Limitations of PCR detection of filarial DNA in human stools from subjects non-infected with soil-transmitted helminths

The standard techniques for diagnosis of human filariasis are the microscopic examination of blood smears or skin biopsies, which are relatively invasive and poorly sensitive at low levels of infection. Recently, filarial DNA has been detected in fecal samples from non-human primates in Central Africa. The aim of this study was to demonstrate proof-of-concept of a non-invasive molecular diagnosis technique for human filariasis by targeting fragments of 12S rDNA, Cox1, ITS1 and LL20-15kDa ladder antigen-gene by conventional PCR in DNA extracted from stool samples of 52 people infected with Mansonella perstans and/or Loa loa. Of these, 10 patients were infected with soil-transmitted helminths (Trichuris trichiura and/or Ascaris lumbricoides), and none were positive for Necator americanus. Interestingly, no filarial gene fragments were detected in the stools of any of the 52 patients. Future studies should evaluate whether a co-infection with soil-transmitted helminths causing gastrointestinal bleeding and likely allowing (micro)filaria exit into the digestive tract, may facilitate the molecular detection of filarial DNA fragments in stool samples

Etude du mécanisme d'interaction du domaine de liaison à l'ADN de la poly(ADP-ribose)polymerase (PARP-1) avec des cibles nucléiques

L'activation de la poly(ADP-ribose)polymerase (hPARP-1) intervient en réponse aux cassures dans l'ADN. Dans le cadre de la recherche de traitements anti-cancéreux, la protéine PARP-1 constitue une cible de choix de par son implication dans les mécanismes de réparation de l'ADN. Etant donné la conservation du site catalytique au sein de la superfamille PARPs, l'élucidation au niveau mole culaire du processus de liaison à l'ADN de PARP-1 apparaît essentielle pour la compréhension de son rôle biologique et pour le développement d'inhibiteurs spécifiques dirigés contre la fonction de liaison.Dans ce contexte, nous nous sommes intéressés au mécanisme de liaison entre le domaine de liaison (DBD) de la hPARP-1 et des cibles nucléiques.Après avoir caractérisé les propriétés de fluorescence du DBD de PARP-1 nous avons déterminé les paramètres de liaison du DBD. Cette étude a mis en évidence une spécificité de reconnaissance au niveau de la jonction entre un double et un simple brin d'ADN avec une extrémité 5' rentrante. Le rôle des résidus tryptophanes apparaît essentiel dans la reconnaissance de la structure nucléique via des interactions de type empilement. Ces résultats nous ont permis de modéliser la liaison et de démontrer une dimérisation de la PARP-1 sur l'extrémité 5' rentrante. La position asymétrique de la PARP-1 sur l'ADN observée par empreinte à la DNase I semble corrélée avec la formation d'un dimère catalytique. Les mesures de l'activité de poly(ADP-ribosyl)ation combinées aux études par spectroscopie de fluorescence nous ont permis de corréler la stoechiométrie de liaison avec le niveau d'activité. Ainsi, une forte activité est mesurée lors de la formation d'un dimère protéique sur la cible nucléique. Enfin, la génération par mutagenèse dirigée d'un mutant du résidu Trp 51 du doigt à zinc FI, a permis de préciser le rôle de ce résidu dans le maintien conformationnel du DBD et son importance dans la conservation des propriétés de liaison du DBD.Activation of Poly(ADP-ribose)polymerase (hPARP-1) is an immediate response to DNA damage. For the research of antitumor agents in addition of actual chemotherapies and radiotherapies, the protein PARP-1 is a good target since it is implicated in facilitating DNA repair. Due to the highly conserved catalytic domain into the whole PARPs family, the elucidation of the molecular binding mechanism appears essential not only for a better knowledge of its biological function but also for developing specific inhibitors.In this context, we have analyzed the binding mechanism of PARP-1 in the presence of nucleic acid targets in order to better understand the protein's function.After the characterization of the fluorescence properties of the PARP-1 DBD, we have determined, the binding parameters of the PARP-1 DBD. This study showed that the recognition of a DNA junction between a double strand and a single strand bearing a 5' recessed end by DBD is highly specific. Trp residues seem to be directly implicated in the nucleic acids binding by stacking interactions. We also modelized the binding and demonstrated a protein PARP-1 dimerization. The asymmetric protection of DBD to DNA seems to be correlated with the formation of a catalytic dimer.Poly(ADP-ribose)activity measurements associated with fluorescence spectroscopy revealed that the activity levels of PARP-1 is strongly correlated with the binding stochioemetry. Even, a tightly relation was established between the binding stochiometry and the nucleic acids structures. Indeed, a great activity is measured when a DBD dimer is formed. Finally, by generating a mutant of DBD PARP-1 in position Trp51 in the zinc finger FI, we have detailed the implication of this Trp residue in the DBD structures maintain. By this work, the Trp51 seems to be relevant for keeping the binding properties of the DBD PARP-1.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

