Séparation, détection et caractérisation de nanoparticules manufacturées dans des eaux naturelles et usées avec la chromatographie hydrodynamique et de multiples détecteurs

L’utilisation accrue des nanomatériaux manufacturés (NM) fait en sorte que les différents acteurs de réglementation se questionnent de plus en plus par rapport à leur destin et leurs impacts sur les écosystèmes et la santé humaine suite à leur rejet dans l’environnement. Le développement de techniques analytiques permettant de détecter et de caractériser les NM en matrice environnementale est impératif étant donné la nécessité d’évaluer le risque relié à ces polluants émergents. Une des approches de plus en plus favorisée est d’utiliser une technique chromatographique et un ou plusieurs détecteurs sensibles dans les buts de réduire les effets de matrice, d’identifier des nanoparticules (NP) selon leurs temps de rétention et de les quantifier à des concentrations représentatives de la réalité environnementale. Une technique analytique utilisant la chromatographie hydrodynamique (HDC) et des détecteurs en ligne ou hors ligne (détecteurs de diffusion statique ou dynamique de la lumière, spectromètre de masse par torche à plasma en mode particule unique (SP-ICPMS), l’ultracentrifugation analytique) a donc été développée. Le couplage de la colonne HDC avec ces détecteurs a permis de caractériser des NP standards et l’optimisation des conditions de séparation de ces nanoparticules de polystyrène, d’or et d’argent a permis de confirmer que les NP y sont bel et bien séparées seulement selon leur taille, tel que la théorie le prédit. De plus, l’utilisation de la colonne HDC couplée au SP-ICPMS a permis de séparer un mélange de nanoparticules d’argent (nAg) et de les détecter à des concentrations représentatives de celles rencontrées dans l’environnement, soit de l’ordre du μg L-1 au ng L-1. Par exemple, dans un échantillon d’eau usée (effluent), un mélange de nAg de 80 et de 40 nm a été séparé et les nAg ont été détectées à l’aide du SP-ICPMS connecté à la colonne HDC (temps de rétention de 25.2 et 25.6 minutes et diamètres déterminés de 71.4 nm et 52.0 nm). Finalement, pour plusieurs échantillons environnementaux auxquels aucun ajout de nanoparticules n’a été fait, les analyses HDC-SP-ICPMS effectuées ont permis de déterminer qu’ils ne contenaient initialement pas de nAg.Due to the widespread use of engineered nanoparticles (ENP), regulatory agencies are very concerned about their fate and their impacts on the environment and on human health. The development of analytical techniques, which will allow the detection, characterization and quantification of ENP in environmental matrices, is therefore critical in order to properly evaluate the exposure associated with these emerging pollutants. One promising approach to detect and quantify the nanoparticles is to couple a chromatographic technique to a sensitive detector in order to: (i) reduce matrix effects; (ii) identify nanoparticles from their retention times and (iii) quantify the ENP at environmentally relevant concentrations. Consequently, the coupling of hydrodynamic chromatography (HDC) was performed with both on-line and off-line detectors (light scattering detectors, inductively coupled plasma mass spectrometer in its single particle mode (SP-ICPMS) and an analytical ultracentrifuge). HDC was first used for the characterization of ENP standards. Separation conditions were optimized for standard nanoparticle suspensions of polystyrene, gold and silver, which allowed us to confirm that the separation was occurring, based on hydrodynamic size, as predicted by theory. By coupling the HDC column to the ICPMS detector in its ‘‘single particle’’ mode, it was possible to separate an ENP mixture and to detect the nanoparticles at environmental concentrations, i.e., in the μg L-1 to ng L-1 range. For example, in a wastewater sample (effluent wastewater), a mixture of two silver nanoparticles (nAg) of 40 and 80 nm were separated and the nAg were detected by SP-ICPMS at retention times of 25.2 and 25.6 minutes. Diameters of 71.4 nm and 52.0 nm were found. HDC-SP-ICPMS analysis carried out on different non-spiked wastewater samples allowed us to conclude that nAg was below the detection limit of 0.1 µg L-1

Separation, detection and characterization of nanomaterials in municipal wastewaters using hydrodynamic chromatography coupled to ICPMS and single particle ICPMS

