Optimizing Western Blots for the Detection of Endogenous α-Synuclein in the Enteric Nervous System

Background:Alpha-synuclein containing inclusions in neurons, the characteristic pathological lesions of Parkinson’s disease (PD), are not limited to the central nervous system, but also affect the enteric nervous system (ENS). This suggests that the ENS offer some potential as a surrogate of central nervous system pathology and that it may represent an original source of biomarkers for PD. However, the usefulness of α-synuclein detection in gastrointestinal biopsies as a biomarker for PD is still unclear, as the different immunohistochemical methods employed to date have led to conflicting results. Objective:Our aim is to propose an optimized immunoblotting method for the detection of endogenous α-synuclein in the healthy ENS that may be used to supplement the immunohistochemical analysis. Methods:Primary culture of rat ENS and homogenates of human small intestine were analyzed by Western Blot using seven different α-synuclein and phospho-α-synuclein antibodies along with two methods that increase α-synuclein retention on blot membranes, namely incubation of the membranes with paraformaldehyde (PFA) or treatment of samples with the crosslinker dithiobis[succinimidylpropionate] (DSP). Results:A moderate improvement in the detection of endogenous enteric α-synuclein was observed following membrane fixation with PFA for only two of the seven antibodies we tested. Immunodetection of total and phosphorylated α-synuclein in the ENS was markedly improved when samples were treated with DSP, regardless of the antibody used. Conclusions:Our results demonstrate that the detection of α-synuclein in the gut by Western Blot can be optimized by using methods for enhanced membrane retention of the protein along with the appropriate antibody. Such an optimized protocol opens the way to the development of novel biomarkers for PD that will enable a quantification of α-synuclein in gastrointestinal biopsies

Int. J. Mol. Sci.

To face the increasing demand for organ transplantation, currently the development of tissue engineering appears as the best opportunity to effectively regenerate functional tissues and organs. However, these approaches still face the lack of an efficient method to produce an efficient vascularization system. To answer these issues, the formation of an intra-volume channel within a three-dimensional, scaffold free, mature, and cell-covered collagen microfibre is here investigated through laser-induced cavitation. An intra-volume channel was formed upon irradiation with a near-infrared, femtosecond laser beam, focused with a high numerical aperture lens. The laser beam directly crossed the surface of a dense and living-cell bilayer and was focused behind the bilayer to induce channel formation in the hydrogel core while preserving the cell bilayer. Channel formation was assessed through confocal microscopy. Channel generation inside the hydrogel core was enhanced by the formation of voluminous cavitation bubbles with a lifetime longer than 30 s, which also improved intra-volume channel durability. Twenty-four hours after laser processing, cellular viability dropped due to a lack of sufficient hydration for processing longer than 10 min. However, the processing automation could drastically reduce the cellular mortality, this way enabling the formation of hollowed microfibres with a high density of living-cell outer bilayer

Systemic α-synuclein injection triggers selective neuronal pathology as seen in patients with Parkinson’s disease

Abstract: Parkinson’s disease (PD) is an α-synucleinopathy characterized by the progressive loss of specific neuronal populations. Here, we develop a novel approach to transvascularly deliver proteins of complex quaternary structures, including α-synuclein preformed fibrils (pff). We show that a single systemic administration of α-synuclein pff triggers pathological transformation of endogenous α-synuclein in non-transgenic rats, which leads to neurodegeneration in discrete brain regions. Specifically, pff-exposed animals displayed a progressive deterioration in gastrointestinal and olfactory functions, which corresponded with the presence of cellular pathology in the central and enteric nervous systems. The α-synuclein pathology generated was both time dependent and region specific. Interestingly, the most significant neuropathological changes were observed in those brain regions affected in the early stages of PD. Our data therefore demonstrate for the first time that a single, transvascular administration of α-synuclein pff can lead to selective regional neuropathology resembling the premotor stage of idiopathic PD. Furthermore, this novel delivery approach could also be used to deliver a range of other pathogenic, as well as therapeutic, protein cargos transvascularly to the brain

La consommation d’aliments issus de l’agriculture biologique réduit-elle l’incidence du cancer ? : une revue systématique de la littérature

