Histone deacetylase inhibitors impair antibacterial defenses of macrophages.

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.

Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.

Fecundity and survival in relation to resistance to oxidative stress in a free-living bird.

Major life history traits, such as fecundity and survival, have been consistently demonstrated to covary positively in nature, some individuals having more resources than others to allocate to all aspects of their life history. Yet, little is known about which resources (or state variables) may account for such covariation. Reactive oxygen species (ROS) are natural by-products of metabolism and, when ROS production exceeds antioxidant defenses, organisms are exposed to oxidative stress that can have deleterious effects on their fecundity and survival. Using a wild, long-lived bird, the Alpine Swift (Apus melba), we examined whether individual red cell resistance to oxidative stress covaried with fecundity and survival. We found that males that survived to the next breeding season tended to be more resistant to oxidative stress, and females with higher resistance to oxidative stress laid larger clutches. Furthermore, the eggs of females with low resistance to oxidative stress were less likely to hatch than those of females with high resistance to oxidative stress. By swapping entire clutches at clutch completion, we then demonstrated that hatching failure was related to the production of low-quality eggs by females with low resistance to oxidative stress, rather than to inadequate parental care during incubation. Although male and female resistance to oxidative stress covaried with age, the relationships among oxidative stress, survival, and fecundity occurred independently of chronological age. Overall, our study suggests that oxidative stress may play a significant role in shaping fecundity and survival in the wild. It further suggests that the nature of the covariation between resistance to oxidative stress and life history traits is sex specific, high resistance to oxidative stress covarying primarily with fecundity in females and with survival in males.

The adaptive function of melanin-based coloration in the tawny owl (Strix aluco) ; the proximate role of the melanocortin system

