Role of HOXA7 to HOXA13 and PBX1 genes in various forms of MRKH syndrome (congenital absence of uterus and vagina)

The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome refers to the congenital absence or severe hypoplasia of the female genital tract, often described as uterovaginal aplasia which is the prime feature of the syndrome. It is the second cause of primary amenorrhea after gonadal dysgenesis and occurs in ~1 in 4500 women. Aetiology of this syndrome remains poorly understood. Frequent association of other malformations with the MRKH syndrome, involving kidneys, skeleton and ears, suggests the involvement of major developmental genes such as those of the HOX family. Indeed mammalian HOX genes are well known for their crucial role during embryogenesis, particularly in axial skeleton, hindbrain and limb development. More recently, their involvement in organogenesis has been demonstrated notably during urogenital differentiation. Although null mutations of HOX genes in animal models do not lead to MRKH-like phenotypes, dominant mutations in their coding sequences or aberrant expression due to mutated regulatory regions could well account for it. Sequence analysis of coding regions of HOX candidate genes and of PBX1, a likely HOX cofactor during Müllerian duct differentiation and kidney morphogenesis, did not reveal any mutation in patients showing various forms of MRKH syndrome. This tends to show that HOX genes are not involved in MRKH syndrome. However it does not exclude that other mechanisms leading to HOX dysfunction may account for the syndrome

Analysis of biofilm-nanoparticles interaction using microscopy (fluorescence, MEB, STEM, MET, EDS)

International audienceAmong biofilm's properties, the ability to interact with/catch pollutants can have applications in bioremediation. Here, biofilm interactions with metals (as iron nanoparticles (NanoFer 25S)) was evaluated using various approaches in microscopy. For this, biofilm growth, sampling, labelling and treatment were developed for each type of microscopy to access the surface or inside of the biofilm, biofilm composition, and metal location. Multispecies biofilms were grown on sand or in PVC tubes inoculated with aquifer water spiked with a nutritive solution to enhance denitrification, and then put in contact with nanoparticles. According to the targeted microscopy, biofilms were (i) sampled as flocs or attached biofilm, (ii) submitted to cells (DAPI) and/or lectins (PNA and ConA coupled to FITC or Au nanoparticles) labelling, and (iii) prepared for observation (fixation, cross-section, freezing…). Fluorescent microscopy revealed that nanoparticles were embedded in the biofilm structure as 0.5-5µm size aggregates. SEM observations also showed NP aggregates closed to microorganisms but it was not possible to conclude a potential interaction between nanoparticles and the biological membranes. STEM-in-SEM analysis showed NP aggregates could enter inside the biofilm over a depth of 7-11µm. Moreover, microorganisms were circled by an EPS ring that prevented the direct interaction between NP and membrane. TEM(STEM)/EDS revealed that NP aggregates were co-localized with lectins suggesting a potential role of exopolysaccharides in NP embedding. The combination of several approaches in microscopy is thus a good tool to better understandi and characterize biofilm/pollutant interaction

Simultaneous Quantification and Visualization of Titanium Dioxide Nanomaterial Uptake at the Single Cell Level in an In Vitro Model of the Human Small Intestine

International audienceUseful properties render titanium dioxide nanomaterials (NMs) to be one of the most commonly used NMs worldwide. TiO2 powder is used as food additives (E171), which may contain up to 36% nanoparticles. Consequently, humans could be exposed to comparatively high amounts of NMs that may induce adverse effects of chronic exposure conditions. Visualization and quantification of cellular NM uptake as well as their interactions with biomolecules within cells are key issues regarding risk assessment. Advanced quantitative imaging tools for NM detection within biological environments are therefore required. A combination of the label-free spatially resolved dosimetric tools, microresolved particle induced X-ray emission and Rutherford backscattering, together with high resolution imaging techniques, such as time-of-flight secondary ion mass spectrometry and transmission electron microscopy, are applied to visualize the cellular translocation pattern of TiO2 NMs and to quantify the NM-load, cellular major, and trace elements in differentiated Caco-2 cells as a function of their surface properties at the single cell level. Internalized NMs are not only able to impair the cellular homeostasis by themselves, but also to induce an intracellular redistribution of metabolically relevant elements such as phosphorus, sulfur, iron, and copper

