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Objective: Binding to angiotensin-converting enzyme 2 (ACE2) receptor is a critical step for severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) to mediate its entry into target cells ACE2 is expressed in many human tissues, including the lungs However, no research has demonstrated that SARS-CoV-2 can infect human breast tissue This study aimed to investigate ACE2 gene expression in human breast tissue using a public database Materials and Methods: A search of a public gene expression database was performed to investigate ACE2 gene expression in in human breast tissue Results: The gene expression profile demonstrated that ACE2 gene expression was higher in human breast tissue than human lung tissue Conclusion: Our knowledge about coronavirus disease-2019 (COVID-19) is expanding rapidly Clinicians are eager for vetted information regarding all aspects of this new illness, and this study demonstrates that the level of ACE2 expression in human breast tissue is higher than that in the lung tissue, a major target tissue affected by SARS-CoV-2 infection This finding strongly suggests that SARS-CoV-2 infection causes breast pathology

Significance: Despite their intrinsic cytotoxic properties, mounting evidence indicates that reactive oxygen species (ROS) physiologically produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) of epithelial cells (NOX1, dual oxidase [DUOX]2) and phagocytes (NOX2) are critical for innate immune response and homeostasis of the intestinal mucosa. However, dysregulated ROS production could be a driving factor in inflammatory bowel diseases (IBDs). Recent Advances: In addition to NOX2, recent studies have demonstrated that NOX1- and DUOX2-derived ROS can regulate intestinal innate immune defense and homeostasis by impacting many processes, including bacterial virulence, expression of bacteriostatic proteins, epithelial renewal and restitution, and microbiota composition. Moreover, the antibacterial role of DUOX2 is a function conserved in evolution as it has been described in invertebrates, and lower and higher vertebrates. In humans, variants of the NOX2, NOX1, and DUOX2 genes, which are associated with impaired ROS production, have been identified in very early onset IBD, but overexpression of NOX/DUOX, especially DUOX2, has also been described in IBD, suggesting that loss-of-function or excessive activity of the ROS-generating enzymes could contribute to disease progression. Critical Issues: Therapeutic perspectives aiming at targeting NOX/DUOX in IBD should take into account the two sides of NOX/DUOX-derived ROS in intestinal inflammation. Hence, NOX/DUOX inhibitors or ROS inducers should be considered as a function of the disease context. Future Directions: A thorough understanding of the physiological and pathological regulation of NOX/DUOX in the gastrointestinal tract is an absolute pre-requisite for the development of therapeutic strategies that can modulate ROS levels in space and time.

Patients with autosomal recessive protein kinase C delta (PKC delta) deficiency suffer from childhood-onset autoimmunity, including systemic lupus erythematosus. They also suffer from recurrent infections that overlap with those seen in patients with chronic granulomatous disease (CGD), a disease caused by defects of the phagocyte NADPH oxidase and a lack of reactive oxygen species (ROS) production. We studied an international cohort of 17 PKC delta-deficient patients and found that their EBV-B cells and monocyte-derived phagocytes produced only small amounts of ROS and did not phosphorylate p40(phox) normally after PMA or opsonized Staphylococcus aureus stimulation. Moreover, the patients' circulating phagocytes displayed abnormally low levels of ROS production and markedly reduced neutrophil extracellular trap formation, altogether suggesting a role for PKC delta in activation of the NADPH oxidase complex. Our findings thus show that patients with PKC delta deficiency have impaired NADPH oxidase activity in various myeloid subsets, which may contribute to their CGD-like infectious phenotype. Yale Center for Mendelian Genomics - National Human Genome Research Institute [UM1HG006504]; National Institute of Allergy and Infectious DiseasesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [5R01AI089970, 5R37AI095983, R01AI127564-01]; National Center for Research ResourcesUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR); National Center for Advancing Translational Sciences of the National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Advancing Translational Sciences (NCATS) [8UL1TR001866]; Rockefeller University; St. Giles Foundation; INSERMInstitut National de la Sante et de la Recherche Medicale (Inserm)European Commission; University of Paris; French Foundation for Medical ResearchFondation pour la Recherche Medicale [EQU201903007798]; Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence [ANR-10-LABX-62-IBEID]; SCOR Corporate Foundation for Science; French National Research Agency (ANR) under the Investments for the future programFrench National Research Agency (ANR) [ANR-10-IAHU-01]; ANR-IFNPHOX [ANR-13-ISV3-0001-01]; ANR-GENMSMDFrench National Research Agency (ANR) [ANR-16-CE17-0005-01]; ANR-GENCMCD [ANR-11-BSV3-005-01]; ANR-HGDIFDFrench National Research Agency (ANR) [ANR-14-CE15-0006-01]; Bettencourt Schueller Foundation; International PhD program of the Imagine Institute; Japanese Foundation; EURO-CMC project [ANR-14-RARE-0005-02]; Societe Nationale Francaise de Medecine Interne (Bourse Marcel Simon); INSERM PhD program for medical doctors (poste d'accueil INSERM); Fulbright grant (Franco-American commission); MD-PhD program of Imagine Institute; Consejo Nacional de Ciencia y Tecnologa National PhD Fellowship Program; National Institute for Health Research Biomedical Research CentreNational Institute for Health Research (NIHR); Great Ormond Street Hospital Charity; Deutsche Forschungsgemeinschaft under Germany's Excellence StrategyGerman Research Foundation (DFG) [EXC 2155, 390874280]; REBIRTH Center for Translational Regenerative Medicine through the State of Lower Saxony [MWK: ZN3440] This paper is dedicated to the memory of Prof. Asghar Agha-mohammadi, who passed away in November 2020, and made immense contributions to the understanding and treatment of primary immunodeficiencies. We thank all patients, their relatives, and the treatment teams for their cooperation in this study. We thank all the members of the Laboratory of Human Genetics of Infectious Diseases for helpful discussions. We also thank Christine Rivalain, Cecile Pa-tissier, Lazaro Lorenzo-Diaz, Dominick Papandrea, Dana Liu, and Yelena Nemirovskaya for administrative assistance and Gaspard Kerner for statistical advice. We thank the Necker Institute Imaging Facility for technical advice. The graphical abstract was created with BioRender.com under subscription. This research was supported by the Yale Center for Mendelian Genomics funded by the National Human Genome Research Insti-tute (UM1HG006504) . The Howard Hughes Medical Institute lab-oratory was funded in part by the National Institute of Allergy and Infectious Diseases (grants 5R01AI089970, 5R37AI095983, and R01AI127564-01) , the National Center for Research Resources, and the National Center for Advancing Translational Sciences of the National Institutes of Health (grant 8UL1TR001866 for J-L. Casa-nova) , the Rockefeller University, the St. Giles Foundation, INSERM, the University of Paris, the French Foundation for MedicalResearch (EQU201903007798) , the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID) , the SCOR Corporate Foundation for Science, and the French National Research Agency (ANR) under the Investments for the future program (grant ANR-10-IAHU-01) , ANR-IFNPHOX (grant ANR-13-ISV3-0001-01 for J. Bustamante) , ANR-GENMSMD (grant ANR-16-CE17-0005-01 for J. Bustamante) , ANR-GENCMCD (grant ANR-11-BSV3-005-01 for A. Puel) , and ANR-HGDIFD (grant ANR-14-CE15-0006-01 for J. Bustamante) . A-L. Neehus was sup-ported by the Bettencourt Schueller Foundation and the Interna-tional PhD program of the Imagine Institute, K. Moriya by a Japanese Foundation for Pediatric Research fellowship grant and EURO-CMC project (grant ANR-14-RARE-0005-02 for J. Bustamante) , R. Levy by the Societe Nationale Francaise de Medecine Interne (Bourse Marcel Simon) , the INSERM PhD program for medical doctors (poste d'accueil INSERM) , and the Fulbright grant (Franco-American commission) , T. Le Voyer by the Bettencourt Schueller Foundation and the MD-PhD program of Imagine Institute, and A. Nieto-Patlan by the Consejo Nacional de Ciencia y Tecnologa Na-tional PhD Fellowship Program. All research at the Great Ormond Street Hospital National Health Service Foundation Trust is sup-ported by the National Institute for Health Research Biomedical Research Centre. P.A. Brogan also acknowledges support from the Great Ormond Street Hospital Charity. N. Lachmann acknowledges support from the Deutsche Forschungsgemeinschaft under Ger-many's Excellence Strategy-EXC 2155-project number 390874280 and the REBIRTH Center for Translational Regenerative Medicine funded through the State of Lower Saxony (MWK: ZN3440) . Author contributions: A-L. Neehus, J-L. Casanova, A. Puel, and J. Bustamante conceived and designed the study. A-L. Neehus, K. Moriya, T. Le Voyer, A. Nieto-Patlan, R. Levy, M. Roynard, K. Haake, M. Migaud, and K. Dorgham performed the experiments. A. ozen, E. Karakoc-Aydiner, S. Baris, A. Yildiran, E. Altundag, F. Dogu, S. Has-kologlu, E. Ince, G. Uzel, A. Kiykim, K. Boztug, M.R. Roderick, M. Shahrooei, P.A. Brogan, H. Abolhassani, G. Hancioglu, N. Parvaneh, A.; Belot, and A. Ikinciogullari were the treating physicians respon-sible for clinical diagnosis, deep phenotyping, sample collection, and patient follow-up. G. Gorochov, L. Abel, N. Lachmann, J. El-Benna, and A. Belot provided conceptual advice. A-L. Neehus, J-L. Casanova, A. Puel, and J. Bustamante analyzed the results and wrote the man-uscript. All authors revised the manuscript and approved the final manuscript as submitted. Disclosures: P.A. Brogan reported personal fees from Sobi, No-vartis, Roche, and GSK, and grants from Sobi outside the sub-mitted work. No other disclosures were reported.

BackgroundProcalcitonin (proCt) was recently proposed as an alternative or in addition to calcitonin (Ct) in medullary thyroid cancer (MTC) diagnostics.MethodsSerum basal Ct (bCt) and proCt (bproCt) levels were measured before surgery from a consecutive series of patients with (n=43) and without (n=75) MTC, retrospectively collected in Padua. Serum bproCt, bCt and stimulated proCt and Ct (sproCt and sCt) were measured in another consecutive series of 33 patients seen at three tertiary-level institutions undergoing a calcium stimulation test prior to surgery, 20 of them with a final diagnosis of MTC, and 13 with non-MTC nodular disease.ResultsMedian bproCt levels were higher in MTC than in non-MTC. A positive correlation was found for bproCt with bCt (P2 = 0.75), and with tumor size (P2 = 0.39). The cut-off for bproCt differentiating between MTC and non-MTC patients was >0.07 ng/ml (sensitivity: 85.7%, specificity: 98.9%, positive predictive value [PPV]: 98.2%, negative predictive value [NPV]: 90.6%, P0.07 ng/ml in 38/39 (97.4%) patients with MTC >10 mm, it was above said cut-off only in 15/23 (65.2%) patients with tumors ≤10 mm. A sproCt >0.19 ng/ml was able to identify MTC [sensitivity: 90.0%, specificity:100.0%, PPV: 100.0%, NPV: 86.7% (PConclusionsOur data suggest that bproCt can be a good adjunct to Ct for MTC diagnostic purposes. In consideration of its high specificity, it can be used in combination with Ct in MTC diagnostics, particularly in the case of mildly elevated basal Ct levels.

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.

The occurrence of phlogopite and amphibole in mantle ultramafic rocks is widely accepted as the modal effect of metasomatism in the upper mantle. However, their simultaneous formation during metasomatic events and the related sub-solidus equilibrium with the peridotite has not been extensively studied. In this work, we discuss the geochemical conditions at which the pargasite-phlogopite assemblage becomes stable, through the investigation of two mantle xenoliths from Mount Leura (Victoria State, Australia) that bear phlogopite and the phlogopite + amphibole (pargasite) pair disseminated in a harzburgite matrix. Combining a mineralogical study and thermodynamic modelling, we predict that the P–T locus of the equilibrium reaction pargasite + forsterite = Na-phlogopite + 2 diopside + spinel, over the range 1.3–3.0 GPa/540–1500 K, yields a negative Clapeyron slope of -0.003 GPa K–1 (on average). The intersection of the P–T locus of supposed equilibrium with the new mantle geotherm calculated in this work allowed us to state that the Mount Leura xenoliths achieved equilibrium at 2.3 GPa /1190 K, that represents a plausible depth of ~ 70 km. Metasomatic K-Na-OH rich fluids stabilize hydrous phases. This has been modelled by the following equilibrium equation: 2 (K,Na)-phlogopite + forsterite = 7/2 enstatite + spinel + fluid (components: Na2O,K2O,H2O). Using quantum-mechanics, semi-empirical potentials, lattice dynamics and observed thermo-elastic data, we concluded that K-Na-OH rich fluids are not effective metasomatic agents to convey alkali species across the upper mantle, as the fluids are highly reactive with the ultramafic system and favour the rapid formation of phlogopite and amphibole. In addition, oxygen fugacity estimates of the Mount Leura mantle xenoliths [Δ(FMQ) = –1.97 ± 0.35; –1.83 ± 0.36] indicate a more reducing mantle environment than what is expected from the occurrence of phlogopite and amphibole in spinel-bearing peridotites. This is accounted for by our model of full molecular dissociation of the fluid and incorporation of the O-H-K-Na species into (OH)-K-Na-bearing mineral phases (phlogopite and amphibole), that leads to a peridotite metasomatized ambient characterized by reduced oxygen fugacity.

Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, neutrophil function may be severely altered in conditions of iron overload, as observed in chronically transfused patients. Therefore, a tight regulation of neutrophil iron homeostasis seems to be critical for avoiding iron toxicity. Hepcidin is the key iron regulator in organisms; however, no studies have investigated its role in maintaining neutrophil iron homeostasis or characterized neutrophil function in patients with hereditary hemochromatosis (HH), a common iron overload genetic disorder that results from a defect in hepcidin production. To explore these issues, we studied 2 mouse models of iron overload: an experimentally induced iron overload model (EIO), in which hepcidin is increased, and a genetic HH model of iron overload with a deletion of hepatic hepcidin. We found that iron-dependent increase of hepatic hepcidin results in neutrophil intracellular iron trapping and consecutive defects in oxidative burst activity. In contrast, in both HH mouse models and HH patients, the lack of hepcidin expression protects neutrophils from toxic iron accumulation. Moreover, systemic iron overload correlated with a surprising neutrophil priming and resulted in a more powerful oxidative burst. Indeed, important factors in neutrophil priming and activation, such as tumor necrosis factor α (TNF-α), VCAM-1, and ICAM-1 are increased in the plasma of HH patients and are associated with an increase in HH neutrophil phagocytosis capacity and a decrease in L-selectin surface expression. This is the first study to characterize neutrophil iron homeostasis and associated functions in patients with HH.

Neutrophils are key cells in innate immunity and inflammation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to enhance many neutrophil functions such as reactive oxygen species (ROS) production, degranulation and cell survival via the activation of the ERK1/2 pathway. ERK1/2 pathway activation is redox sensitive and could be modulated by ROS. In order to investigate whether NADPH oxidase NOX2-derived ROS could contribute to GM-CSF-induced ERK1/2 phosphorylation, we tested the effect of two selective NOX2 inhibitors, diphenylene iodonium (DPI) and apocynin. Results showed that, while both DPI and apocynin strongly inhibited neutrophil ROS production, only apocynin, but not DPI, inhibited GM-CSF-induced ERK1/2 phosphorylation, suggesting that ROS are not involved in this process. Apocynin did not affect GM-CSF-induced p38MAPKinase phosphorylation, another redox sensitive kinase. Interestingly, apocynin inhibited GM-CSF-induced MEK1/2 and AKT phosphorylation without affecting fMLF-induced phosphorylation of these proteins. GM-CSF is known to inhibit neutrophils apoptosis and to promote cell survival via the AKT-ERK1/2 pathway. In this regard, we found that apocynin also inhibited GM-CSF-induced anti-apoptotic effect in neutrophils. These results suggest that NADPH oxidase NOX2-derived ROS are not involved in GM-CSF-induced ERK1/2 phosphorylation and that apocynin inhibits GM-CSF-induced ERK1/2 phosphorylation pathway independently of its inhibitory action on NADPH oxidase NOX2. Thus, apocynin can exert an anti-inflammatory effect not only by limiting neutrophil ROS production but also by decreasing neutrophil survival at inflammatory site.

Superoxide anion production by neutrophils is essential for host defense against microbes. Superoxide anion generates other reactive oxygen species (ROS) that are very toxic for microbes and host cells, therefore their excessive production could induce inflammatory reactions and tissue injury. Cyclic adenosine monophosphate (cAMP) elevating agents are considered to be physiological inhibitors of superoxide production by neutrophils but the mechanisms involved in this inhibitory effect are poorly understood. Superoxide is produced by the phagocyte NADPH oxidase, a complex enzyme composed of two membrane subunits, gp91phox or NOX2 and p22phox, and four cytosolic components p47phox, p67phox, p40phox, and Rac2. Except Rac2, these proteins are known to be phosphorylated upon neutrophil stimulation. Here we show that forskolin, an activator of the adenylate cyclase-cAMP-PKA pathway, induced phosphorylation of gp91phox/NOX2 and inhibited fMLF-induced NADPH oxidase activation in human neutrophils. H89, a PKA inhibitor prevented the forskolin-induced phosphorylation of gp91phox and restored NADPH oxidase activation. Furthermore, PKA phosphorylated the recombinant gp91phox/NOX2-cytosolic C-terminal region in vitro only on a few specific peptides containing serine residues, as compared to PKC. Interestingly, phosphorylation of NOX2-Cter by PKA alone did not induce interaction with the cytosolic components p47phox, p67phox and Rac2, however it induced inhibition of PKC-induced interaction. Furthermore, PKA alone did not induce NOX2 electron transfer activity, however it inhibited PKC-induced activation. These results suggest that PKA phosphorylates NOX2 in human neutrophils, a process essential to limit ROS production and inflammation under physiological conditions. Our data identify the cAMP-PKA-NOX2-axis as a critical gatekeeper of neutrophil ROS production.

The two most common polymorphs of MnO2, ramsdellite and pyrolusite, are often found in natural association. Our starting sample is from the Mistake mine (Arizona) containing macroscopic crystals of both ramsdellite (a = 4.5131(6), b = 9.2689(13), c = 2.8610(4) A, V = 119.69(3) A3; S.G. Pbmn) and pyrolusite (a = 4.4030(2), c = 2.87392(16) A, V = 55.715(5) A3; S.G. P42/mnm), along with a smaller amount of “groutellite”. A mixed powder was used to study the ramsdellite→pyrolusite transformation by in situ high-temperature X-ray powder diffraction. Our results reveal that this transformation is not a direct transition, but it occurs in two steps, as a function of temperature; ramsdellite transforms into an amorphous phase, which then recrystallizes into pyrolusite. Amorphization of ramsdellite and crystallization of pyrolusite kinetics were studied by the universal equation for solid–solid reactions. The two activation energies are comparable, but the pre-exponential factor of the ramsdellite amorphization is two orders of magnitude larger than pyrolusite crystallization’s. As a consequence, ramsdellite→pyrolusite transformation implies the formation of an amorphous transition, due to a mismatch between the conversion rates, that reaches its maximum at around 630 K and then decreases at higher T, when pyrolusite crystallization is strongly promoted.

Noble gas plasmas are commonly used as ion and excitation sources in inductively coupled plasma (ICP) optical emission and mass spectrometry for organic and inorganic chemical analysis. However, the high power (∼kW) and voluminous gas flow rate (∼15 L min−1) of commercial plasmas limit their potential deployment in remote terrestrial and planetary environments. Here, using argon and helium gas supplies, we investigate the fundamental characteristics of low power and reduced-pressure microwave plasmas for organic and elemental analysis. These plasmas require a fraction of the power ( 99%) organic molecules or elements with high ionization potentials (e.g. glycine, 8.9 eV, or S, 10.4 eV); in comparison, helium can achieve similar performance metrics with as little as 5 W of forward power.

The active targeting strategy has emerged as a promising approach to achieve selectivity in nanobiotechnology applications. Peptides are particularly suited as targeting moieties because the multiv...

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.

Volatiles and refractories represent the two end-members in the volatility range of species in any surface-bounded exosphere. Volatiles include elements that do not interact strongly with the surface, such as neon (detected on the Moon) and helium (detected both on the Moon and at Mercury), but also argon, a noble gas (detected on the Moon) that surprisingly adsorbs at the cold lunar nighttime surface. Refractories include species such as calcium, magnesium, iron, and aluminum, all of which have very strong bonds with the lunar surface and thus need energetic processes to be ejected into the exosphere. Here we focus on the properties of species that have been detected in the exospheres of inner Solar System bodies, specifically the Moon and Mercury, and how they provide important information to understand source and loss processes of these exospheres, as well as their dependence on variations in external drivers.

International audience; Context: Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), commonly known as clove, originally found in the Muluku Islands in East Indonesia, is widely used as a spice and has numerous medicinal properties.Objective: This study investigated the antioxidant potential of S. aromaticum aqueous extract (SAAE) in vitro and its protective effects on lipopolysaccharide (LPS)-induced lung inflammation in mice.Material and methods: Neutrophils were isolated from healthy donors and reactive oxygen species (ROS) generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by cytochrome c reduction assay. H 2 O 2 was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was mesured by tetramethyl benzidine oxidation method. To study the anti-inflammatory activity of SAAE, lung inflammation was induced in mice (BALB/c) by intra-tracheal instillation of lypopolysaccharide (5 mg/mouse), and SAAE (200 mg/kg body weight) was injected intraperitoneally prior to LPS administration. Bronchoalveolar lavage and lung tissue were collected to assess inflammatory cells count and total protein content. Metalloproteinases activity was detected by zymography technique.Results: SAAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formylmethionyl-leucyl-phenylalanine-or phorbol myristate acetate-stimulated neutrophils, with an inhibitory effect starting at a concentration as low as 0.5 mg/mL. Moreover, SAAE reduced significantly MPO activity and it exhibits a dose-dependent action (IC 50 ¼ 0.5 mg/mL). In vivo results showed that SAAE decreased markedly neutrophil count (From 61% to 15%) and proteins leakage into bronchoalveolar lavage fluid. Gelatin zymography assay showed that S. aromaticum inhibited MMP-2 (15%) and MMP-9 (18%) activity in lung homogenates.Discussion and conclusion: Our results suggest that the anti-inflammatory activity of SAAE, in vivo, is due to the inhibition of ROS production and metalloproteinases activity via its action on MPO. According to these findings, SAAE could be a potential source of new compounds with anti-inflammatory activity.

International audience; Excessive reactive oxygen species (ROS) production can induce tissue injury involved in a variety of neurodegenerative disorders such as neurodegeneration observed in pilocarpine-induced temporal lobe epilepsy. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist has beneficial effects in pilocarpine-induced temporal lobe epilepsy when administered within minutes of seizure to avoid the harmful neurological lesions induced by pilocarpine. However, the enzymes involved in ROS productions and the effect of ketamine on this process remain less documented. Here we show that during pilocarpine-induced epilepsy in mice, the expression of the phagocyte NADPH oxidase NOX2 subunits (NOX2/gp91 phox , p22 phox , and p47 phox) and the expression of myeloperoxidase (MPO) were dramatically increased in mice brain treated with pilocarpine. Interestingly, treatment of mice with ketamine before or after pilocarpine administration decreased this process mainly when injected before pilocarpine. Finally, our results showed that pilocarpine induced p47 phox phosphorylation and H 2 O 2 production in mice brain and ketamine was able to inhibit these processes. Our results show that pilocarpine-induced NOX2 activation to produce ROS in mice brain and that administration of ketamine before or after the induction of temporal lobe epilepsy by pilocarpine inhibited this activation in mice brain. These results suggest a key role of the phagocyte NADPH oxidase NOX2 and MPO in epilepsy and identify a novel effect of ketamine.

International audience

In this study, we report the synthesis and characterization of novel nanocomposite namely doubly functionalized chloromethyl-calix[4]arene-methoxy grafted silylated clay (SC-C[4]). The silylation of nanoclay (thickness of the sheets ≈ 1 nm) was performed using (3-aminopropyl)trimethoxysilane (APTMS) (height ≈ 4.55 A) coupling agent. The resulting organoclay was characterized by various techniques such as X-ray diffraction (XRD), infrared spectroscopy (IR), thermal gravimetric analysis (TGA), and 29Si and 13C solid-state NMR. The XRD results suggested the presence of a double layer of silanes molecules into the clay galleries of nanoclay minerals by increasing in the basal spacing. While the IR, 29Si and 13C solid-state NMR, and TGA analysis demonstrated the successful silylation of clay by the appearance of novel absorption bands, the changes in the chemical environment and the peaks degradation in the range 200 to 500 ° C related to aminosilane molecules, respectively. The calix[4]arenes derivatives were synthesized and characterized by IR spectroscopy and 1HNMR. The results showed that calix[4]arene was functionalized at the lower and upper rim. The grafting of the doubly functionalized calix[4]arene onto clay surfaces was performed through nucleophilic reaction. Structure, thermal properties of the nanocomposite SC-C[4] were also investigated. All data approved the successful grafting of para-chloromethyl-calix[4]arene-OMe onto clay surfaces.

International audience; Neutrophils are key cells of the innate immune and inflammatory responses. They are the first blood cells to migrate to the infection site where they release high amounts of reactive oxygen species (ROS) and several peptides and enzymes required for microbial killing. However, excessive neutrophil activation can induce tissue injury participating in inflammation, thus the characterization of the enzymes involved in neutrophil activation could help to identify new pharmacological targets to treat inflammation. The prolyl-isomerase Pin1 is a ubiquitous enzyme involved in several functions, however, its role in neutrophil functions is less known. In this study, we show that the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP or fMLF), a G-protein coupled receptor (GPCR) agonist-induced Pin1 activation in human neutrophils. PiB and juglone, two Pin1 inhibitors inhibited Pin1 activity in neutrophils and consequently inhibited fMLP-induced chemotaxis and -degranulation of azurophil and specific granules as measured by myeloperoxidase and neutrophil gelatinase-associated lipocalin (NGAL) release respectively. We also showed that PiB inhibited TNFα + fMLP-induced superoxide production, confirming the effect of juglone. These data show that inhibitors of Pin1 impaired key pro-inflammatory neutrophil functions elicited by GPCR activation and suggest that Pin1 could control neutrophil inflammatory functions.

International audience; We report seasonal and dust-related variations in neutral and ion species (CO2, O, and N2, and CO2+, O2+, O+, and N+, respectively) in the dayside Martian upper atmosphere between altitudes of ∼150 and ∼250 km observed by the Neutral Gas and Ion Mass Spectrometer aboard the Mars Atmosphere and Volatile Evolution spacecraft. The sinusoidal seasonal variations in CO2+ and O2+ densities are clearly identified, while that of O+ is less discernible. These observed variations in ion densities are well reproduced by a photochemical equilibrium model for CO2+ and O+ densities when we combine them with solar cycle variations. Furthermore, we find a decrease in O, O+, and O2+ densities in the whole altitude range at Ls = 342 - 346 in MY 33 during a regional dust event. The decrease in O density would lead to decreases in O+ and O2+ densities in the ionosphere through ion-neutral reactions. Observed variations in ion and neutral species associated with the season and a regional dust storm are also confirmed in pressure coordinates. Observations show that the CO2+/O+ ratio at a given pressure level in the ionosphere varies by a factor of ∼3, which can modify the composition of ion outflow from the Martian atmosphere.

Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

The structural behaviour of cassiterite (SnO2), pyrolusite (MnO2) and sellaite (MgF2), i.e. AX2-minerals, has been investigated at room temperature by in situ high-pressure single-crystal diffraction, up to 14 GPa, using a diamond anvil cell. Such minerals undergo a ferroelastic phase transition, from rutile-like structure (SG: P42/mnm) to CaCl2-like structure (SG: Pnnm), at ≈ 10.25, 4.05 and 4.80 GPa, respectively. The structural evolution under pressure has been described by the trends of some structure parameters that are other than zero in the region of the low-symmetry phase’s stability. In particular, three tilting-angles (ω, ω′, ABS) and the metric distortion of the cation-centred octahedron (quantified via the difference between apical-anion and equatorial-anion distances Δ|Xax−Xeq|) are used to express the atoms’ readjustment, i.e. relaxation, taking place in the CaCl2-like structures under pressure. The crystallographic investigation presented is complemented with an analysis of the energy involved in the phase transition using the Landau formalism and adopting the following definition for the order parameter: Q = η11–η22, ηij being the spontaneous strain tensor. The dependence of ω, ω′, ABS and Δ|Xax−Xeq| on Q allows determination of a correlation between geometrical deformation parameter and energy. Lastly, the relaxation mechanisms that exploits ω, ω′, ABS and Δ|Xax−Xeq| may be related to the ionic degree of bonding, the latter modelled via quantum mechanics and Bader theory. Sellaite, the mineral exhibiting the highest degree of ionic bonding among those investigated, tends to accomplish relaxation through pure rotation of the octahedron, rather than a metric distortion (Δ|Xax−Xeq|), which would shorten inter-atomic distances thus increasing repulsion between anions.

The mechanical behavior of carbonate-bearing fluorapatite (CFAP) (with up to 5.5 wt% CO3) was investigated at high pressure up to 7 GPa. The incorporation of carbonate in CFAP samples was investigated by FTIR spectroscopy. The chemical formulae and cell parameters are Ca4.90Fe0.04 (PO4)2.87 (CO3)0.13 F1.23 and a = 9.3527(1), c = 6.8752(1) A, V = 520.83(1) A3 for the FOW CFAP (Fowey Consols area, UK), and Ca4.97Sr0.03 (PO4)2.55 (CO3)0.45 F1.42 and a = 9.3330(1), c = 6.8984(1) A, V = 520.38(1) A3 for the FRA CFAP (Framont region, France). Preliminary characterization at ambient conditions was done by single-crystal X-ray diffraction study. The structure refinements, in space group P63/m, confirm a type-B substitution of the phosphate (PO4)3− group by the carbonate ion (CO3)2−. The site occupancies for the C atom are 0.04 for FOW and 0.11 for FRA CFAP, in quite good agreement with the 1.6 and 5.5 wt% CO3 amount obtained by analytical methods. Single-crystal high-pressure XRD study on the two type-B CFAP samples was performed. The FOW and FRA crystals were mounted concurrently in a ETH-type DAC and cell parameters were determined at 26 different pressures up to 6.86 GPa at room T. The variation with pressure of the unit-cell parameters and volume shows no discontinuity that could be related to any possible phase transition in the P range investigated. The linear compressibility coefficients are βa = 3.63 × 10−3 GPa−1 and βc = 2.47 × 10−3 GPa−1 for FOW, and βa = 3.67 × 10−3 GPa−1 and βc = 2.65 × 10−3 GPa−1 for FRA, giving an axial anisotropy of βa:βc = 1.47:1 and 1.38:1, respectively. The P-V data were fitted by a second-order Birch–Murnaghan EoS and the resulting BM2-EoS coefficients are V0 = 519.81(7) A3, KT0 = 92.1(3) GPa for FOW, and V0 = 518.95(9) A3, KT0 = 89.1(4) GPa for FRA CFAP. The results obtained indicate that a 5.5 wt% CO3 content (type-B) reduces the isothermal bulk modulus by about 9%.

ObjectiveCirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients’ susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients’ neutrophils and blood.DesignNeutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR.ResultsThe impaired superoxide production by patients’ neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91phox/NOX2 and p22phox), its cytosolic partner p47phoxbut not p67phox. NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients’ plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients’ neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients’ neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production.ConclusionImpairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.

Place: Oxford Publisher: Pergamon-Elsevier Science Ltd WOS:000551658300034; Phagocytes, especially neutrophils, can produce reactive oxygen species (ROS), through the activation of the NADPH oxidase (NOX2). Although this enzyme is crucial for host-pathogen defense, ROS production by neutrophils can be harmful in several pathologies such as cardiovascular diseases or chronic pulmonary diseases. The ROS production by NOX2 involves the assembly of the cytosolic subunits (p67(phox), p47(phox), and p40(phox)) and Rac with the membrane subunits (gp91(phox) and p22(phox)). Many studies are devoted to the activation of NOX2. However, the mechanisms that cause NADPH oxidase deactivation and thus terminate ROS production are not well known. Here we investigated the ability of class I phosphoinositide 3-kinases (PI3Ks) to sustain NADPH oxidase activation. The NADPH oxidase activation was triggered by seeding neutrophil-like PLB-985 cells, or human neutrophils on immobilized fibrinogen. Adhesion of the neutrophils, mediated by beta 2 integrins, induced activation of the NADPH oxidase and translocation of the cytosolic subunits at the plasma membrane. Inhibition of class I PI3Ks, and especially PI3K beta, terminated ROS production. This deactivation of NOX2 is due to the release of the cytosolic subunits, p67(phox) and p47(phox) from the plasma membrane. Overexpression of an active form of Rac 1 did not prevent the drop of ROS production upon inhibition of class I PI3Ks. Moreover, the phosphorylation of p47(phox) at S328, a potential target of kinases activated by the PI3K pathway, was unchanged. Our results indicate that the experimental downregulation of class I PI3K products triggers the plasma membrane NADPH oxidase deactivation. Release of p47(phox) from the plasma membrane may involve its PX domains that bind PI3K products.

Dust storms cause Mars to lose water Mars was once a wet planet, but it has lost most of its water through reactions that produce hydrogen, which escapes from the upper atmosphere into space. Stone et al. used data from the Mars Atmosphere and Volatile Evolution spacecraft to study how water is transported to the upper atmosphere and converted to hydrogen. They found that water can reach higher altitudes than previously thought, especially during global or regional dust storms. Photochemical modeling shows that this process dominates the current loss of water from Mars and influenced the evolution of its climate. Science , this issue p. 824

Eugenol is a polyphenol extracted from Syzygium aromaticum essential oil. It is known to have anti-inflammatory and chemoprotective properties as well as a potent anti-oxidant activity due the presence of its phenolic group. In this study, we examined the effects of eugenol on neutrophil superoxide production, a key process involved in innate immunity and inflammation. Superoxide anion generationin human neutrophils was measured by cytochrome c reduction assay. Western blotting was used to analyze the phosphorylation of, p47phox, MAPKinases (p38 and ERK1/2), MEK1/2 and Raf, key proteins involved in the activation of NADPH oxidase. Pretreatment of neutrophils by increasing concentrations (2.5 µg/mL–20 µg/mL) of eugenol for 30 min, inhibited significantly (p − production was affected only at the highest eugenol concentration (20 µg/mL). Results showed that eugenol decreased the phosphorylation of p47phox onSer-345 and Ser-328, the translocation of p47phox to the membranesand the phosphorylation of Raf, MEK1/2 and ERK1/2 proteins. Taken together, our results suggest that eugenol inhibits the generation of superoxide anion by neutrophils via the inhibition of Raf/MEK/ERK1/2/p47phox-phosphorylation pathway.

The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22phox and gp91phox (renamed NOX2), which form the cytochrome b558, and four cytosolic proteins, p47phox, p67phox, p40phox, and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91phox/NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47phox, p67phox, p40phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

The aim of the present study is to investigate the potential protective role of Ceratonia siliqua L. against dextran sodium sulfate (DSS)-induced oxidative damage and inflammation in liver and kidney of rats. The hepatotoxicity and nephrotoxicity were induced in rats by oral administration of synthetic DSS (5%) in the drinking water for over 7 days. However, carob pods aqueous extract (CPAE; 50 and 100 mg/kg body weight) was given by oral administration for 21 days. Myeloperoxidase (MPO) activity, malondialdehyde, H2O2 content, as well as the levels of antioxidant enzymes in organs were measured to observe the possible mechanisms. As a result, the CPAE counteracted DSS-induced increase of MPO activity, lipoperoxidation, and the activity of antioxidant enzymes, such as superoxide dismutase and catalase (CAT). DSS administration increased also in the organs hydrogen peroxide (H2O2) and free iron levels, whereas the CPAE pretreatment reversed all intracellular mediator perturbations. It was concluded that the CPAE exerted a potential protective effect against DSS-induced inflammation and oxidative stress in the rat organs. Consequently, it is essential that adequate care is taken when we use carob pods for patients with hepatotoxicity and nephrotoxicity.

Introduction Obese patients have an increased risk of COVID-19 critical illness leading to ICU admission or death compared to normal weight individuals. SARS-CoV-2 binding to angiotensin-converting enzyme 2 (ACE2) receptor is a critical step mediate virus entry into target cells. Articles have alluded that the level of ACE2 gene expression in adipose tissue is higher than lung tissue, but a PubMed search found no results in articles to demonstrate this. The aim of this study was to investigate ACE2 gene expression in adipose tissue and lung tissue using a public database. Material and Methods A search of a public gene expression database to investigate ACE2 gene expression in human tissues. Results ACE2 gene expression was present in both visceral and subcutaneous adipose tissues. The gene expression profile demonstrated that ACE2 gene expression was higher in human visceral and subcutaneous adipose tissues than human lung tissue. Conclusion This study demonstrates that ACE2 gene expression is higher in visceral and subcutaneous adipose tissue than that in lung tissue, a major target tissue affected by SARS-CoV-2 infection. This suggests a mechanism by which excess adiposity may drive greater infection severity in patients with COVID-19., Highlights • SARS-CoV-2 binding to angiotensin-converting enzyme 2 (ACE2) receptor is a critical step mediating virus entry into target cells. • Articles have alluded that the level of ACE2 gene expression in adipose tissue is higher than lung tissue but a PubMed search found no article to demonstrate this. • The study demonstrates that ACE2 gene expression is higher in human visceral and subcutaneous adipose tissues than human lung tissue. • This critical discovery implies that adipose tissue is susceptible to SARS-Cov-2 infection via the ACE2 receptor.

The preparation of a new drug delivery system (DDS) based on the combination of two natural polymers chitosan (CS) and high-esterified pectin (HEP) and beidellite (Bd) was proposed. Diclofenac sodium was chosen as model drug and was loaded in the nanocomposite by means of intercalation solution technique. The samples were characterized by X-ray diffraction, Thermal analysis, Infrared spectroscopy and high resolution transmission electron microscopy coupled with energy dispersive X-Ray analysis. Drug loading capacity and encapsulation efficiency of the clay polymer nanocomposite was quantified. UV–Vis spectroscopy (276 nm) was used as the drug quantification technique throughout the whole study. In vitro drug release profile in simulated intestinal fluid was also evaluated. Results demonstrated the formation of diclofenac polymorph during the drug loading process, thus obtaining a new crystal form of the drug possessing different physico-chemical properties with respect to the diclofenac sodium salt form. Moreover, the in vitro release study showed that the bionanocomposite elaborated could form a network aiming for a modified release of diclofenac. Consequently, Na-Bd/CS/HEP nanocomposite proved to be a promising formulation to control anionic drug release process., This work was supported by a grant from the Tunisian Ministry of Higher Education and Scientific Research. The authors thank the Andalusian group CTS-946 and the CGL2016-80833-R project for providing necessary facilities and financial support for the characterization of the nanocomposites.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

Nanostructures can strongly interact with cells or other biological structures; furthermore when they are functionalized with targeting units, they are of great interest for a variety of applications in the biotechnology field like those for efficient imaging, diagnosis and therapy and in particular for cancer theranostics. Obtaining targeting with good specificity and sensitivity is a key necessity, which, however, is affected by the complexity of the interactions between the nanostructures and the biological components. In this work we report the study of specificity and sensitivity of gold nanoparticles functionalized with the peptide GE11 for the targeting of the epidermal growth factor receptor, expressed on many cells and, in particular, on many types of cancer cells. We show how a combination of spectroscopic measurements and molecular dynamics simulations allows the comprehension of the targeting activity of peptides linked to the surface of gold nanostructures and how the targeting is tuned by the presence of polyethylene glycol chains.

NMDA-type glutamate receptors (NMDAR) trigger superoxide production by neuronal NADPH oxidase-2 (NOX2), which if sustained leads to cell death. This process involves Ca2+ influx through NMDAR channels. By contrast, comparable Ca2+ influx by other routes does not induce NOX2 activation or cell death. This contrast has been attributed to site-specific effects of Ca2+ flux through NMDAR. Here we show instead that it stems from non-ionotropic signaling by NMDAR GluN2B subunits. To evaluate non-ionotropic effects, mouse cortical neurons were treated with NMDA together with 7-chlorokynurenate, L-689,560, or MK-801, which block Ca2+ influx through NMDAR channels but not NMDA binding. NMDA-induced superoxide formation was prevented by the channel blockers, restored by concurrent Ca2+ influx through ionomycin or voltage-gated calcium channels, and not induced by the Ca2+ influx in the absence of NMDAR ligand binding. Neurons expressing either GluN2B subunits or chimeric GluN2A/GluN2B C-terminus subunits exhibited NMDA-induced superoxide production, whereas neurons expressing chimeric GluN2B/GluN2A C-terminus subunits did not. Neuronal NOX2 activation requires phosphoinositide 3-kinase (PI3K), and NMDA binding to NMDAR increased PI3K association with NMDA GluN2B subunits independent of Ca2+ influx. These findings identify a non-ionotropic signaling pathway that links NMDAR to NOX2 activation through the C-terminus domain of GluN2B.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47(PHOX), a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91(PHOX) and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47(PHOX) phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential.

In pathological situations such as ischemia-reperfusion and acute respiratory distress syndrome, reactive oxygen species (ROS) are produced by different systems which are involved in endothelial cells injury, ultimately leading to severe organ dysfunctions. The aim of this work was to study the effect of ROS produced by hypoxanthine-xanthine oxidase (Hx-XO) on the adhesion of human umbilical vein endothelial cells (HUVEC) and on the signaling pathways involved. Results show that Hx-XO-derived ROS induced an increase in HUVEC adhesion in the early stages of the process (less than 30 min), followed by a decrease in adhesion in the later stages of the process. Interestingly, Hx-XO-derived ROS induced the same biphasic effect on the phosphorylation of the focal adhesion kinase (FAK), a nonreceptor tyrosine kinase critical for cell adhesion, but not on ERK1/2 phosphorylation. The biphasic effect was not seen with ERK1/2 where a decrease in phosphorylation only was observed. Wortmannin, a PI3-kinase inhibitor, inhibited ROS-induced cell adhesion and FAK phosphorylation. Orthovanadate, a protein tyrosine phosphatase inhibitor, and Resveratrol (Resv), an antioxidant agent, protected FAK and ERK1/2 from dephosphorylation and HUVEC from ROS-induced loss of adhesion. This study shows that ROS could have both stimulatory and inhibitory effects on HUVEC adhesion and FAK phosphorylation and suggests that PI3-kinase and tyrosine phosphatase control these effects.

Inflammation and oxidative stress are a common mechanism of many gastrointestinal diseases such ulcerative colitis. Polyphenols are micronutrients with antioxidant and anti-inflammatory properties and may play an interesting role in the prevention of intestinal inflammation. In this context, we examined the protective effect of carob pods aqueous extract (CPAE) against dextran sulfate sodium (DSS)-induced sub-acute experimental ulcerative colitis in a rat model. Colon inflammation was induced in rats by oral administration of synthetic DSS (5%) in the drinking water over 7 days. However, CPAE (50 and 100 mg kg−1 body weight) was given by oral administration for 21 days. At the end of the experimental period, the colon and plasma were examined for lipid peroxidation and cytokine determination as well as myeloperoxidase (MPO) and antioxidant enzyme activities. Our result indicated that DSS caused severe histopathological damage in the colon mucosa. The lesions were associated with increased MPO activity, cytokines and oxidative damage. The CPAE treatment restored dose-dependent body weight gain, prevented colonic shortening and reduced the severity of colonic lesions and biochemical alterations. It was concluded that CPAE consumption limits DSS-induced colonic damage in rats. The antioxidant and anti-inflammatory properties of polyphenols may play a role in this effect.

The current study was performed to assess the effects of carob pod aqueous extracts (CPAE, pulp, seeds or mixture) on gastrointestinal transit (GIT) and intestinal epithelium permeability. In this aspect, male rats fasted for 16 hours were used and given by oral administration NaCl (0.9%, control group) or various doses of CPAE (50, 100 and 200 mg kg−1, b.w.). Two other groups of rats (batch tests) received clonidine (an alpha-2 adrenergic agonist, 1 mg kg−1) or yohimbine (an alpha-2 adrenergic antagonist, 2 mg kg−1). Two hours later, all animals were given a test meal containing charcoal and gum arabic in water. 30 minutes later, rats were anesthetized, a laparotomy was performed and the distance traveled by the meal compared to the total length of the small intestine was measured. Regarding the effect of CPAE on diarrhoea, the extract was administered orally to three groups of rats (ten in each group). Two other groups received normal saline (10 mL kg−1) and loperamide (10 mg kg−1) as a negative and standard group. Compared with the control group, the animals treated with the CPAE of pulp, seeds or a mixture (50% pulp and 50% seeds) of mature carob, showed a significant increase (3–25%) of GIT in a dose-dependent manner. By contrast, the CPAE of immature carob pods significantly and dose dependently decreased (3–19%) the GIT and diarrhoea (66–87%). However, clonidine and yohimbine respectively decreased (58%) and increased (30%) the GIT. More importantly, using the Ussing chamber system, we found that aqueous extracts of mature and immature carob pods significantly and dose-dependently increased or decreased intestinal epithelium permeability. The results indicate that carob possesses significant laxative and anti-diarrheal activities due to its opposite effects both on gastrointestinal propulsion and permeability. These findings confirm that the degree of maturity of carob characterized by a different phytochemical composition may be responsible for these actions.

Chronic inflammation and oxidative stress are induced by biological, chemical and physical factors which are, in turn, associated with an increased risk of several human diseases. Plants are a large source of new bioactive molecules with therapeutic potential. In this respect, the present study was designed to investigate the effects of a Ceratonia siliqua L. leaf aqueous extract (CSLAE) on human neutrophil reactive oxygen species (ROS) production, in vitro myeloperoxidase (MPO) activity and expression as well as the small intestinal fluid and electrolyte secretion. Neutrophils were isolated from whole human blood of healthy volunteers using the ficoll–dextran method and ROS generation and H2O2 were measured by luminol amplified chemiluminescence. Superoxide anion generation was also detected by chemiluminescence using the lucigenin method. MPO activity and quantity were measured by the tetramethylbenzidine oxidation method and Western blotting analysis. Concerning the in vivo part, fasted male rats received by gavage either the vehicle (NaCl, 0.9%), the extract at various doses (50, 100 and 200 mg kg−1) or clonidine (1 mg kg−1). An activated charcoal suspension was administered by oral gavage. Thirty minutes after receiving the charcoal meal, rats were euthanized and the small intestine was removed. The length of the small intestine and the distance traveled by the charcoal were recorded. Castor oil-induced hypersecretion in Wistar rats was treated with administration of CSLAE (50, 100 and 200 mg kg−1) and antidiarrheal drug, Atropine (0.1 mg kg−1 i.p.). CSLAE inhibited luminol-amplified chemiluminescence of (PMA)-stimulated neutrophils in a concentration-dependent manner and is able to scavenge superoxide anions and hydrogen peroxide. The CSLAE also reduces significantly and dose-dependently MPO activity and expression. On the other hand, in vivo studies showed that the CSLAE decreased notably and dose-dependently the GIT activity, intestinal fluid and electrolyte concentration. The chemical analysis using a HPLC technique showed that the CSLAE is rich in phenolic compounds, especially kaempferol, tannic acid and catechin hydrate. The intended neutrophil inhibition is introduced as a part of a new strategy for pharmacological modulation of chronic inflammatory and oxidative stress processes. On the basis of these findings, it can be also assumed that CSLAE could be a potential source for novel discovery for antidiarrhoeal drug development.

The aim of the present work was to investigate the mechanism implicated in the protective effects of Myrtus communis leaves essential oils (MCEO) on human neutrophils reactive oxygen species (ROS) production. We also studied its preventive effect against intestinal ischemia reperfusion (IIR)-induced oxidative stress in rat model. Essential oils were obtained from the plant leaves by hydrodistillation and analyzed by GC-MS. Neutrophils were isolated from whole human blood using ficoll–dextran method. ROS generation and H2O2 production were measured by luminol-amplified chemiluminescence. The cytochrome c reduction assay was used for superoxide anion determination and Western blotting analysis was to determine the neutrophils myeloperoxidase (MPO) expression. Rats were divided into four groups: control (C), intestinal IR (IIR), MCEO, and MCEO plus IIR. Animals were pretreated with MCEO (50 mg kg−1) during 7 days. IIR was produced by 75 min of intestinal ischemia followed by reperfusion for 120 min. The GC-MS analysis, allowed to the identification of twenty five bioactive compounds in MCEO. In vitro, we found that MCEO inhibited ROS and H2O2 production and attenuated the neutrophils MPO expression. In vivo, the MCEO administration counteracted IIR-induced small intestine, lung and liver lipid peroxidation as well as the depletion of antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). MCEO pretreatment also corrected IIR-induced non-enzymatic antioxidants levels depletion such as sulfhydryl groups (–SH) and reduced glutathione (GSH). More importantly, IIR was accompanied by H2O2, free iron and calcium increase while MCEO administration reversed all intracellular mediator perturbations. In conclusion, we suggest that MCEO had a potential protective role against intestinal IR injury, in part owing to its antioxidant potential and ROS scavenging activities.

Poly(glycidyl methacrylate) (PGMA)/clay nanocomposites were prepared by free radical in situ photopolymerization using intercalated chain transfer agent (3-mercaptopropyl)trimethoxysilane (MPS) in the layers of bentonite (B) clay. The natural bentonite was treated with MPS (at 1–5% v/v) which acts as both intercalant and chain transfer agent. Glycidyl methacrylate (GMA) was photopolymerized in situ in the presence of the silanized bentonite (B-MPS) using 2,2-azobisisobutyronitrile (AIBN). The intercalation ability of MPS and exfoliated nanocomposite structure were evidenced by both X-ray diffraction spectroscopy (XRD) and transmission electron microscopy (TEM). Surface analysis and morphologies of the resultant nanocomposites were also studied. The mass loading of PGMA reached 81 wt.% while XPS spectra, particularly the high resolution C1s region, resemble those of pure PGMA. The primary B-MPS/PGMA nanocomposite was then used in the preparation of nanocomposite coatings by mixing it with di-glycidyl ether of bisphenol A (DGEBA) epoxy matrix and 4,4-diaminodiphenyl sulfone (DDS) hardener in order to obtain ternary Epoxy/B-MPS/PGMA. The latter was cured and then was compressed into 5 mm thick sheets; it was found to be transparent with mixed intercalated/exfoliated structure. The thermal and mechanical performances of Epoxy/B-MPS/PGMA are by far superior to those of the reference epoxy–pristine bentonite nanocomposite.

Citrillus colocynthis peel aqueous extract (CCPAE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. The objectives of this work were to study the effect of C. colocynthis peel aqueous extract, on human neutrophil reactive oxygen species (ROS) production in vitro, and to evaluate its protective effect on lipopolysaccharide (LPS)-induced lung inflammation in vivo in mice. Neutrophils were isolated from blood of healthy volunteers. ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H2O2 was detected by horseradish peroxidase (HRP)-amplified chemiluminescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethylbenzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. CCPAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbolmyristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. CCPAE also inhibited superoxide anion generation; and did not scavenge H2O2 and superoxide anions nor inhibited MPO activity in vitro suggesting that it inhibits nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. In vivo studies showed that CCPAE attenuated LPS-induced lung inflammation in mice. This study shows that CCPAE inhibits neutrophil ROS production and attenuates LPS-induced lung inflammation in mice. Inhibition of NADPH oxidase activation by CCPAE could explain its anti-inflammatory action. Key words: Citrullus colocynthis, colocynth, inflammation, neutrophils, reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.