Colour polymorphism is common in wild population. One of the main questioning of evolutionary biologists is to understand how different colour variants could have evolved and be maintained in fluctuating environments, a selective process that forces individuals to constantly adapt their strategies in order to survive. This issue is particularly true for traits that are genetically inherited. Natural selection erodes genotypes with lowest fitness (less adapted), reducing in turn global genetic variation within population. In this context, the study of the evolution and maintenance of melanin- based coloration is relevant since inter-individual variation in the deposition of these pigments is common in animal and plant kingdoms and is under strong genetic control. In this thesis, I focus on the specific case of the tawny owl (Strix aluco), a species displaying continuous variation in reddish pheomelanin-based coloration. Interestingly, empirical studies highlighted covariations between melanin-based coloration and important behavioural, physiological and life history traits. Recently, a genetic model pointed out the melanocortin system and their pleiotropic effects as a potential regulator of these covariations. Accordingly, this PhD thesis further investigates colour-specific behavioural, physiological, or life history strategies, while examining the proximate mechanisms underlying these reaction norms. We found that differently coloured tawny owls differently resolve fundamental trade-off between offspring number and quality (Chapter 1), light melanic individuals producing many low- quality offspring and dark, melanic ones producing few high-quality offspring. These reproductive strategies are likely to induce alternative physiological constraints. Indeed, we demonstrated that light melanic individuals produced higher levels of reactive oxygen species (ROS, Chapter 2), but also expressed higher levels of antioxidant (GSH, Chapters 2 & 3). Interestingly, we showed that light melanic breeding females could modulate their POMC prohormone levels according to the environmental conditions, while dark reddish ones produced constant levels of this prohormone {Chapter 4). Finally, we highlighted colour-specific patterns of prohormone convertase 1 (PCI) gene expression (Chapter 5), an enzyme responsible for POMC prohormone processing to ACTH and a- MSH, for instance. Altogether, these results provide strong evidence of colour-specific strategies, light and melanic tawny owls better coping with stressful and relaxed environments, respectively. Variation in melanin-based coloration is likely to be maintained by the heterogeneity of our study area and strong environmental stochasticity within and between years, these process favouring differently coloured tawny owls at different periods of time. From a proximate point of view, this PhD thesis supports the hypothesis that covariations between phenotypic traits and melanin-based coloration stems from the melanocortin system, especially the fundamental role of POMC gene expression and its processing to melanocortin peptides. - Le polymorphisme de couleur est une variation ph��notypique tr��s fr��quente dans la nature. En biologie ��volutive, une des probl��matiques cl��s est donc de comprendre comment diff��rent morphes de couleur peuvent ��tre apparus et maintenus au cours du temps dans des environnements aussi variables que les n��tres, surtout que ces fluctuations forcent ces morphes �� s'adapter constamment pour assurer leur survie. Cette th��matique est particuli��rement r��elle lorsque les variations ph��notypiques sont h��r��ditaires et donc sous forte influence g��n��tique. La s��lection naturelle a en effet le pouvoir d'��roder rapidement la variation g��n��tique en ��liminant les g��notypes mal adapt��s. Dans ce sens, l'��tude de l'��volution, et de la maintenance de la coloration m��lanique est donc tout �� fait pertinente car la variation de coloration entre individus est tr��s r��pandue �� travers les r��gnes animal et v��g��tal et sous forte influence g��n��tique. Dans cette th��se, je me suis concentr�� sur le cas sp��cifique de la chouette hulotte (Strix aluco), une esp��ce pr��sentant une variation continue dans la d��position de pigments pheom��laniques roux. De pr��c��dentes ��tudes ont d��j�� montr�� que cette variation de coloration ��tait associ��e avec des variations de traits comportementaux, physiologiques ou d'histoire de vie. R��cemment, une ��tude a soulign�� l'importance du syst��me des m��lanocortines et de leurs effets pl��iotropes dans la r��gulation de ces covariations. En cons��quence, cette th��se de doctoral a pour but d'��tudier un peu plus les strat��gies comportementales, physiologiques ou d'histoire de vie sp��cifiques �� chaque morphe de couleur, tout en examinant un peu plus les m��canismes proximaux potentiellement �� la base de ces normes de r��actions. Nous constatons tout d'abord que les morphes de couleurs ��taient associ��s �� diff��rentes strat��gies dans la r��solution de compromis telle que la production de beaucoup de jeunes ou des jeunes de qualit�� (Chapitre 1). Les morphes gris (dit peu m��laniques) ont tendance �� produire beaucoup de jeunes mains de moindre qualit��, alors que les morphes roux (dit fortement m��laniques) produisent moins de jeunes mais de meilleure qualit��. Ces strat��gies sont susceptibles alors d'induire certaines contraintes physiologiques. Par exemple, nous montrons que les morphes gris produisent plus de d��riv��s r��actifs de l'oxyg��ne (ROS, Chapitre 2), mais aussi plus d'antioxydants (GSH, Chapitres 2 & 3). Nous montrons ensuite que les femelles grises ont une plus grande capacit�� �� moduler leur niveau de POMC prohormone dans le sang en fonction des conditions environnementales, alors que les femelles rousses gardent un niveau constant (Chapitre 4). Finalement, nous d��montrons que les patterns d'expression du g��ne codant pour la prohormone convertase 1 varient chez des jeunes issus de parents gris ou roux (Chapitre 5). Ceci est particuli��rement int��ressant car cette enzyme permet de scinder la POMC prohormone en plusieurs peptides importants tels que l'ACTH ou l'a-MSH. En conclusion, ces r��sultats d��montrent qu'il y a bel et bien des strat��gies ��volutives diff��rentes entre les morphes de couleurs, les chouettes hulottes grises et rousses ��tant respectivement plus adapt��s �� des environnements stressants ou favorables. L'h��t��rog��n��it�� de notre zone d'��tude et la stochasticit�� environnementale qui caract��rise ses habitats pourraient donc agir comme une source de s��lection temporelle, laquelle favoriserait les diff��rents morphes de couleurs �� diverses p��riodes. D'un point de vue plus proximale maintenant, cette th��se de doctorat soutient l'hypoth��se que les covariations observ��es entre la coloration m��lanique et des traits ph��notypiques importants sont modul��es par les effets pl��iotropes du syst��me des m��lanocortines, et met en avant le r��le pr��pond��rant que pourrait jouer l'expression du g��ne POMC et sa post traduction en m��lanocortines.

