Control of ATP homeostasis during the respiro-fermentative transition in yeast

Respiring Saccharomyces cerevisiae cells respond to a sudden increase in glucose concentration by a pronounced drop of their adenine nucleotide content. Transient accumulation of the purine salvage pathway intermediate inosine accounts for the apparent loss of adenine nucleotides.Inosine formation in response to perturbations of cellular energy balance depends on the presence of a fermentable carbon source. Under respiratory conditions, AMP accumulates instead and no inosine is formed.Conversion of AXPs into inosine is facilitated by AMP deaminase, Amd1, and IMP-specific 5'-nucleotidase, Isn1. Inosine recycling into the AXP pool is facilitated by the purine nucleoside phosphorylase, Pnp1, and joint action of the phosphoribosyltransferases, Hpt1 and Xpt1.Impaired inosine formation results in altered metabolite pool dynamics in response to glucose addition, but does not change glycolytic flux. However, mutants blocked in inosine formation exhibit delayed growth acceleration after glucose addition

An evolutionary approach to modelling the thermo-mechanical behaviour of unsaturated soils

A new data mining approach is presented for modelling of the stress-strain and volume change behaviour of unsaturated soils considering temperature effects. The proposed approach is based on the evolutionary polynomial regression (EPR), which unlike some other data mining techniques, generates a transparent and structured representation of the behaviour of systems directly from raw experimental (or field) data. The proposed methodology can operate on large quantities of data in order to capture nonlinear and complex relationships between contributing variables. The developed models allow the user to gain a clear insight into the behaviour of the system. Unsaturated triaxial test data from literature was used for development and verification of EPR models. The developed models were also used (in a coupled manner) to produce the entire stress path of triaxial tests. Comparison of the EPR model predictions with the experimental data revealed the robustness and capability of the proposed methodology in capturing and reproducing the constitutive thermo-mechanical behaviour of unsaturated soils. More importantly, the capability of the developed models in accurately generalising the predictions to unseen data cases was illustrated. The results of a sensitivity analysis showed that the models developed from data are able to capture and represent the physical aspects of the unsaturated soil behaviour accurately. The merits and advantages of the proposed methodology are also discussed

Hazard Assessment of Abraded Thermoplastic Composites Reinforced with Reduced Graphene Oxide

Graphene-related materials (GRMs) are subject to intensive investigations and considerable progress has been made in recent years in terms of safety assessment. However, limited information is available concerning the hazard potential of GRM-containing products such as graphene-reinforced composites. In the present study, we conducted a comprehensive investigation of the potential biological effects of particles released through an abrasion process from reduced graphene oxide (rGO)-reinforced composites of polyamide 6 (PA6), a widely used engineered thermoplastic polymer, in comparison to as-produced rGO. First, a panel of well-established in vitro models, representative of the immune system and possible target organs such as the lungs, the gut, and the skin, was applied. Limited responses to PA6-rGO exposure were found in the different in vitro models. Only as-produced rGO induced substantial adverse effects, in particular in macrophages. Since inhalation of airborne materials is a key occupational concern, we then sought to test whether the in vitro responses noted for these materials would translate into adverse effects in vivo. To this end, the response at 1, 7 and 28 days after a single pulmonary exposure was evaluated in mice. In agreement with the in vitro data, PA6-rGO induced a modest and transient pulmonary inflammation, resolved by day 28. In contrast, rGO induced a longer-lasting, albeit moderate inflammation that did not lead to tissue remodeling within 28 days. Taken together, the present study suggests a negligible impact on human health under acute exposure conditions of GRM fillers such as rGO when released from composites at doses expected at the workplace

Air–Liquid Interface In Vitro Models for Respiratory Toxicology Research: Consensus Workshop and Recommendations

In vitro air–liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported

Herpes Simplex Virus Dances with Amyloid Precursor Protein while Exiting the Cell

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/−6.7%) and travel together with APP inside living cells (81.1+/−28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/−0.2 to 0.3+/−0.1 µm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/−0.1 to 0.4+/−0.1 µm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease

Predicting the in vivo pulmonary toxicity induced by acute exposure to TiO2 and CeO2 nanoparticules by using in vitro methods