New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

Frontotemporal dementia (FTD) is a fatal neurodegenerative disease characterized by behavioral and language disorders. The main genetic cause of FTD is an intronic hexanucleotide repeat expansion (G4C2)n in the C9ORF72 gene. A loss of function of the C9ORF72 protein associated with the allele-specific reduction of C9ORF72 expression is postulated to contribute to the disease pathogenesis. To better understand the contribution of the loss of function to the disease mechanism, we need to determine precisely the level of reduction in C9ORF72 long and short isoforms in brain tissue from patients with C9ORF72 mutations. In this study, we developed a sensitive and robust mass spectrometry (MS) method for quantifying C9ORF72 isoform levels in human brain tissue without requiring antibody or affinity reagent. An optimized workflow based on surfactant-aided protein extraction and pellet digestion was established for optimal recovery of the two isoforms in brain samples. Signature peptides, common or specific to the isoforms, were targeted in brain extracts by multiplex MS through the parallel reaction monitoring mode on a Quadrupole–Orbitrap high resolution mass spectrometer. The assay was successfully validated and subsequently applied to frontal cortex brain samples from a cohort of FTD patients with C9ORF72 mutations and neurologically normal controls without mutations. We showed that the C9ORF72 short isoform in the frontal cortices is below detection threshold in all tested individuals and the C9ORF72 long isoform is significantly decreased in C9ORF72 mutation carriers

Insectes pollinisateurs dans les paysages agricoles (approche pluri-échelle du rôle des habitats semi-naturels, des pratiques agricoles et des cultures entomophiles)

Cette thèse porte sur les facteurs influençant la composition des communautés d insectes pollinisateurs (abeilles et syrphes) dans les paysages agricoles, en présence d une culture entomophile, le colza. Son originalité réside dans la prise en compte des pratiques agricoles, et non pas uniquement de la quantité d habitats semi-naturels. A l échelle de 14 sites européens, les abeilles sont affectées négativement par l intensification agricole (mesurée par la quantité de pesticides et de fertilisants azotés et par la charge en bétail) et positivement par la quantité d habitats semi-naturels. A l échelle du paysage, nous avons montré que: (1) les abeilles et les syrphes se concentrent au niveau des parcelles de colza ; (2) le rôle des habitats semi-naturels diffère en fonction du type de paysage(bocage vs. openfield); (3) une analyse de l histoire récente met en évidence le rôle positif de la présence de prairies temporaires dans les rotations culturales. Une étude de la répartition spatiale des insectes pollinisateurs au sein des parcelles de colza indique que (1) dans le bocage, abeilles et syrphes utilisent à la fois les bordures et l intérieur des parcelles. Les bourdons fréquentent préférentiellement le centre des parcelles, où la quantité de ressources est maximale; (2) dans l openfield, où la surface en colza est plus élevée, nous observons un effet dilution , qui rend plus difficile la compréhension du comportement des insectes.En améliorant les connaissances sur l utilisation de l espace par les in sectes pollinisateurs, ce travail permet de mieux estimer les flux de pollen, et donc les risques associés à la mise en place de cultures transgéniques.This study deals with the composition of pollinator insect communities (bees and hoverflies) in agricultural landscapes where oilseed rape, a mass flowering crop, is produced. It is original as it considers agricultural practices and not only the amount of semi-natural habitats. In 14 European sites, the bees are adversely affected by agricultural intensification (measured by the amount of pesticides and nitrogen fertilizers and by the livestock density) and positively affected by the amount of semi-natural habitats. At the landscape scale, we find that: (1) bees and hoverflies are more abundant on oilseed rape (OSR) field margins than on other types of field margins; (2) the role of semi-natural habitats can differ according to the landscape structure ( bocage vs. openfield); (3) in the bocage , accounting for the recent past use of crop fields shows that temporary grasslands in crop rotations have a positive impact on solitary bee species richness. An analysis of the spatial distribution of pollinator insects in OSR fields indicates that (1) in the bocage , all insects use both the edges and the interior of fields. Bumblebees use preferentially the interior of fields, where the amount of resources is maximum; (2) in the openfield where the amount of OSR is higher, we observe a "dilution" effect, which makes more difficult the understanding of insect behavior. By improving knowledge about spatial distribution of pollinator insects, our study permits to better estimate pollen flow, and therefore the potential impacts of transgenic crops.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Spatial arrangement of casein micelles and whey protein aggregate in acid gels: Insight on mechanisms