Biologie oxydative et implications cliniques du peroxynitrite [Oxidative biology and clinical implications of peroxynitrite]

Peroxynitrite is a strong biological oxidant formed from the reaction between two free radicals, superoxide and nitric oxide. It inflicts serious damages to most biomolecules, including proteins, lipids and nucleic acids, either through direct oxidation or through the secondary generation of highly reactive free radicals. When such damage reaches a critical threshold, cells eventually die by necrosis or apoptosis. An excessive production of peroxynitrite is instrumental in the development of organ damage and dysfunction in conditions such as circulatory shock and ischemia-reperfusion. In such circumstances, various synthetic metalloporphyrins, able to degrade peroxynitrite, disclose important beneficial effects in animal models, and might therefore represent novel pharmacological agents in the future.

Biomarkers of oxidative stress and its association with the urinary reducing capacity in bus maintenance workers.

BACKGROUND: Exposure to particles (PM) induces adverse health effects (cancer, cardiovascular and pulmonary diseases). A key-role in these adverse effects seems to be played by oxidative stress, which is an excess of reactive oxygen species relative to the amount of reducing species (including antioxidants), the first line of defense against reactive oxygen species. The aim of this study was to document the oxidative stress caused by exposure to respirable particles in vivo, and to test whether exposed workers presented changes in their urinary levels for reducing species.METHODS: Bus depot workers (n = 32) exposed to particles and pollutants (respirable PM4, organic and elemental carbon, particulate metal content, polycyclic aromatic hydrocarbons, NOx, O3) were surveyed over two consecutive days. We collected urine samples before and after each shift, and quantified an oxidative stress biomarker (8-hydroxy-2'-deoxyguanosine), the reducing capacity and a biomarker of PAH exposure (1-hydroxypyrene). We used a linear mixed model to test for associations between the oxidative stress status of the workers and their particle exposure as well as with their urinary level of reducing species.RESULTS: Workers were exposed to low levels of respirable PM4 (range 25-71 &#956;g/m3). However, urinary levels of 8-hydroxy-2'-deoxyguanosine increased significantly within each shift and between both days for non-smokers. The between-day increase was significantly correlated (p < 0.001) with the concentrations of organic carbon, NOx, and the particulate copper content. The within-shift increase in 8OHdG was highly correlated to an increase of the urinary reducing capacity (Spearman &#961; = 0.59, p < 0.0001).CONCLUSIONS: These findings confirm that exposure to components associated to respirable particulate matter causes a systemic oxidative stress, as measured with the urinary 8OHdG. The strong association observed between urinary 8OHdG with the reducing capacity is suggestive of protective or other mechanisms, including circadian effects. Additional investigations should be performed to understand these observations.

Increased vulnerability of neurones and glial cells to low concentrations of methylmercury in a prooxidant situation.

Using reaggregating rat brain cell cultures at two different stages of differentiation, we examined the biochemical effects of a 10-day treatment with nanomolar concentrations of methylmercuric chloride (monomethylmercury), in the presence or absence of promoters of hydroxyl radical formation (10 microM copper sulphate plus 100 microM ascorbate). A decrease in total protein content accounted for the general cytotoxicity of these compounds, whereas selective effects were assessed by determining the activities of cell type-specific enzymes. Methylmercury, up to 100 nM, as well as the copper ascorbate mixture, when applied separately, induced no general cytotoxicity, and only slight effects on neuronal parameters. However, when applying 100 nM methylmercury and the copper-ascorbate mixture together, a drastic decrease in neuronal and glial parameters was found. Under these conditions, the content of reactive oxygen species, assessed by 2',7'-dichlorofluorescin oxidation, increased greatly, while the activities of antioxidant enzymes decreased. In the presence of copper and ascorbate, differentiated cultures appeared more resistant than immature ones to low methylmercury concentrations (1-10 mM), but did undergo similar changes in both cell type-specific and antioxidant enzyme activities at 100 nM methylmercury. These results suggest that in prooxidant conditions low doses of mercury can become much more deleterious for the central nervous system.