Les nanoparticules (NPs) représentent un danger potentiel pour la santé des travailleurs et du grand public, notamment en cas d’exposition par voie respiratoire. Si une NP est évaluée in vivo comme toxique chez l’animal, cela peut inciter à prendre des mesures pour réduire l’exposition de l’Homme à celle-ci, avant qu’il y ait des conséquences sanitaires graves. Les études in vivo sont donc d’une importance capitale afin de diminuer les potentiels risques sanitaires des NPs pour l’Homme. Néanmoins, dans un contexte de réduction du nombre d’animaux utilisés et compte tenu du nombre important de NPs existantes et de leur grande diversité physico-chimique, la toxicologie a besoin de modèles alternatifs, comme le in vitro, permettant de prédire de manière fiable les potentiels effets pulmonaires chez l’Homme. Des progrès importants ont été faits pour développer des modèles in vitro pulmonaires plus physiologiques et des méthodes d’exposition permettant de simuler l’inhalation de NPs in vitro. Cependant, des incertitudes existent quant à la capacité de ces nouveaux modèles in vitro à prédire les réponses observées in vivo dans les poumons après exposition à des NPs. Dans ce contexte, l’objectif de ce travail a été d’évaluer la capacité de plusieurs méthodes in vitro, de complexité différente, à prédire les effets toxiques observés in vivo chez le rat après exposition aiguë (24h) pulmonaire à des NPs métalliques faiblement solubles de TiO2 et de CeO2. Dans un premier temps, des expérimentations in vitro ont été effectuées afin d’évaluer si exposer des cellules alvéolaires à l’interface air-liquide (ALI) à des aérosols de NPs de TiO2 et de CeO2, générait des résultats différents par rapport à des expositions classiques à des suspensions en submergé. Dans un second temps, des expérimentations in vivo par aspiration intratrachéale ont été réalisées afin de comparer les réponses toxiques pulmonaires in vitro avec celles obtenues in vivo. Pour comparer les réponses pulmonaires in vivo et in vitro, des référentiels de dose similaires, notamment la masse par unité de surface ou par macrophage, ont été utilisés. Après 24h d’exposition, des réponses biologiques significatives (inflammation principalement) ont été observées in vitro à des doses inférieures à l’ALI par rapport au submergé. Nous avons par ailleurs souligné la nécessité de prendre en compte les doses réellement déposées sur les cellules ainsi que le débit de dose pour effectuer les comparaisons entre les deux méthodes d’exposition in vitro utilisées. Nous avons ensuite comparé les résultats in vitro avec ceux obtenus in vivo. Nous avons constaté que la méthode ALI générait des résultats plus prédictifs du in vivo, en termes de niveau d’activation des réponses toxiques à 24h. Finalement, nous avons établi un classement des quatre NPs utilisées dans notre étude et celles-ci ont été classées similairement in vivo et in vitro et quelle que soit la méthode utilisée in vitro. Nous avons par ailleurs montré l’importance de considérer la surface active des NPs pour établir ce classement. En conclusion, notre approche nous a permis de mieux évaluer le fossé existant entre le in vivo et le in vitro. Nos résultats soulignent l’intérêt d’utiliser des méthodes in vitro plus réalistes et plus proches de la physiologie humaine dans le but de modéliser les potentiels effets indésirables des NPs pour l’Homme. Cela ouvre des perspectives quant à l’utilisation et au développement de méthodologies in vitro de plus en plus représentatives des conditions d’exposition in vivo.Nanoparticles (NPs) represent a potential danger for workers and public, especially after inhalation. When a NP is shown toxic for the lungs in vivo in animals, this can incite regulators to implement measures to reduce human exposure risks. The in vivo studies are thus of utmost importance in reducing the potential health risks for humans. However, in a context of a diminution in the number of animals used in experimentations and considering the high number of NPs used and their physicochemical diversity, there is an urgent need for alternative methods, like the in vitro, which could be used to predict the potential health effects of NPs in human. Many progresses have been made recently to develop more physiological cell models and exposure methods simulating the inhalation of NPs in vitro. Nevertheless, uncertainties remain regarding the capacity of these new in vitro methods to predict the biological responses observed in vivo into the lungs after exposure to NPs. In this context, the aim of our study was to assess the ability of several in vitro methods, differing in complexity, to predict the adverse responses observed in vivo in rat lungs after acute exposure (24h) to several metallic and poorly soluble TiO2 and CeO2 NPs. For this, in vitro experimentations were first performed to assess if exposing alveolar cells in monoculture or in co-culture at the ALI interface to aerosols of NPs, generated different results compared to classic exposure in submerged conditions to suspensions. In a second step, rats were exposed by intratracheal aspiration of NP suspensions to compare the biological responses in vitro to those obtained in vivo. To compare the pulmonary responses in vivo and in vitro, similar dose metrics were selected, including the mass per surface unit or per macrophage. After 24h of exposure, significant biological responses (mostly inflammation) were observed at lower doses at the ALI compared to in submerged conditions. Moreover, we highlighted the necessity to take into account the deposited dose on the cells and the timing of the dose delivery in order to compare the two exposure methods used in vitro. When we compared the responses in vitro to those observed in vivo, we noticed that the ALI methods generated more predictive results than the submerged one, in term of biological activation levels after 24h of exposure. Finally, a ranking of the four NPs used in our study was provided and the NPs were ranked similarly both in vivo and in vitro and whatever the exposure method used in vitro. We also showed the importance of the surface area when ranking the poorly soluble NPs. In conclusion, the gap existing between the in vivo and the in vitro has been evaluated in our study. Our results highlighted the relevance of using more realistic in vitro exposure methods to model the potential adverse effects of NPs for human. This brings perspectives about using and developing in vitro methods mimicking more closely the in vivo exposure conditions