Nouvelle méthode statistique pour l'analyse de données de ChIP-chip

International audienceLa méthode de Chromatin ImmunoPrecipitation on chip (ChIP on chip ou ChIP-chip) a pour but de détecter les sites de fixation des protéines (généralement des facteurs de transcription) sur la molécule d'ADN. L'analyse statistique des données consiste a rechercher des régions de pics significatifs synonymes de sites de fixation. La méthode que nous avons élaborée est issue de la théorie des valeurs extrêmes et particulièrement de la méthode POT (Peaks Over Threshold). Cette méthode consiste à modéliser les données de queues de distribution, en ne retenant que les valeurs dépassant un certain seuil, elle a la particularité de modéliser d'une part les intensités de dépassement de seuil, mais aussi les positions d'occurrences de ces dépassements de seuil. Cette méthode va nous permettre de déterminer un seuil au delà duquel les pics pourront être considérés comme significatifs

Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia

Background: Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008–2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. Methods: To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010–2011 from 16 health centres in malaria endemics areas in Cambodia. Results: Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. Conclusions: The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia

Functional analysis of Plasmodium falciparum subpopulations associated with artemisinin resistance in Cambodia

Background: Plasmodium falciparum malaria is one of the most widespread parasitic infections in humans and remains a leading global health concern. Malaria elimination efforts are threatened by the emergence and spread of resistance to artemisinin-based combination therapy, the first-line treatment of malaria. Promising molecular markers and pathways associated with artemisinin drug resistance have been identified, but the underlying molecular mechanisms of resistance remains unknown. The genomic data from early period of emergence of artemisinin resistance (2008–2011) was evaluated, with aim to define k13 associated genetic background in Cambodia, the country identified as epicentre of anti-malarial drug resistance, through characterization of 167 parasite isolates using a panel of 21,257 SNPs. Results: Eight subpopulations were identified suggesting a process of acquisition of artemisinin resistance consistent with an emergence-selection-diffusion model, supported by the shifting balance theory. Identification of population specific mutations facilitated the characterization of a core set of 57 background genes associated with artemisinin resistance and associated pathways. The analysis indicates that the background of artemisinin resistance was not acquired after drug pressure, rather is the result of fixation followed by selection on the daughter subpopulations derived from the ancestral population. Conclusions: Functional analysis of artemisinin resistance subpopulations illustrates the strong interplay between ubiquitination and cell division or differentiation in artemisinin resistant parasites. The relationship of these pathways with the P. falciparum resistant subpopulation and presence of drug resistance markers in addition to k13, highlights the major role of admixed parasite population in the diffusion of artemisinin resistant background. The diffusion of resistant genes in the Cambodian admixed population after selection resulted from mating of gametocytes of sensitive and resistant parasite populations. (Résumé d'auteur

Spatial distribution of cerebral white matter lesions predicts progression to mild cognitive impairment and dementia

CONTEXT White matter lesions (WML) increase the risk of dementia. The relevance of WML location is less clear. We sought to determine whether a particular WML profile, based on the density and location of lesions, could be associated with an increased risk of mild cognitive impairment (MCI) or dementia over the following 7 years. METHODS In 426 healthy subjects from a cohort of community-dwelling people aged 65 years and over (ESPRIT Project), standardized cognitive and neurological evaluations were repeated after 2, 4 and 7 years. Patterns of WML were computed with a supervised data mining approach (decision trees) using the regional WML volumes (frontal, parietal, temporal, and occipital regions) and the total WML volume estimated at baseline. Cox proportional hazard models were then constructed to study the association between WML patterns and risk of MCI/dementia. RESULTS Total WML volume and percentage of WML in the temporal region proved to be the best predictors of progression to MCI and dementia. Specifically, severe total WML load with a high proportion of lesions in the temporal region was significantly associated with the risk of developing MCI or dementia. CONCLUSIONS Above a certain threshold of damage, a pattern of WML clustering in the temporal region identifies individuals at increased risk of MCI or dementia. As this WML pattern is observed before the onset of clinical symptoms, it may facilitate the detection of patients at risk of MCI/dementia.The ESPRIT Project is financed by the regional government of Languedoc-Roussillon (http://www.laregion.fr), the Agence Nationale de la Recherche (ANR: http://www.agence-nationale-recherche.fr) and an unconditional grant from Novartis (http://www.novartis.fr). This study is also supported by France Alzheimer (http://www.francealzheimer.org/)

