Renal and cardiac oxidative/nitrosative stress in salt-loaded pregnant rat

Sodium supplementation given for 1 wk to nonpregnant rats induces changes that are adequate to maintain renal and circulatory homeostasis as well as arterial blood pressure. However, in pregnant rats, proteinuria, fetal growth restriction, and placental oxidative stress are observed. Moreover, the decrease in blood pressure and expansion of circulatory volume, normally associated with pregnancy, are prevented by high-sodium intake. We hypothesized that, in these pregnant rats, a loss of the balance between prooxidation and antioxidation, particularly in kidneys and heart, disturbs the normal course of pregnancy and leads to manifestations such as gestational hypertension. We thus investigated the presence of oxidative/nitrosative stress in heart and kidneys following high-sodium intake in pregnant rats. Markers of this stress [8-isoprostaglandin F(2alpha) (8-iso-PGF(2alpha)) and nitrotyrosine], producer of nitric oxide [nitric oxide synthases (NOSs)], and antioxidants [superoxide dismutase (SOD) and catalase] were measured. Then, molecules (Na(+)-K(+)-ATPase and aconitase) or process [apoptosis (Bax and Bcl-2), inflammation (monocyte chemoattractant protein-1, connective tissue growth factor, and TNF-alpha)] susceptible to free radicals was determined. In kidneys from pregnant rats on 1.8% NaCl-water, NOSs, apoptotic index, and nitrotyrosine expression were increased, whereas Na(+)-K(+)-ATPase mRNA and activity were decreased. In the left cardiac ventricle of these rats, heightened nitrotyrosine, 8-iso-PGF(2alpha), and catalase activity together with reduced endothelial NOS protein expression and SOD and aconitase activities were observed. These findings suggest that oxidative/nitrosative stress in kidney and left cardiac ventricle destabilizes the normal course of pregnancy and could lead to gestational hypertension.     

Aminoguanidine and N-acetyl-cysteine supress oxidative and nitrosative stress in EAE rat brains

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). We have evaluated the role of oxidative and nitrosative stress, as the causal factors in the development of EAE, responsible for the damage of cardinal cellular components, such as lipids, proteins and nucleic acids, resulting in demyelination, axonal damage, and neuronal death. EAE was induced in female Sprague-Dawley rats, 3 months old (300 ± 20 g), by immunization with myelin basic protein in combination with Complete Freund's adjuvant (CFA). The animals were divided into seven groups: control, EAE, CFA, EAE + aminoguanidine (AG), AG, EAE + N-acetyl-l-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the levels of nitrosative and oxidative stress were determined in 10% homogenate of the whole encephalitic mass. In EAE rats, brain NO production and MDA level were significantly increased (P < 0.001) compared to the control values, whereas AG and NAC treatment decreased both parameters in EAE rats compared to EAE group (P < 0.001). Glutathione (GSH) was reduced (P < 0.001) in EAE rats in comparison with the control and CFA groups, but increased in EAE + AG and EAE + NAC group compared to the EAE group (P < 0.01). Superoxide dismutase (SOD) activity was significantly decreased (P < 0.001) in the EAE group compared to all other experimental groups. The clinical expression of EAE was significantly decreased (P < 0.05) in the EAE groups treated with AG and NAC compared to EAE rats, during disease development.

The obtained results prove an important role of oxidative and nitrosative stress in the pathogenesis of EAE, whereas AG and NAC protective effects offer new possibilities for a modified combined approach in MS therapy.     

UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in mouse myotubes

The deleterious effects of oxidants on proteins may be modified by overexpression of uncoupling protein 3 (UCP3) in skeletal muscle cells exposed to hyperoxia or H₂O₂. UCP3 overexpression significantly attenuated the increase in protein carbonylation in response to hyperoxia and H₂O₂ exposures. However, antioxidant enzyme content and activity (superoxide dismutases, peroxiredoxins, glutathione peroxidase-I, and catalase) were reduced or not modified in UCP3-overexpressing myotubes exposed to oxidants. Protein nitration increased in UCP3-overexpressing cells exposed to hyperoxia, but not to H₂O₂. We conclude that protein oxidation rather than nitration is neutralized by UPC3 overexpression in mouse myotubes exposed to abundant reactive oxygen species.     

Physiology and pathophysiology of nitrosative and oxidative stress in osteoarthritic joint destruction

Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synovial tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.     