DNA-binding regulates site-specific ubiquitination of IRF-1

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.</jats:p

Poly(ADP-ribose) polymerase-1 dimerizes at a 5' recessed DNA end in vitro: a fluorescence study

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation, or oxidants. The resulting formation of protein-bound poly(ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA

DNA-Induced Dimerization of Poly(ADP-ribose) Polymerase-1 Triggers Its Activation †

International audienc

Limites de la détection par PCR d’ADN de filaires dans les selles humaines de sujets non-infectés par les géohelminthes

International audienceThe standard techniques for diagnosis of human filariasis are the microscopic examination of blood smears or skin biopsies, which are relatively invasive and poorly sensitive at low levels of infection. Recently, filarial DNA has been detected in fecal samples from non-human primates in Central Africa. The aim of this study was to demonstrate proof-of-concept of a non-invasive molecular diagnosis technique for human filariasis by targeting fragments of 12S rDNA, Cox1, ITS1 and LL20-15kDa ladder antigen-gene by conventional PCR in DNA extracted from stool samples of 52 people infected with Mansonella perstans and/or Loa loa . Of these, 10 patients were infected with soil-transmitted helminths ( Trichuris trichiura and/or Ascaris lumbricoides ), and none were positive for Necator americanus . Interestingly, no filarial gene fragments were detected in the stools of any of the 52 patients. Future studies should evaluate whether a co-infection with soil-transmitted helminths causing gastrointestinal bleeding and likely allowing (micro)filaria exit into the digestive tract, may facilitate the molecular detection of filarial DNA fragments in stool samples.Les techniques standards de diagnostic des filarioses humaines (examen microscopique de gouttes épaisses ou de biopsies cutanées) sont relativement invasives et peu sensibles à de faibles niveaux d’infection. De l’ADN de filaires a été récemment détecté dans des échantillons de fèces de primates non-humains en Afrique centrale. L’objectif de cette étude était de démontrer la preuve de concept d’un diagnostic moléculaire non invasif des filarioses chez l’homme en ciblant des fragments d’ADNr 12S, Cox1, ITS1 et l’antigène LL20-15kDa par PCR classique. L’ADN a été extrait d’échantillons de selles de 52 personnes infectées par Mansonella perstans et/ou Loa loa . Parmi ces patients, dix étaient infectés par des géohelminthes ( Trichuris trichiura et/ou Ascaris lumbricoides ) et aucun n’était positif pour Necator americanus . De manière intéressante, aucun fragment de gène de filaires n’a été détecté dans les selles des 52 patients. Des études futures devraient être menées pour évaluer si une coinfection avec des géohelminthes (provoquant des hémorragies gastro-intestinales et permettant probablement l’effraction de (micro)filaires dans le tube digestif) facilite la détection moléculaire de fragments d’ADN de filaires dans les selles

Objective Evaluation of Clinical Actionability for Genes Involved in Myopathies: 63 Genes with a Medical Value for Patient Care

International audienceThe implementation of high-throughput diagnostic sequencing has led to the generation of large amounts of mutational data, making their interpretation more complex and responsible for long delays. It has been important to prioritize certain analyses, particularly those of “actionable” genes in diagnostic situations, involving specific treatment and/or management. In our project, we carried out an objective assessment of the clinical actionability of genes involved in myopathies, for which only few data obtained methodologically exist to date. Using the ClinGen Actionability criteria, we scored the clinical actionability of all 199 genes implicated in myopathies published by FILNEMUS for the “National French consensus on gene Lists for the diagnosis of myopathies using next generation sequencing”. We objectified that 63 myopathy genes were actionable with the currently available data. Among the 36 myopathy genes with the highest actionability scores, only 8 had been scored to date by ClinGen. The data obtained through these methodological tools are an important resource for strategic choices in diagnostic approaches and the management of genetic myopathies. The clinical actionability of genes has to be considered as an evolving concept, in relation to progresses in disease knowledge and therapeutic approaches