Skim milk used for the yoghurt manufacture contains 2 main colloidal particles, the native micellarcasein (NMC) and the heat-induced whey protein aggregates (WPA). The aim of this study was to understandhow these 2 colloids organize in space to form binary acid gels. While acid milk gels wereconsidered homogeneous for scales typically above ~10 mm, shorter length scales were examined toinvestigate the early stages of particle aggregation. The NMC and WPA were dispersed in milk permeateat different protein concentrations, separately or in an 80/20 w/w mixture (MIX). Acidification wasperformed at 35 C with glucono-d-lactone to achieve a final pH at ~4.5 in 6 h. Acid gelation was studiedby rheology, while the microstructure of the gels at pH 4.5 was studied by confocal scanning laser microscopyand transmission electron microscopy (TEM). Using Shih et al.'s (1990) model on the rheologicaldata, it seemed that aggregation in the NMC and MIX mixtures was driven by the casein micelles andtherefore differed from that of the WPA suspension. The different organizations were confirmed usingimage analysis of confocal or TEM images. The differences in gel formation were discussed in terms of thedifferent interactive properties of the surface of these 2 colloids, together with their different internalstructure

Adaptation of cryo-sectioning for immuno-EM-labeling of asymmetric samples: a study using C. elegans.

International audienceCryo-sectioning procedures, initially develop by Tokuyasu have been successfully improved for tissues and cultured cells, enabling efficient protein localization on the ultrastructural level. Without a standard procedure applicable to any sample, currently existing protocols must be individually modified for each model organism or asymmetric sample. Here, we describe our method that enables reproducible cryo-sectioning of C. elegans larvae/adults and embryos. We have established a chemical fixation procedure in which flat embedding considerably simplifies manipulation and lateral orientation of larvae or adults. To bypass the limitations of chemical fixation, we have improved the hybrid cryo-immobilization-rehydration technique, and reduced the overall time required to complete this procedure. Using our procedures precise cryo-sectioning orientation can be combined with good ultrastructural preservation and efficient immuno-electron microscopy protein localization. Also, GFP fluorescence can be efficiently preserved, permitting a direct correlation of the fluorescent signal and its subcellular localization. Though developed for C. elegans samples, our method addresses the challenge of working with small asymmetric samples in general, and thus could be used to improve the efficiency of immuno-electron localization in other model organisms

Réalisations phares en microscopie des colonies bactériennes en matrices fromagères

Contexte de l’étude : Les bactéries jouent un rôle clé dans l’affinage et la flaveur du fromage et se développent en colonies.Questions : Quels sont les outils et les méthodes les mieux adaptés en microscopie pour observer les interfaces entre colonie de bactéries (Lactococcus Lactis (coque de 1μm de diamètre)) et matrice solide ainsi que l’organisation des bactéries dans les colonies

Root uptake and phytotoxicity of nanosized molybdenum octahedral clusters

CMEBA; Plateforme ONIS/EuropIA; NanoSIMSInternational audienceHere are examined the root uptake and phytotoxicity of octahedral hexamolybdenum clusters on rapeseed plants using the solid state compound Cs2Mo6Br14 as cluster precursor. [Mo6Br14]2− cluster units are nanosized entities offering a strong and stable emission in the near-infrared region with numerous applications in biotechnology. To investigate cluster toxicity on rapeseed plants, two different culture systems have been set up, using either a water-sorbing suspension of cluster aggregates or an ethanol-sorbing solution of dispersed nanosized clusters. Size, shape, surface area and state of clusters in both medium were analyzed by FE-SEM, BET and XPS. The potential contribution of cluster dissolution to phytotoxicity was evaluated by ICP-OES and toxicity analysis of Mo, Br and Cs. We showed that the clusters did not affect seed germination but greatly inhibited plant growth. This inhibition was much more important when plants were treated with nanosized entities than with microsized cluster aggregates. In addition, nanosized clusters affected the root morphology in a different manner than microsized cluster aggregates, as shown by FE-SEM observations. The root penetration of the clusters was followed by secondary ion mass spectroscopy with high spatial resolution (NanoSIMS) and was also found to be much more important for treatments with nanosized clusters