Aminoguanidine and N-acetyl-cysteine supress oxidative and nitrosative stress in EAE rat brains

Experimental autoimmune encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). We have evaluated the role of oxidative and nitrosative stress, as the causal factors in the development of EAE, responsible for the damage of cardinal cellular components, such as lipids, proteins and nucleic acids, resulting in demyelination, axonal damage, and neuronal death. EAE was induced in female Sprague-Dawley rats, 3 months old (300±20 g), by immunization with myelin basic protein in combination with Complete Freund's adjuvant (CFA). The animals were divided into seven groups: control, EAE, CFA, EAE+aminoguanidine (AG), AG, EAE+N-acetyl-L-cysteine (NAC) and NAC. The animals were sacrificed 15 days after EAE induction, and the levels of nitrosative and oxidative stress were determined in 10% homogenate of the whole encephalitic mass. In EAE rats, brain NO production and MDA level were significantly increased (P<0.001) compared to the control values, whereas AG and NAC treatment decreased both parameters in EAE rats compared to EAE group (P<0.001). Glutathione (GSH) was reduced (P<0.001) in EAE rats in comparison with the control and CFA groups, but increased in EAE+AG and EAE+NAC group compared to the EAE group (P<0.01). Superoxide dismutase (SOD) activity was significantly decreased (P<0.001) in the EAE group compared to all other experimental groups. The clinical expression of EAE was significantly decreased (P<0.05) in the EAE groups treated with AG and NAC compared to EAE rats, during disease development. The obtained results prove an important role of oxidative and nitrosative stress in the pathogenesis of EAE, whereas AG and NAC protective effects offer new possibilities for a modified combined approach in MS therapy.     

A discussion of the chemistry of oxidative and nitrosative stress in cytotoxicity

Nitric oxide (NO) has been shown to be a key bioregulatory agent in a wide variety of biological processes, yet cytotoxic properties have been reported as well. This dichotomy has raised the question of how this potentially toxic species can be involved in so many fundamental physiological processes. We have investigated the effects of NO on a variety of toxic agents and correlated how its chemistry might pertain to the observed biology. The results generate a scheme termed the chemical biology of NO in which the pertinent reactions can be categorized into direct and indirect effects. The former involves the direct reaction of NO with its biological targets generally at low fluxes of NO. Indirect effects are reactions mediated by reactive nitrogen oxide species, such as those generated from the NO/O2 and NO/O2- reactions, which can lead to cellular damage via nitrosation or oxidation of biological components. This report discusses several examples of cytotoxicity involved with these stresses.     

Differential susceptibilities of serine/threonine phosphatases to oxidative and nitrosative stress

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are signal-transducing molecules that regulate the activities of a variety of proteins. In the present investigation, we have compared the effects of superoxide (O2-), nitric oxide (NO), and hydrogen peroxide (H2O2) on the activities of three highly homologous serine/threonine phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), and calcineurin (protein phosphatase type 2B). Although superoxide, generated from xanthine/xanthine oxidase or paraquat, and NO, generated from (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide or sodium nitroprusside, potently inhibited the phosphatase activity of calcineurin in neuroblastoma cell lysates, they had relatively little effect on the activities of PP1 or PP2A. In contrast, H2O2 inhibited the activities of all three phosphatases in lysates but was not a potent inhibitor for any of the enzymes. Calcineurin inactivated by O2-, NO, and H2O2 could be partially reactivated by the reducing agent ascorbate or by the thiol-specific reagent dithiothreitol (DTT). Maximal reactivation was achieved by the addition of both reagents, which suggests that ROS and RNS inhibit calcineurin by oxidizing both a catalytic metal(s) and a critical thiol(s). Reactivation of H2O2-treated PP1 also required the combination of both ascorbate and DTT, whereas PP2A required only DTT for reactivation. These results suggest that, despite their highly homologous structures, calcineurin is the only major Ser/Thr phosphatase that is a sensitive target for inhibition by superoxide and nitric oxide and that none of the phosphatases are sensitive to inhibition by hydrogen peroxide.     