Background: The links between diet and cancer are now established. The literature tends to observe that environmental exposure to pesticides is carcinogenic and that we are exposed to it daily through food. Studies show less exposure to pesticides when eating organic food. Since consumers eat organic food out of belief that they can improve their health, we wanted to know if this consumption could reduce the incidence of cancer. Methods: We carried out an exhaustive search of the literature in 8 search engines including PUBMED. All types of studies, descriptive or analytical, were sought. Results: We reviewed 974 publications and two of which met the inclusion criteria. A first study observed a reduction in the incidence of all cancers combined among organic consumers (RR 0.75), while the second study showed no difference. Results for breast cancer were inconsistent (RR 0.91 vs RR 0.66). The NHL incidence was significantly reduced among organic consumers in both studies (RR 0,79 and 0,14). Conclusions: The search results do not allow us to conclude. The aim of this thesis was to expose a major lack of data. It is necessary to promote further studies analysing this link. In the meantime, we must continue to encourage our patients to eat conventional fruits and vegetables if they cannot access organic food, and to follow the PNNS4 recommendations (physical activity, food balance).Introduction : les liens entre alimentation et cancer sont maintenant établis. Les publications tendent à observer que l’exposition environnementale aux pesticides est cancérigène et que nous y sommes quotidiennement exposés via l’alimentation. Des études montrent une moindre exposition aux pesticides lors de la consommation d’aliments bio. Le consommateur s’alimentant bio par conviction d’accéder à une meilleure santé, nous avons voulu savoir si cette consommation pouvait réduire l’incidence du cancer. Matériel et méthode : nous avons effectué une recherche exhaustive de la littérature dans 8 moteurs de recherche dont PUBMED. Tous les types d’études, descriptives ou analytiques, ont été recherchés. Résultats : nous avons passé en revue 974 publications dont deux ont répondu aux critères d’inclusion. Une première étude observe une réduction d’incidence de tous les cancers confondus chez les consommateurs de bio (RR 0,75), la seconde ne montre pas de différence. Les résultats concernant le cancer du sein sont discordants (RR 0,91 vs RR 0,66). L’incidence du LNH est significativement réduite chez les consommateurs de bio dans les deux études (RR 0,79 et 0,14). Conclusion : les résultats de notre recherche ne nous permettent pas de conclure. Cette thèse aura eu pour finalité d'exposer un manque de données majeur. Il est nécessaire de promouvoir d’autres études analysant ce lien. En attendant, nous devons continuer à encourager nos patients à manger des fruits et légumes conventionnels si ils ne peuvent pas avoir accès au bio, et à suivre les recommandations du PNNS4 (activité physique, équilibre alimentaire)

Etude d'un arc transfere dans le methane en atmosphere non oxydante et a pression normale

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 81298 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Développement d'un modèle in vitro d'exposition chronique aux forces de cisaillement pariétal sur primocultures de cellules endothéliales coronaires de rat pour l'évaluation de la dysfonction endothéliale in vitro