Engineered nanoparticles (ENP) are increasingly being incorporated into consumer products and reaching the environment at a growing rate. Unfortunately, few analytical techniques are available that allow the detection of ENP in complex environmental matrices. The major limitations with existing techniques are their relatively high detection limits and their inability to distinguish ENP from other chemical forms (e.g. ions, dissolved) or from natural colloids. Of the matrices that are considered to be a priority for method development, ENP are predicted to be found at relatively high concentrations in wastewaters and wastewater biosolids. In this paper, we demonstrate the capability of hydrodynamic chromatography (HDC) coupled to inductively coupled plasma mass spectrometry (ICPMS), in its classical and single particle modes (SP ICPMS), to identify ENP in wastewater influents and effluents. The paper first focuses on the detection of standard silver nanoparticles (Ag NP) and their mixtures, showing that significant dissolution of the Ag NP was likely to occur. For the Ag NP, detection limits of 0.03 μg L−1 were found for the HDC ICPMS whereas 0.1 μg L−1 was determined for the HDC SP ICPMS (based on results for the 80 nm Ag NP). In the second part of the paper, HDC ICPMS and HDC SP ICPMS were performed on some unspiked natural samples (wastewaters, river water). While nanosilver was below detection limits, it was possible to identify some (likely natural) Cu nanoparticles using the developed separation technology

Impact of TNF and IL-1β on capillary networks within engineered human adipose tissues

Inflammation is a normal phase of the wound healing process, which likely occurs following tissue transplantation. For reconstructive surgery purposes, engineered adipose tissues represent promising alternatives to autologous fat grafts. It is therefore important to study the impact of an inflammatory microenvironment on the cellular functions of the different cell types comprised within matrix-rich reconstructed tissues. In this study, human reconstructed adipose tissues (hrATs) featuring a preformed capillary network formed by microvascular endothelial cells (hMVECs) were produced from adipose-derived stem/stromal cells (ASCs) by the self-assembly approach of tissue engineering. We hypothesized that a prolonged inflammatory context, mediated by tumor necrosis factor (TNF) and interleukin-1β (IL-1β), would impact hrATs' secretory profile and mediate detrimental effects on the microvascular network in vitro. Analysis of conditioned media established tissue responsiveness through the increased secretion of monocyte chemoattractant protein-1 (up to 23 fold), interleukin-6 (up to 69 fold) and angiopoietin-1 (up to 2.7 fold) after 3 and 6 days of cytokine exposure, along with a significant reduction in adiponectin secretion. Imaging of the preformed capillary network within the hrATs revealed increased disorganization in the presence of TNF/IL-1β, featuring a less extended and less ramified network with apoptotic hMVECs in the remaining capillary structures. These results indicate that a prolonged inflammatory context can be deleterious to the capillary network featured by in vitro engineered tissues. Strategies aiming at preserving the integrity of the vascular network will help develop substitutes that are better suited to face inflammatory conditions upon grafting

Short-term post-implantation dynamics of in vitro engineered human microvascularized adipose tissues

Engineered adipose tissues are developed for their use as substitutes for tissue replacement in reconstructive surgery. To ensure a timely perfusion of the grafted substitutes, different strategies can be used such as the incorporation of an endothelial component. In this study, we engineered human adipose tissue substitutes comprising of functional adipocytes as well as a natural extracellular matrix using the self-assembly approach, without the use of exogenous scaffolding elements. Human microvascular endothelial cells (hMVECs) were incorporated during tissue production in vitro and we hypothesized that their presence would favor the early connection with the host vascular network translating into functional enhancement after implantation into nude mice in comparison to the substitutes that were not enriched in hMVECs. In vitro, no significant differences were observed between the substitutes in terms of histological aspects. After implantation, both groups presented numerous adipocytes and an abundant matrix in addition to the presence of host capillaries within the grafts. The substitutes thickness and volume were not significantly different between groups over the short-term time course of 14 days. For the microvascularized adipose tissues, human CD31 staining revealed a human 3 capillary network connecting with the host microvasculature as early as 3 days after grafting. The detection of murine red blood cells within human CD31+ structures confirmed the functionality of the human capillary network. By analyzing the extent of the global vascularization achieved, a tendency towards increased total capillary network surface and volume was revealed for prevascularized tissues over 14 days. Therefore, applying this strategy on thicker reconstructed adipose tissues with rate-limiting oxygen diffusion might procure added benefits and prove useful to provide voluminous substitutes for patients suffering from adipose tissue loss or defects

Magnetic resonance imaging of human tissue-engineered adipose substitutes

Adipose tissue (AT) substitutes are being developed to answer the strong demand in reconstructive surgery. To facilitate the validation of their functional performance in vivo, and to avoid resorting to excessive number of animals, it is crucial at this stage to develop biomedical imaging methodologies, enabling the follow-up of reconstructed AT substitutes. Until now, biomedical imaging of AT substitutes has scarcely been reported in the literature. Therefore, the optimal parameters enabling good resolution, appropriate contrast, and graft delineation, as well as blood perfusion validation, must be studied and reported. In this study, human adipose substitutes produced from adipose-derived stem/stromal cells using the self-assembly approach of tissue engineering were implanted into athymic mice. The fate of the reconstructed AT substitutes implanted in vivo was successfully followed by magnetic resonance imaging (MRI), which is the imaging modality of choice for visualizing soft ATs. T1-weighted images allowed clear delineation of the grafts, followed by volume integration. The magnetic resonance (MR) signal of reconstructed AT was studied in vitro by proton nuclear magnetic resonance (1H-NMR). This confirmed the presence of a strong triglyceride peak of short longitudinal proton relaxation time (T1) values (200±53 ms) in reconstructed AT substitutes (total T1=813±76 ms), which establishes a clear signal difference between adjacent muscle, connective tissue, and native fat (total T1 ∼300 ms). Graft volume retention was followed up to 6 weeks after implantation, revealing a gradual resorption rate averaging at 44% of initial substitute's volume. In addition, vascular perfusion measured by dynamic contrast-enhanced-MRI confirmed the graft's vascularization postimplantation (14 and 21 days after grafting). Histological analysis of the grafted tissues revealed the persistence of numerous adipocytes without evidence of cysts or tissue necrosis. This study describes the in vivo grafting of human adipose substitutes devoid of exogenous matrix components, and for the first time, the optimal parameters necessary to achieve efficient MRI visualization of grafted tissue-engineered adipose substitutes

Data mining in conservation research using Latin and vernacular species names

In conservation science, assessments of trends and priorities for actions often focus on species as the management unit. Studies on species coverage in online media are commonly conducted by using species vernacular names. However, the use of species vernacular names for web-based data search is problematic due to the high risk of mismatches in results. While the use of Latin names may produce more consistent results, it is uncertain whether a search using Latin names will produce unbiased results as compared to vernacular names. We assessed the potential of Latin names to be used as an alternative to vernacular names for the data mining within the field of conservation science. By using Latin and vernacular names, we searched for species from four species groups: diurnal birds of prey, Carnivora, Primates and marine mammals. We assessed the relationship of the results obtained within different online sources, such as Internet pages, newspapers and social media networks. Results indicated that the search results based on Latin and vernacular names were highly correlated, and confirmed that one may be used as an alternative for the other. We also demonstrated the potential of the number of images posted on the Internet to be used as an indication of the public attention towards different species

Dynamic MAIT cell response with progressively enhanced innateness during acute HIV-1 infection.

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality

Interleukin-1β sequesters hypoxia inducible factor 2α to the primary cilium.

BACKGROUND: The primary cilium coordinates signalling in development, health and disease. Previously we have shown that the cilium is essential for the anabolic response to loading and the inflammatory response to interleukin-1β (IL-1β). We have also shown the primary cilium elongates in response to IL-1β exposure. Both anabolic phenotype and inflammatory pathology are proposed to be dependent on hypoxia-inducible factor 2 alpha (HIF-2α). The present study tests the hypothesis that an association exists between the primary cilium and HIFs in inflammatory signalling. RESULTS: Here we show, in articular chondrocytes, that IL-1β-induces primary cilia elongation with alterations to cilia trafficking of arl13b. This elongation is associated with a transient increase in HIF-2α expression and accumulation in the primary cilium. Prolyl hydroxylase inhibition results in primary cilia elongation also associated with accumulation of HIF-2α in the ciliary base and axoneme. This recruitment and the associated cilia elongation is not inhibited by blockade of HIFα transcription activity or rescue of basal HIF-2α expression. Hypomorphic mutation to intraflagellar transport protein IFT88 results in limited ciliogenesis. This is associated with increased HIF-2α expression and inhibited response to prolyl hydroxylase inhibition. CONCLUSIONS: These findings suggest that ciliary sequestration of HIF-2α provides negative regulation of HIF-2α expression and potentially activity. This study indicates, for the first time, that the primary cilium regulates HIF signalling during inflammation

Phenotypic robustness can increase phenotypic variability after non-genetic perturbations in gene regulatory circuits

Non-genetic perturbations, such as environmental change or developmental noise, can induce novel phenotypes. If an induced phenotype confers a fitness advantage, selection may promote its genetic stabilization. Non-genetic perturbations can thus initiate evolutionary innovation. Genetic variation that is not usually phenotypically visible may play an important role in this process. Populations under stabilizing selection on a phenotype that is robust to mutations can accumulate such variation. After non-genetic perturbations, this variation can become a source of new phenotypes. We here study the relationship between a phenotype's robustness to mutations and a population's potential to generate novel phenotypic variation. To this end, we use a well-studied model of transcriptional regulation circuits. Such circuits are important in many evolutionary innovations. We find that phenotypic robustness promotes phenotypic variability in response to non-genetic perturbations, but not in response to mutation. Our work suggests that non-genetic perturbations may initiate innovation more frequently in mutationally robust gene expression traits.Comment: 11 pages, 5 figure