Neuronal nicotinic receptors as new targets foramphetamine-induced oxidative damage and neurotoxicity

Amphetamine derivatives such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are drugs widely abused in a recreational context. This has led to concern because of the evidence that they are neurotoxic in animal models and cognitive impairments have been described in heavy abusers. The main targets of these drugs are plasmalemmal and vesicular monoamine transporters, leading to reverse transport and increased monoamine efflux to the synapse. As far as neurotoxicity is concerned, increased reactive oxygen species (ROS) production seems to be one of the main causes. Recent research has demonstrated that blockade of ���7 nicotinic acetylcholine receptors (nAChR) inhibits METH- and MDMA-induced ROS production in striatal synaptosomes which is dependent on calcium and on NO-synthase activation. Moreover, ���7 nAChR antagonists (methyllycaconitine and memantine) attenuated in vivo the neurotoxicity induced by METH and MDMA, and memantine prevented the cognitive impairment induced by these drugs. Radioligand binding experiments demonstrated that both drugs have affinity to ���7 and heteromeric nAChR, with MDMA showing lower Ki values, while fluorescence calcium experiments indicated that MDMA behaves as a partial agonist on ���7 and as an antagonist on heteromeric nAChR. Sustained Ca increase led to calpain and caspase-3 activation. In addition, modulatory effects of MDMA on ���7 and heteromeric nAChR populations have been found.

Endoplasmic reticulum stress promotes inflammation through activation of the NLRP3 inflammasome

SummaryMulticellular organisms have evolved an immune system in order to cope with the constant threats they are facing. Foreign pathogens or endogenous danger signals released by injured or dying host cells can be readily detected through a set of germline- encoded pattern-recognition receptors. The NOD-like receptors are a cytoplasmic family of pattern-recognition receptors that have recently attracted considerable attention due to their ability to form inflammasomes, which are molecular complexes responsible for the activation of caspase-1 and the subsequent processing of the pro��inflammatory cytokines IL-IB and 11-18 into their mature, bioactive form.In this study, we describe a novel pro-inflammatory signaling pathway, whereby the endoplasmic reticulum promotes inflammation through activation of the NLRP3 inflammasome. This was shown to be independent of the classical endoplasmic reticulum stress response pathway constituted by the effectors IREla, PERK and ATF6a. In keeping with other known NLRP3 activators, generation of reactive oxygen species and potassium efflux were required. We also provide evidence that calcium signaling is critical to this pathway, and possibly integrates signaling triggered by various NLRP3 inflammasome activators. Moreover, the mitochondrial channel VDAC1 was instrumental in mediating this response. We thus propose that the endoplasmic reticulum acts as an integrator of stress and is able to activate the mitochondria in a calcium-dependent manner in order to promote NLRP3 inflammasome activation in response to a wide range of activators.Given the role played by inflammation in the pathogenesis of atherosclerosis, we decided to investigate a possible role for the NLRP3 inflammasome in the progression of the disease. Using an ApoE mouse model, we find that deficiency in the NLRP3 inflammasome components NLRP3, ASC or Caspase-1 does not impair atherosclerosis progression, nor does it impact plaque stability. While previous studies have clearly shown a role for the interleukin-1 family of ligands in atherosclerosis, our results suggest that its contribution might be more complex than previously appreciated, and further research is thus warranted in this field.R��sum��Les organismes multicellulaires ont d��velopp�� un syst��me immunitaire pour faire face aux menaces qui les entourent. Des pathog��nes ��trangers ou des signaux de danger rel��ch��s par des cellules de l'h��te en d��tresse peuvent ��tre rapidement d��tect��s via un assemblage de r��cepteurs sp��cifiques qui sont pr��sents d��s la naissance. Certains membres de la famille de r��cepteurs NOD ont r��cemment attir�� beaucoup d'attention au vu de leur capacit�� �� former des inflammasomes, complexes mol��culaires responsables de l'activation de la easpase-1 et de la maturation des cytokines pro-inflammatoires IL- 1&#946; et IL-18 en leur forme bioactive.Dans cette ��tude, nous d��crivons une nouvelle voie de signalisation pro-inflammatoire, par laquelle le r��ticulum endoplasmique induit l'inflammation via l'activation de l'inflammasome NLRP3. Cette voie est ind��pendante de la voie classique de r��ponse au stress du r��ticulum endoplasmique, qui comprend les effecteurs IRE1, PERK et ATF6. Comme pour d'autres activateurs de NLRP3, la g��n��ration de radicaux libres d'oxyg��ne ainsi que &#915; efflux de potassium sont requis. Nous montrons ��galement que le calcium joue un r��le critique dans cette voie, et int��gre possiblement la signalisation provoqu��e par divers activateurs de l'inflammasome NLRP3. De plus, le canal mitochondrial VDAC1 est essentiel dans cette r��ponse. Nous proposons donc que le r��ticulum endoplasmique agit comme un int��grateur de stress, activant la mitochondrie d'une fa��on calcium-d��pendante pour promouvoir l'activation de l'inflammasome NLRP3 en r��ponse �� divers activateurs.Au vu du r��le jou�� par l'inflammation dans la pathogen��se de l'ath��roscl��rose, nous avons ��tudi�� un possible r��le pour l'inflammasome NLRP3 dans la progression de la maladie. Dans un mod��le de souris ApoE, l'absence des composants de l'inflammasome NLRP3 que sont NLRP3, ASC et Caspase-1 n'influence pas la progression des plaques ni leur stabilit��. Alors que d'autres ��tudes ont d��montr�� un r��le pour les membres de la famille de l'interleukine-1 dans l'ath��roscl��rose, nos r��sultats sugg��rent que leur contribution pourrait ��tre plus complexe que pr��c��demment appr��ci��, et d'autres recherches dans ce domaine sont donc n��cessaires.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-&#954;&#914; transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-&#954;&#914; activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-&#954;&#914; and JNK triggered by TNF-&#945;, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-&#945; induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. R��sum�� Au cours de l'��volution, les organismes multicellulaires ont d��velopp�� le syst��me immunitaire afin de se prot��ger contre les pathog��nes. Une ��tape cruciale pour le d��clenchement des r��ponses protectrices est la reconnaissance par les cellules du syst��me immunitaire de mol��cules propres aux microbes gr��ce �� des r��cepteurs sp��cifiques. Les r��sultats pr��sent��s dans cette th��se d��crivent des nouveaux aspects concernant les voies de signalisation impliqu��es dans la d��tection des virus. Le premier projet d��crit un m��canisme de r��gulation des voies activ��es par la d��tection d'ARN virale. Nos r��sultats montrent que TRIF et Cardif, des prot��ines adaptatrices des voies d��clench��es par la reconnaissance de ces acides nucl��iques au niveau des endosomes et du cytoplasme, sont cliv��s et inactiv��s par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activ��e par la d��tection d'ADN au niveau du cytoplasme. La prot��ine DAI a ��t�� r��cemment d��crite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-&#954;&#914; et d'induire ainsi la production des interf��rons de type I. Ici on d��montre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appel��s RHIM et que ce complexe est responsable de l'activation de NF-&#954;&#914;. On a aussi identifi�� une prot��ine du cytom��galovirus de la souris, M45, qui contient ce m��me domaine et on a pu d��montrer qu'elle a la capacit�� d'interf��rer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-&#954;&#914;. Enfin on d��crit ici la g��n��ration de souris d��ficientes pour le g��ne qui code pour la prot��ine NLRX1. Cette prot��ine fait partie de la famille des r��cepteurs NOD et est localis��e dans la mitochondrie. Une ��tude a sugg��r�� que NLRX1 agit comme un inhibiteur des voies antivirales activ��es par les r��cepteurs du type RIG-I (RLR) en interagissant avec la prot��ine adaptatrice Cardif. Une autre ��tude propose par contre que NLRX1 participe �� la production des d��riv��s r��actifs de l'oxyg��ne et contribue ainsi �� augmenter l'activation de NF- &#954;&#914; et JNK induite par le TNF-&#945; ou le poly(I:C). Nos r��sultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les r��ponses induites par le TNF-&#945;. Des analyses ult��rieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la prot��ine UQCRC2, une des sous-unit��s qui composent le complexe III de la cha��ne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des d��riv��s r��actifs de l'oxyg��ne au niveau de cette organelle.

Development of a dose-controlled multiculture cell exposure chamber for efficient delivery of airborne and engineered nanoparticles

In order to study the various health influencing parameters related to engineered nanoparticles as well as to soot emitted b diesel engines, there is an urgent need for appropriate sampling devices and methods for cell exposure studies that simulate the respiratory system and facilitate associated biological and toxicological tests. The objective of the present work was the further advancement of a Multiculture Exposure Chamber (MEC) into a dose-controlled system for efficient delivery of nanoparticles to cells. It was validated with various types of nanoparticles (diesel engine soot aggregates, engineered nanoparticles for various applications) and with state-of-the-art nanoparticle measurement instrumentation to assess the local deposition of nanoparticles on the cell cultures. The dose of nanoparticles to which cell cultures are being exposed was evaluated in the normal operation of the in vitro cell culture exposure chamber based on measurements of the size specific nanoparticle collection efficiency of a cell free device. The average efficiency in delivering nanoparticles in the MEC was approximately 82%. The nanoparticle deposition was demonstrated by Transmission Electron Microscopy (TEM). Analysis and design of the MEC employs Computational Fluid Dynamics (CFD) and true to geometry representations of nanoparticles with the aim to assess the uniformity of nanoparticle deposition among the culture wells. Final testing of the dose-controlled cell exposure system was performed by exposing A549 lung cell cultures to fluorescently labeled nanoparticles. Delivery of aerosolized nanoparticles was demonstrated by visualization of the nanoparticle fluorescence in the cell cultures following exposure. Also monitored was the potential of the aerosolized nanoparticles to generate reactive oxygen species (ROS) (e.g. free radicals and peroxides generation), thus expressing the oxidative stress of the cells which can cause extensive cellular damage or damage on DNA.

Selective ion changes during spontaneous mitochondrial transients in intact astrocytes.

The bioenergetic status of cells is tightly regulated by the activity of cytosolic enzymes and mitochondrial ATP production. To adapt their metabolism to cellular energy needs, mitochondria have been shown to exhibit changes in their ionic composition as the result of changes in cytosolic ion concentrations. Individual mitochondria also exhibit spontaneous changes in their electrical potential without altering those of neighboring mitochondria. We recently reported that individual mitochondria of intact astrocytes exhibit spontaneous transient increases in their Na(+) concentration. Here, we investigated whether the concentration of other ionic species were involved during mitochondrial transients. By combining fluorescence imaging methods, we performed a multiparameter study of spontaneous mitochondrial transients in intact resting astrocytes. We show that mitochondria exhibit coincident changes in their Na(+) concentration, electrical potential, matrix pH and mitochondrial reactive oxygen species production during a mitochondrial transient without involving detectable changes in their Ca(2+) concentration. Using widefield and total internal reflection fluorescence imaging, we found evidence for localized transient decreases in the free Mg(2+) concentration accompanying mitochondrial Na(+) spikes that could indicate an associated local and transient enrichment in the ATP concentration. Therefore, we propose a sequential model for mitochondrial transients involving a localized ATP microdomain that triggers a Na(+)-mediated mitochondrial depolarization, transiently enhancing the activity of the mitochondrial respiratory chain. Our work provides a model describing ionic changes that could support a bidirectional cytosol-to-mitochondria ionic communication.

The Oncogene ATF3 Is Potentiated by Cyclosporine A and Ultraviolet Light A.

Cutaneous squamous cell carcinoma (SCC) represents the most important cutaneous complication following organ transplantation. It develops mostly on sun-exposed areas. A recent study showed the role of activating transcription factor 3 (ATF3) in SCC development following treatment with calcineurin inhibitors. It has been reported that ATF3, which may act as an oncogene, is under negative calcineurin/nuclear factor of activated T cells (NFAT) control and is upregulated by calcineurin inhibitors. Still, these findings do not fully explain the preferential appearance of SCC on chronically sun-damaged skin. We analyzed the influence of UV radiation on ATF3 expression and its potential role in SCC development. We found that ATF3 is a specifically induced AP1 member in SCC of transplanted patients. Its expression was strongly potentiated by combination of cyclosporine A and UVA treatment. UVA induced ATF3 expression through reactive oxygen species-mediated nuclear factor erythroid 2-related factor 2 (NRF2) activation independently of calcineurin/NFAT inhibition. Activated NRF2 directly binds to ATF3 promoter, thus inducing its expression. These results demonstrate two mechanisms that independently induce and, when combined together, potentiate the expression of ATF3, which may then force SCC development. Taking into account the previously defined role of ATF3 in the SCC development, these findings may provide an explanation and a mechanism for the frequently observed burden on SCCs on sun-exposed areas of the skin in organ transplant recipients treated by calcineurin inhibitors.

Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. Methodology/Principal Findings: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. Conclusion/Significance: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.