lac/F3Pyr couple, so we tested the reduction of F3Pyr by FCB2 and HAO under transhydrogenation conditions, and found the reaction to be quite rapid under these conditions.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Caractérisation moléculaire de la laforine impliquée dans la maladie de Lafora (une protéine phosphatase humaine à double spécificité associée à un domaine de fixation à l'amidon)

Certaines mutations dans le gène EPM2A provoquent la maladie de Lafora, une épilepsie myoclonique progressive dont l'issue est fatale, caractérisée par la présence de corps d'inclusion de polyglucosan dans les tissus nerveux. L'analyse de la séquence du produit du gène (laforine) suggère la présence de deux domaines : un site de liaison à l'amidon (CBM20) en N-terminal, identifié pour la première fois dans une protéine humaine et un site catalytique de phosphatase à double spécificité (DSPc) en C-terminal. La partie codante du gène a été clonée a partir d'une banque de cDNA humain. Différentes constructions ont été réalisées et ont permis d'obtenir la laforine pure et soluble. Une première caractérisation a montré que la laforine se liait à l'amidon, conformément aux prédictions, mais également au glycogène. Des expériences de compétition entre ces deux polysaccharides mettent en évidence que la fixation aux sucres est spécifique. L'activité catalytique phosphatase a été mesurée pour deux substrats : le p-nitrophényl phosphate et l'O-méthylfluorescéinyl phosphate (OMFP). Les paramètres cinétiques montrent une meilleure efficacité catalytique vis-à-vis de l'OMFP, indiquant que la laforine porte une fonction de DSP. Ni le glycogène ni de petits saccharides n'affectent la fonction catalytique. Ceci est à comparer aux données sur les MAP kinases phosphatases (MKP) dont le site catalytique est homologue à celui de la laforine mais dont l'activité est régulée par la fixation au substrat. Par conséquent, il apparaît que le rôle du domaine CBM20 de la laforine soit de localiser la protéine au glycogène où elle pourrait jouer un rôle, qui reste à élucider, dans son métabolisme.Mutations in the EPM2A gene cause Lafora disease, a fatal progressive myoclonus epilepsy characterized by the presence of polyglucosan inclusion bodies in nervous tissues. Sequence analysis of the gene product (laforin), suggested two domains: an N-terminal starch-binding domain (CBM20), found for the first time in a human protein, and a C-terminal dual specificity phosphatase catalytic domain (DSPc). The gene was cloned from human muscle cDNA. Different constructs were assayed to express and purify the laforin which aggregated easily. Adsorption experiments demonstrate that laforin binds granular starch as predicted, and ultracentrifugation studies showed the protein also binds to glycogen. Competition experiments between granular starch with either glycogen or beta-cyclodextrin indicated the binding to be specific. To demonstrate the second predicted function, the catalytic activity was tested with p-nitrophenyl phosphate and O-methylfluorescein phosphate (OMFP), two non physiological substrates. Laforin exhibited a better efficiency toward OMFP, indicating that laforin bears a DSP function. Further experiments showed that neither glycogen nor smaller sugars affected the laforin catalytic activity. This is in contrast with MAP kinases phosphatases (MKP). Indeed, the laforin catalytic domain is homologous to that of MKPs but CBM20 is different from their regulatory domain. For MKPs, the catalytic activity is increased when the regulatory domain binds to their substrates. Thus it appears that the role of the laforin CBM20 domain is to target the protein to glycogen in the cells. In conclusion laforin may play a role in glycogen metabolism which has to be elucidated.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF