Approaching prosthesis infection environment: Development of an innovative in vitro Staphylococcus aureus biofilm model

The major role and implication of bacterial biofilms in the case of bone and prosthesis infections have been highlighted and often linked to implant colonization. Management strategies of these difficult-to-treat infections consist in surgeries and antibiotic treatment, but the rate of relapse remains high, especially if Staphylococcus aureus, a high-virulent pathogen, is involved. Therapeutic approaches are not adapted to the specific features of biofilm in bone context whereas infectious environment is known to importantly influence biofilm structure. In the present study, we aim to characterize S. aureus SH1000 (methicillin-sensitive strain, MSSA) and USA300 (methicillin-resistant strain, MRSA) biofilm on different surfaces mimicking the periprosthetic environment. As expected, protein adsorption on titanium enhanced the number of adherent bacteria for both strains. On bone explant, USA300 adhered more than SH1000. The simultaneous presence of two different surfaces was also found to change the bacterial behaviour. Thus, proteins adsorption on titanium and bone samples (from bank or directly recovered after an arthroplasty) were found to be key parameters that influence S. aureus biofilm formation: adhesion, matrix production and biofilm-related gene regulation. These results highlighted the need for new biofilm models, more relevant with the infectious environment by using adapted culture medium and presence of surfaces that are representative of in situ conditions to better evaluate therapeutic strategies against biofilm

L’alimentation des premiers agropasteurs du Néolithique : apport de l’étude des microrestes du tartre dentaire des individus de la Roussille (Auvergne)

National audienceLe Néolithique, épisode majeur de l’histoire Humaine est marqué par la domestication des espèces végétales et animales, les débuts de l’agriculture et une croissance démographique significative. Le projet ANR WomenSOFar (21-CE03-0008) vise à identifier le statut des femmes à travers la façon dont elles se déplacent, se nourrissent, travaillent et prennent soin des jeunes. Dans le cadre de ce projet de recherche, une approche novatrice et exploratoire, consistant en l’analyse des microrestes contenus dans le tartre dentaire, a été réalisée. Les objectifs étaient de tester l’intérêt de cette approche jusqu’à présent embryonnaire en France et de proposer des éléments de réponse au régime alimentaire des communautés néolithiques de La Roussille (Vertaizon, Puys-de-Dôme) en addition aux données d’autres disciplines paléoécologiques et archéobiologiques. Le site étudié, La Roussille, se situe en Auvergne et il présente la particularité d’être composé d’un habitat et d’un zone funéraire, datée d’environ 4600 à 4300 ans avant notre ère. Les échantillons de tartre dentaire de 7 individus, dont 2 hommes, 4 femmes, et 1 de sexe inconnu, ont été analysés (University College of Dublin, Irlande). Les échantillons de tartre dentaire ont été décalcifiés selon la méthode EDTA, avant d’être étudiés au microscope optique. Les microrestes ont été identifiés grâce à des publications et livres de référence. Les principaux éléments retrouvés étaient des phytolithes, corps microscopiques siliceux produits par les plantes en guise de défense contre des attaques telle que l’herbivorie, mais aussi des cellules animales, des micro-charbons, des pollen ou encore des spores de champignon. Ces premiers résultats interrogent sur la représentativité des différents types d’éléments identifiés dans cette matrice et l’utilisation des dents en tant qu’outil

L’alimentation des premiers agropasteurs du Néolithique : apport de l’étude des microrestes du tartre dentaire des individus de la Roussille (Auvergne)

National audienceThe Neolithic, a major episode in human history, is marked by the domestication of plant and animal species, the beginnings of agriculture, and significant demographic growth. The ANR WomenSOFar project (ANR-21-CE03-0008) aims to identify the status of women through how they move, eat, work, and care for the young. As part of this research project, an innovative and exploratory approach involving the analysis of micro-remains contained in dental calculus was carried out. The objectives were to test the interest of this still embryonic approach in France and to provide insights into the dietary habits of Neolithic communities in La Roussille (Vertaizon, Puy-de-Dôme), in addition to data from other paleoecological and bioarchaeological disciplines.The studied site, La Roussille, is located in Auvergne and has the particularity of being composed of a habitation area and a burial zone, dated to approximately 4600 to 4300 years BCE. Dental calculus samples from 7 individuals, including 2 males, 4 females, and 1 of unknown sex, were analyzed at the University College of Dublin, Ireland. Dental calculus samples were decalcified using the EDTA method before being studied under an optical microscope. Micro-remains were identified through publications and reference books. The main elements found were phytoliths, microscopic siliceous bodies produced by plants as a defense against attacks such as herbivory, as well as animal cells, micro-charcoal, pollen, and fungal spores. These initial results raise questions about the representativeness of the different types of elements identified in this matrix and the use of teeth as a tool.Wolfgang Haak, Stéphane Rottier, Guillaume Leduc, Gwenaëlle GoudeLe Néolithique, épisode majeur de l’histoire Humaine est marqué par la domestication des espèces végétales et animales, les débuts de l’agriculture et une croissance démographique significative. Le projet ANR WomenSOFar (21-CE03-0008) vise à identifier le statut des femmes à travers la façon dont elles se déplacent, se nourrissent, travaillent et prennent soin des jeunes. Dans le cadre de ce projet de recherche, une approche novatrice et exploratoire, consistant en l’analyse des micro-restes contenus dans le tartre dentaire, a été réalisée. Les objectifs étaient de tester l’intérêt de cette approche jusqu’à présent embryonnaire en France et de proposer des éléments de réponse au régime alimentaire des communautés néolithiques de La Roussille (Vertaizon, Puy-de-Dôme) en addition aux données d’autres disciplines paléoécologiques et archéobiologiques. Le site étudié, La Roussille, se situe en Auvergne et il présente la particularité d’être composé d’un habitat et d’un zone funéraire, datée d’environ 4600 à 4300 ans avant notre ère. Les échantillons de tartre dentaire de 7 individus, dont 2 hommes, 4 femmes, et 1 de sexe inconnu, ont été analysés (University College of Dublin, Irlande). Les échantillons de tartre dentaire ont été décalcifiés selon la méthode EDTA, avant d’être étudiés au microscope optique. Les microrestes ont été identifiés grâce à des publications et livres de référence. Les principaux éléments retrouvés étaient des phytolithes, corps microscopiques siliceux produits par les plantes en guise de défense contre des attaques telle que l’herbivorie, mais aussi des cellules animales, des microcharbons, des pollen ou encore des spores de champignon. Ces premiers résultats interrogent sur la représentativité des différents types d’éléments identifiés dans cette matrice et l’utilisation des dents en tant qu’outil

L’alimentation des premiers agropasteurs du Néolithique : apport de l’étude des microrestes du tartre dentaire des individus de la Roussille (Auvergne)

National audienceThe Neolithic, a major episode in human history, is marked by the domestication of plant and animal species, the beginnings of agriculture, and significant demographic growth. The ANR WomenSOFar project (ANR-21-CE03-0008) aims to identify the status of women through how they move, eat, work, and care for the young. As part of this research project, an innovative and exploratory approach involving the analysis of micro-remains contained in dental calculus was carried out. The objectives were to test the interest of this still embryonic approach in France and to provide insights into the dietary habits of Neolithic communities in La Roussille (Vertaizon, Puy-de-Dôme), in addition to data from other paleoecological and bioarchaeological disciplines.The studied site, La Roussille, is located in Auvergne and has the particularity of being composed of a habitation area and a burial zone, dated to approximately 4600 to 4300 years BCE. Dental calculus samples from 7 individuals, including 2 males, 4 females, and 1 of unknown sex, were analyzed at the University College of Dublin, Ireland. Dental calculus samples were decalcified using the EDTA method before being studied under an optical microscope. Micro-remains were identified through publications and reference books. The main elements found were phytoliths, microscopic siliceous bodies produced by plants as a defense against attacks such as herbivory, as well as animal cells, micro-charcoal, pollen, and fungal spores. These initial results raise questions about the representativeness of the different types of elements identified in this matrix and the use of teeth as a tool.Le Néolithique, épisode majeur de l’histoire Humaine est marqué par la domestication des espèces végétales et animales, les débuts de l’agriculture et une croissance démographique significative. Le projet ANR WomenSOFar (21-CE03-0008) vise à identifier le statut des femmes à travers la façon dont elles se déplacent, se nourrissent, travaillent et prennent soin des jeunes. Dans le cadre de ce projet de recherche, une approche novatrice et exploratoire, consistant en l’analyse des micro-restes contenus dans le tartre dentaire, a été réalisée. Les objectifs étaient de tester l’intérêt de cette approche jusqu’à présent embryonnaire en France et de proposer des éléments de réponse au régime alimentaire des communautés néolithiques de La Roussille (Vertaizon, Puy-de-Dôme) en addition aux données d’autres disciplines paléoécologiques et archéobiologiques. Le site étudié, La Roussille, se situe en Auvergne et il présente la particularité d’être composé d’un habitat et d’un zone funéraire, datée d’environ 4600 à 4300 ans avant notre ère. Les échantillons de tartre dentaire de 7 individus, dont 2 hommes, 4 femmes, et 1 de sexe inconnu, ont été analysés (University College of Dublin, Irlande). Les échantillons de tartre dentaire ont été décalcifiés selon la méthode EDTA, avant d’être étudiés au microscope optique. Les microrestes ont été identifiés grâce à des publications et livres de référence. Les principaux éléments retrouvés étaient des phytolithes, corps microscopiques siliceux produits par les plantes en guise de défense contre des attaques telle que l’herbivorie, mais aussi des cellules animales, des microcharbons, des pollen ou encore des spores de champignon. Ces premiers résultats interrogent sur la représentativité des différents types d’éléments identifiés dans cette matrice et l’utilisation des dents en tant qu’outil

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.This study was supported by the CNRS (Centre National de la Recherche Scientifique), the Brittany Region (France) (PhD funding for JP), the European Commission (FP7-LUPA, GA-201370), the Rosembloom Family and the Companion animal health fund from the Faculté de Médecine Vétérinaire, Université de Montréal, and the CRB-Anim infrastructure, ANR-11-INBS-0003, funded by the French National Research Agency in the frame of the ‘Investing for the Future’ program.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

RNA-Seq Data for Reliable SNP Detection and Genotype Calling: Interest for Coding Variant Characterization and Cis-Regulation Analysis by Allele-Specific Expression in Livestock Species

International audienceIn addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis -regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which ∼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, ∼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to ( i ) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), ( ii ) study, on a large scale, cis -regulations of gene expression, with ∼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with ∼29% of them that were cis -regulated, and ( iii ) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies

Identification of PCPE-2 as the endogenous specific inhibitor of human BMP-1/tolloid-like proteinases

International audienceAbstractBMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-β superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.</jats:p

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.This study was supported by the CNRS (Centre National de la Recherche Scientifique), the Brittany Region (France) (PhD funding for JP), the European Commission (FP7-LUPA, GA-201370), the Rosembloom Family and the Companion animal health fund from the Faculté de Médecine Vétérinaire, Université de Montréal, and the CRB-Anim infrastructure, ANR-11-INBS-0003, funded by the French National Research Agency in the frame of the ‘Investing for the Future’ program.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Summary of the SNPs found in the targeted sequencing.

<p>Only variants that are homozygous for a rare allele in all affected dogs and not homozygous in controls were considered to perfectly segregate. Nb SNPs: Number of SNPs; Hom: Nb Homozygous variants; HTZ: Nb Heterozygous variants.</p

The AMS variant affect binding of nuclear complex.

<p>EMSAs were performed with wild type (WT) or mutated (Mut) duplex and nuclear extract (NE). (A) Detection of a mobility shift after incubation of 40 fmoles of radiolabelled WT-duplex (lane 1 to 4) or Mut-duplex (lane 5 to 8) with increasing amounts (0,3–0,6–1,2–2,4 μl) of HeLa NE. (B) 40 fmoles of radiolabelled WT-duplex were incubated without (lane 9 and 13) or with 1μl of HeLa NE (lane 10 to 12) or 5 μl of SY5Y (lane 14 to 16) NE and in the presence of 2 pmol of competitor WT-duplex (lane 11 and 15) or Mut-duplex (lane 12 and 16). Black bar: specific complex associated with the radioactive duplex, asterisk: non specific complex.</p