Cellular models and assays to study NLRP3 inflammasome biology

The NLRP3 inflammasome is a multi-protein complex that initiates innate immunity responses when exposed to a wide range of stimuli, including pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Inflammasome activation leads to the release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 and to pyroptotic cell death. Over-activation of NLRP3 inflammasome has been associated with several chronic inflammatory diseases. A deep knowledge of NLRP3 inflammasome biology is required to better exploit its potential as therapeutic target and for the development of new selective drugs. To this purpose, in the past few years, several tools have been developed for the biological characterization of the multimeric inflammasome complex, the identification of the upstream signaling cascade leading to inflammasome activation, and the downstream effects triggered by NLRP3 activation. In this review, we will report cellular models and cellular, biochemical, and biophysical assays that are currently available for studying inflammasome biology. A special focus will be on those models/assays that have been used to identify NLRP3 inhibitors and their mechanism of action

cigarette smoke inhibits the nlrp3 inflammasome and leads to caspase 1 activation via the tlr4 trif caspase 8 axis in human macrophages

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In silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design

Electrochemical sensor for evaluating oxidative stress in airway epithelial cells

Cigarette smoke exposure induces oxidative stress within the airways. Increased oxidative burden contributes to the pathogenesis of chronic lung disorders and is associated with aging and chronic inflammation. Airway epithelial cells highly contribute to Reactive Oxygen Species (ROS) generation within injured and inflamed lung tissues. Among ROS, hydrogen peroxide (H2O2) can be monitored in the extracellular space. Herein, we present an amperometric/voltammetric sensor based on gold nanoparticles and graphene oxide able to detect H2O2 with good sensitivity and selectivity. Using this sensor, H2O2 release was measured in conditioned medium from primary bronchial epithelial cells (PBEC), bronchial epithelial cell line, 16HBE, and adenocarcinoma alveolar basal epithelial cell line, A549, exposed to cigarette smoke extracts (CSE). 16HBE were also treated with resveratrol, an anti-oxidant compound. The results were compared with those obtained by flow cytometry using the same cells stained with Carboxy-H2DCFDA and MitoSOX Red, which detect intracellular ROS and mitochondrial superoxide, respectively. The exposure to CSE resulted in a significant increase of the cathodic current due to the reduction of H2O2 indicating an increased release. Addition of resveratrol decreased CSE-induced release of H2O2 in 16HBE. All the results paralleled those obtained by flow cytometry. The proposed sensor is highly sensitive and selective, fast and cost effective and can potentially be applied for real time and easy monitoring of oxidative stress

Au royaume des discours incomplets : ambigüité et malentendu dans la conversation entre mafieux

Im Reich der Andeutungen : Bewusst mehrdeutige und missverständliche Aussagen in Mafiosi-Gesprächen. Beim mündlichen Austausch müssen sich Mitglieder der Cosa Nostra gleichzeitig an die Schweigepflicht nach außen und die Wahrheitspflicht nach innen halten. Die Sprache der Mafiosi muss daher eine semantisch indirekte Form annehmen, die dem Sprechenden seinen Spielraum lässt, um die Unvorhersehbarkeit seiner Absichten zu bewahren. Diese Sprachform hat Tommaso Buscetta dazu gebracht, dieses Milieu als „das Reich der Andeutungen“ zu bezeichnen. Die Ordnung der Mafia beruht auf einem wohl verstandenen doppelten Spiel bewusst missverständlicher Aussagen, im Sinne von Jankélévitch, die keine falsche Beziehung, sondern eine falsche Situation darstellt, in der ein gegenseitiges Wissen um die Täuschung aufrechterhalten wird, die ein unendliches Spiel der Dialektik erlaubt. Dieses Spiel wird hier untersucht anhand der Gespräche -im Beisein ihrer Richter und Rechtsanwälte -zwischen einem ehemaligen Ehrenmann einer Mafiafamilie, der zum Kollaborateur des Justiz wurde, und einem Angeklagten, der mit seinem Bruder für den Kreis der gleichen Familie verantwortlich war.In the realm of incomplete speech : ambiguity and misunderstandings in conversations between Mafiosi. Spoken exchanges between members of the Cosa Nostra are governed by the twofold restriction of the law of silence towards the outside and the obligation of speaking the truth inside. To this purpose, the Mafia’s language obeys a semantic obliqueness which provides speakers with a margin of freedom by preserving the unpredictability of their intentions, which led Tommaso Buscetta to qualify the setting as “the realm of incomplete speech”. The Mafia order is based on a practice of misunderstandings doubly understood, in the meaning of Jankélévitch, which comprises not only a false relationship but also a false situation maintaining mutual knowledge of deception thereby permitting an infinite, dialectic game. This game is explored here through exchanges, in the presence of their judges and lawyers, between a former man of honour from a Mafia family who switched sides to become an informer, and the accused who, with his brother, was the district leader of the same family.Les échanges parlés entre membres de la Cosa Nostra sont régis par la double contrainte de la loi du silence vis-à-vis de l’extérieur et de l’obligation de dire la vérité à l’intérieur. Le langage mafieux obéit de ce fait à une obliquité sémantique qui fournit au locuteur sa marge de liberté en préservant l’imprévisibilité de ses intentions, et qui a conduit Tommaso Buscetta à qualifier le milieu de «royaume des discours incomplets». L’ordre mafieux se fonde sur une pratique du malentendu doublement bien entendu, au sens de Jankélévitch, qui n’est pas qu’une fausse relation, mais une fausse situation entretenant une connaissance réciproque de la tromperie qui autorise un jeu dialectique infini. Ce jeu est ici exploré à partir des échanges, en présence de leurs juges et de leurs avocats, entre un ex-homme d’honneur d’une famille mafieuse passé collaborateur de justice et d’un prévenu qui avec son frère a été responsable de circonscription de la même famille.Dino Alessandra, Buscetta Roberto. Au royaume des discours incomplets : ambigüité et malentendu dans la conversation entre mafieux. In: Revue des sciences sociales, N°50, 2013. Malentendus. pp. 52-59

Development of a nanostructured sensor for monitoring oxidative stress in living cells

Oxidative burden is elevated in the lung of COPD patients and is associated with aging and chronic inflammation. When overcoming physiological levels, reactive oxygen species (ROS) cause cell damage and sustain inflammation. Both lung epithelium and alveolar macrophages contribute to ROS generation. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric assays. We present an amperometric nanostructured sensor for real-time detection of hydrogen peroxide (H2O2) released by living cells. The H2O2 sensing performance was evaluated through the current vs time response of platinum rod at a working potential of −0.45 V vs saturated calomel electrode acting as a reference. The detection current was related to the bulk concentration of H2O2 in solution. Using this sensor, H2O2 release was measured in conditioned medium from THP-1 macrophages and 16-HBE bronchial epithelial cells exposed to different stimuli (lipopolysaccharide (LPS), cigarette smoke extract (CSE), nigericin). Results were compared with those obtained by flow cytometry using the same cells stained with Carboxy-H2DCFDA and MitoSOXTM Red. The addition of LPS, CSE and nigericin resulted in a significant increase of the cathodic current due to the reduction of H2O2 indicating an increase in H2O2 release. The results paralleled those obtained by flow cytometry. The proposed nanostructured sensor offers several advantages over current methods: (i) real time release of H2O2 is monitored without disturbing cell growth; (ii) quantitative data can be generated using a calibration curve; (iii) the method is highly sensitive, fast and cost effective; (iv) potential use for in vivo monitoring of oxidative stress

Severe case of rhabdomyolysis following jellyfish envenomation in the Mediterranean Sea

Jellyfish envenomation is a common problem in coastal areas all over the world; usually symptoms are self-limited with no long-lasting complications. Despite that, some jellyfish species, mainly populating the Indian Ocean, are renown to be potentially lethal and in some cases may cause severe myopathy. We report the first case of rhabdomyolysis following a jellyfish sting in the Mediterranean Sea. A 17-year-old patient was admitted to the intensive care unit of our hospital in life-threatening conditions. He was dyspnoeic and dysphagic with pain and functional impairment of upper and lower limbs. The evidence of a red mark in his face and the clinical presentation, coupled with the diagnostic test performed, allowed the diagnosis of toxidrome from jellyfish venom. Treatment with hydration, ventilatory support and steroids led to a progressive improvement of patient conditions. Our case report stresses the importance of prompt identification and treatment of potential rhabdomyolysis determined by jellyfish and rises awareness on the presence of such venomous species in the Mediterranean Sea

Electrochemical sensor based on rGO/Au nanoparticles for monitoring H2O2 released by human macrophages

Increased oxidative burden contributes to the pathogenesis of most inflammatory diseases and is associated with aging and chronic inflammation. Macrophages contribute to the generation of reactive oxygen species (ROS) within inflamed tissues. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric biochemical assays. Hydrogen peroxide (H2O2) diffuses through the cell membrane and can be monitored in the extracellular space. Herein, we present a sensor for H2O2 detection released by cells in culture supernatants. H2O2 sensing performance was evaluated using chronoamperometric detection. A sensitivity of 0.0641 μA μM−1 cm−2 with a limit of detection of 6.55 μM and excellent selectivity against many interferents was found. H2O2 release was also measured in conditioned medium from human THP-1 macrophages exposed to pro-oxidant and anti-oxidant treatments. The results were compared with those obtained by flow cytometry using the same cells stained with carboxy-H2DCFDA and MitoSOX Red, which detect intracellular ROS and mitochondrial superoxide, respectively. The addition of pro-oxidants lipopolysaccharide (LPS) and nigericin resulted in a significant increase in the cathodic current due to the H2O2 reduction, indicating an increased release of H2O2. The addition of 17-oxo-DHA, which inhibits LPS- and nigericin-dependent responses, decreased the LPS- and nigericin-induced release of H2O2. All the results obtained with the sensor were consistent with those obtained using flow cytometry. The operation of the sensor directly in the cell culture growth medium had no impact on cell viability. The sensor is highly sensitive, fast, and cost effective, and it can potentially be used for real time monitoring of oxidative stress