Le rôle de l'endothélium vasculaire dans les pathologies cardiovasculaires a gagné un intérêt croissant au cours des dernières années de recherche. Autrefois considéré comme un tissu inerte, celui-ci s'est peu à peu doté de propriétés d'interaction et de régulation qui sont à la base même de l'homéostasie du système cardiovasculaire. Les capacités d'interaction de l'endothélium avec son environnement direct ne facilitent pas son étude in vitro lorsque les cellules endothéliales sont cultivées hors de tout contexte physiologique. Le développement d'un modèle in vitro permettant de recréer et de moduler de façon indépendante les paramètres physico-chimiques du micro-environnement endothélial (forces de cisaillement pariétal, pression hydrostatique, pression partielle en oxygène) s'avère nécessaire afin d'évaluer finement les relations qui les lient. Le système des rollers permet d'évaluer de façon chronique et de façon indépendante des paramètres physiques (forces de cisaillement pariétal), chimiques (pression partielle en oxygène) et physiologiques (substances vasoactives). Les études récentes ayant mis l'accent sur le rôle primordial de la balance oxydo-réductrice de la cellule endothéliale dans la survenue ou non de phénomènes pathologiques, nous nous sommes intéressés aux réponses cellulaires pour différentes conditions micro-environnementales. Nos résultats montrent que l'augmentation des forces de cisaillement pariétal induisent un stress oxydant transitoire ainsi qu'une induction pérenne des systèmes de défense anti-oxydant alors qu'une diminution chronique semble provoquer un déséquilibre de la balance oxydo-réductrice cellulaire. L'hypoxie ne provoque pas la génération d'espèces radicalaires mais module le stress oxydant induit par le flux et augmente la biodisponibilité du monoxyde d'azote produit. L'étude de l'angiotensine II a montré un effet anti-oxydant prédominant de la voie des récepteurs AT2 sur les cellules endothéliales coronaires alors que les effets pro-inflammatoires et pro-oxydant du TNF alpha sont plus marquées sur les cellules ayant subi une diminution chronique de flux. Ainsi, il apparaît que le micro-environnement physico-chimique des cellules endothéliales module fortement leur physiologie et leurs réponses aux stress exogènes. Le rôle de l'hypoxie au niveau tissulaire ainsi que le type cellulaire impliqué dans l'oxyception restent à déterminer au moyen de systèmes de co-culture cardiomyocytes/cellules endothéliales. Par ailleurs, l'action prédominante de la voie AT2 au niveau de l'endothélium coronaire ouvre une voie de recherche supplémentaire pour le développement de stratégies thérapeutiques. En conclusion, le modèle développé permet d'évaluer finement et de façon chronique les interrelations qui existent entre l'endothélium et son environnement tant physico-chimique qu'humoral. Le concept de culture dynamique des cellules endothéliales apparaît alors primordial pour le développement d'études offrant une alternative à l'expérimentation animale.Vascular endothelium role in cardiovascular diseases has gained more and more interest for a few years. Being considered just as a barrier, research gave it many properties which became the foundations of cardiovascular homeostasis. As endothelium may interact with its micro-environment, in vitro studies are not easy since endothelial cells are grown under non-physiological conditions. Development of an in vitro model which could simulate and modulate independently physical, chemical and physiological parameters of endothelium micro-environment appears to be necessary for the in vitro studies relevance. The "Roller" system allows study in a chronic and independent fashion those parameters. As recent studies pointed out the crucial role of oxidative status of endothelial cells into the outcome of physio-pathological events, we have been interested in endothelial oxidative status cellular responses when exposed to various micro-environmental conditions. Our results show that shear stress increase induces transient oxidative stress as well as long-term anti-oxidant enzymatic systems; chronic decrease in shear stress seems to induce an imbalance in endothelial cell oxidative state. Hypoxia did not induce reactive oxygen species production but modulates flow-induced oxidative stress and enhances NO bioavailability. Angiotensin II showed a dominant anti-oxidant effect mediated by AT2 receptors and pro-inflammatory and pro-oxidant TNF alpha effects were stronger when cells had undergone chronic flow decrease. Then, it appears that micro-environment strongly affects endothelial cell in culture and their response to exogenous stresses. Hypoxia role and mechanism at the tissue level remains to be assessed with co-culture systems (cardiomyocytes/endothelial cells). Furthermore, AT2 receptor presence at the coronary level could lead to novel research in terms of therapeutical strategies. To conclude, this model allows fine and chronic evaluation of endothelium interaction with its environment. Dynamic culture concept appears to be uncircumventable for development of studies which could offer an alternative to animal experimentation.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

A new analytical methodology for a fast evaluation of semi-volatile polycyclic aromatic hydrocarbons in the vapor phase downstream of a diesel engine particulate filter.

International audienceA new sampling method was developed to collect vapor-phase polycyclic aromatic compounds (PAHs) downstream of a diesel engine equipped with a diesel particulate filter (DPF). This configuration allowed us to collect separately the particulate phase, which was trapped inside the DPF, and the vapor phase, which was sampled downstream of the DPF. PAHs, which were not predominantly absorbed into the poor organic fraction of the diesel soot, but were rather physically sorbed on high energetic adsorption sites, should be extracted using very drastic extraction conditions Microwave-assisted extraction using solvent mixtures composed of pyridine and diethylamine were used to desorb particulate PAHs, and the total PAH amounts corresponded to a very low value, i.e., 8 μg g⁻¹ or 0.24 μg km⁻¹, with a predominance of low weight PAHs. For collection of the vapor phase, gas bubbling in an aqueous medium was preferred to conventional methods, e.g., trapping on solid sorbents, for several reasons: aqueous trapping allowed us to use a solid phase enrichment process (SPE) that permitted PAH sampling at the sub-picogram levels. Consequently, low volume sampling was possible even if the sampling duration was very short (20 min). Additionally, the amount of time saved for the analysis was considerable when coupling SPE to the analytical system (liquid chromatography with fluorimetric detection). Solvent consumption for the overall sampling and analytical processes was also drastically reduced. Experiments on a diesel engine showed that vapor phase samples collected downstream of the DPF contained all of the 15 target priority PAHs, even the heaviest ones. The total vapor-phase PAH amount was 6.88 μg N m⁻³ or 10.02 μg km⁻¹, which showed that the gaseous fraction contains more PAHs than the particulate fraction. Partitioning coefficients (K(p)) were estimated showing the predominance in the vapor phase of all the PAHs. However, the DPF technology effects a considerable decrease in the total PAH emission when compared to non-equipped diesel vehicles

Experimental Investigation on the Characteristics and on the Reproducibility of the Flow issuing from a High-Pressure Direct-Injection Nozzle

International audienc