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution

Prédiction de la structure 3D des boucles protéiques

International audienceLa connaissance de la structure tridimensionnelle des protéines est essentielle pour comprendre leur fonction. Devant la complexité de l'obtention expérimentale de ces informations, la mise au point de méthodes permettant leur prédiction à partir des séquences en acides aminés est un défi majeur. Par ailleurs, dans ce contexte, l'étude des boucles est particulièrement importante car leur rôle fonctionnele est prépondérant et leur structure tridimensionnelle particulièrement variable. Nous proposons une méthode reposant sur l'encodage des structures 3D par alphabet structural. Cette méthode comporte une première étape de prédiction locale d'une lettre structurale à partir de quatre acides aminés par une méthode d'arbre. La prédiction est ensuite affinée en tenant compte des informations telles que les ressemblances entre lettres structurales et la succession non aléatoire des lettres dans les séquences. Un modèle de Markov caché est utilisé dans ce but. La méthode sera testée sur une large base de données de boucles contenant un jeu d'apprentissage ainsi qu'un jeu de validation

Pilot Study of Whole Blood MicroRNAs as Potential Tools for Diffuse Low-Grade Gliomas Detection

International audienceEarlier diagnosis and longitudinal monitoring of diffuse low-grade gliomas (DLGG) increase overall survival by maximizing surgery efficacy and optimizing time for an adjuvant treatment when resection is incomplete. Presently, only imaging permits the non-invasive detection and monitoring of DLGG, but it lacks sensitivity. Measure of circulating microRNAs levels could represent a non-invasive alternative. We hypothesized that slow-growing DLGG induce overtime a systemic reaction impacting blood cells microRNA profiles, while the intact blood-brain barrier restricts the passage of tumor microRNAs into bloodstream. In 15 DLGG patients and 15 healthy controls, expression levels of 758 microRNAs were measured by the TaqMan OpenArray RT-qPCR platform, on preoperative whole blood, containing both cell-free and blood cells microRNAs. Normalized data were computed by a Student t test with a p value threshold allowing a 10% rate of false positive. Statistical analysis retained fifteen microRNAs, all overexpressed in patients. MiR-20a, miR-106a, miR-20b, and miR-93 belong to clusters genetically related. As miR-223 and miR-let7e, they target the transcription factor STAT3. MicroRNA expression levels were not correlated to preoperative tumor volume. A signature composed of miR-93, miR-590-3p, and miR-454 enabled to nearly perfectly separate patients from controls. Our study performed on a homogeneous cohort was designed accordingly to DLGG particularities and provided the first microRNAs signature proposal. Functional convergence on STAT3 and overexpression of miR-223, factors respectively involved in myeloid-derived suppressor cells and granulocytes, argued for a systemic peripheral response. Overexpressed microRNAs and tumor volume were uncorrelated, making a tumor origin elusive

Development of an HRM-based tool for the automated identification of nucleotide sequences in large datasets

International audienceNucleic acid characterization by High Resolution Melting (HRM) is a simple, flexible, low-cost and powerful technique for identify- ing sequence variations, making it attractive for a broad range of diagnostic and research applications including infectious diseases, oncology, epigenetics and even metabarcoding. Current procedures for analyzing HRM curves mostly rely on unsupervised methods, principally via subtraction (difference) plots against a known con- trol sample, and less commonly on supervised methods through the use of discrimination analyses. If these procedures have proven useful for discriminating a small number of variants, they are yet limiting for analyzing large HRM data sets and do not provide pre- cise feedback to the user.In this context, we have developed an innovative method that enables the simultaneous discrimination of a large number of variants from their HRM profile. This method relies on the estab- lishment of a melting profile library, computes new descriptors from the HRM curves for an optimal discrimination and offers a fully automated analysis of melting profiles. The output consists in the possibility to assign a given melting profile to an existing group included in the reference library (assorted with a confidence index) or to reject any assignment in case of an unknown profile.This method was first validated on a set of 19 nontuberculous mycobacterial species. Each species was represented by 3–20 bio- logical samples consisting of genomic DNA extracted either from animal tissues or from cultivated isolates. Each sample was ampli- fied with a unique pair of primers targeting the 16S-ITS region and yielding amplicons with sizes ranging from ∼230 to ∼350 bp. Melt- ing profiles of the corresponding amplicons were generated using 3-9 replicates per sample. On a total of 95 samples, 91 were allot- ted to the right species. Automatic group rationalization led to split two species into two subgroups, suggesting that this method is able to integrate intraspecific sequence variations.The method was then applied to develop a diagnostic tool tar- geting five different pathogens responsible for abortive diseases in cattle. Each pathogen was represented by 10 to 22 different biological samples consisting of genomic DNA extracted from dif- ferent matrices (e.g. feces, swabs, whole blood). Each biological sample was amplified and melted in 4 replicates with a ready-to- use mastermix containing 5 sets of primers, specific to the targeted pathogens. The limit of detection of this multiplex test was equiva- lent to that of the current individual tests using hydrolysis probes, around 10 copies/PCR. Moreover, all samples containing at least 10 copies of pathogen(s) were correctly identified with high confi- dence.These results underline the high potential of this novel HRM- based method for the simultaneous detection and identification of a large number of nucleotide sequences, in both simplex and multiplex formats