Mitochondrial response to oxidative and nitrosative stress in early stages of diabetes

Diabetes mellitus (DM) is associated with increased production of reactive oxygen and nitrogen species; consequently, an increase in lipid peroxidation and a decrease in antioxidants resulting in mitochondrial dysfunction. Using a rat model of DM induced by streptozotocin, we show the opposite: an increase in NO levels, S-nitrosylation, aconitase activity, and total glutathione and a decrease in lipid peroxidation at early stages of diabetes. These data imply that the decrease in lipid peroxidation is a vital early response to hyperglycemia to prevent escalation of ROS generation in mitochondria. These results also suggest a need for novel therapeutic targets to prevent the neurological consequences of diabetes.     

Reducing oxidative/nitrosative stress: a newly-discovered genre for melatonin

The discovery of melatonin and its derivatives as antioxidants has stimulated a very large number of studies which have, virtually uniformly, documented the ability of these molecules to detoxify harmful reactants and reduce molecular damage. These observations have clear clinical implications given that numerous age-related diseases in humans have an important free radical component. Moreover, a major theory to explain the processes of aging invokes radicals and their derivatives as causative agents. These conditions, coupled with the loss of melatonin as organisms age, suggest that some diseases and some aspects of aging may be aggravated by the diminished melatonin levels in advanced age. Another corollary of this is that the administration of melatonin, which has an uncommonly low toxicity profile, could theoretically defer the progression of some diseases and possibly forestall signs of aging. Certainly, research in the next decade will help to define the role of melatonin in age-related diseases and in determining successful aging. While increasing life span will not necessarily be a goal of these investigative efforts, improving health and the quality of life in the aged should be an aim of this research.     

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

Cytochrome bd is a prokaryotic respiratory quinol:O₂ oxidoreductase, phylogenetically unrelated to the extensively studied heme–copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O₂, NO and H₂O₂ is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Local oxidative and nitrosative stress increases in the microcirculation during leukocytes-endothelial cell interactions

Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O(2) (?-)) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O(2)(?-) and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O(2)(?-) and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O(2)(?-) and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ~0.6 fold, O(2)(?-) increased ~27 fold and peroxynitrite increased ~30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase in O(2)(?-) and peroxynitrite concentration in the lumen. The increased O(2) (?-) and peroxynitrite can cause leukocytes priming through peroxynitrite and leukocytes activation through secondary stimuli of O(2)(?-) in bloodstream without endothelial interaction. This finding supports that leukocyte rolling/adhesion and activation are independent events.     

Gamma-glutamylcysteine protects ergothioneine-deficient Mycobacterium tuberculosis mutants against oxidative and nitrosative stress

Mycobacterium tuberculosis (M.tb.), the causative agent of tuberculosis (TB), cannot synthesize GSH, but synthesizes two major low molecular weight thiols namely mycothiol (MSH) and ergothioneine (ERG). Gamma-glutamylcysteine (GGC), an intermediate in GSH synthesis, has been implicated in the protection of lactic acid bacteria from oxidative stress in the absence of GSH. In mycobacteria, GGC is an intermediate in ERG biosynthesis, and its formation is catalysed by EgtA (GshA). GGC is subsequently used by EgtB in the formation of hercynine-sulphoxide-GGC. In this study, M.tb. mutants harbouring unmarked, in-frame deletions in each of the fives genes involved in ERG biosynthesis (egtA, egtB, egtC, egtD and egtE) or a marked deletion of the mshA gene (required for MSH biosynthesis) were generated. Liquid chromatography tandem mass spectrometry analyses (LC-MS) revealed that the production of GGC was elevated in the MSH-deficient and the ERG-deficient mutants. The ERG-deficient ΔegtB mutant which accumulated GGC was more resistant to oxidative and nitrosative stress than the ERG-deficient, GGC-deficient ΔegtA mutant. This implicates GGC in the detoxification of reactive oxygen and nitrogen species in M.tb.     

Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats

Reactive oxygen and nitrogen species (ROS and RNS) have been proposed as mechanisms of cancer-induced cachexia. In this study, we assessed using Western blot analysis the levels of total protein carbonylation (2,4-dinitrophenylhydrazine assay), both malondialdehyde- (MDA-) and 2-hydroxy-4-nonenal- (HNE-) protein adducts, Mn-superoxide dismutase (Mn-SOD), catalase, heme oxygenase-1 (HO-1) and 3-nitrotyrosine formation in gastrocnemius muscles of rats bearing the Yoshida AH-130 hepatoma. In the muscles of the tumour-bearing animals, protein carbonylation as measured by total levels of carbonyl group formation and both HNE and MDA-protein adducts, and protein tyrosine nitration were significantly greater than in control muscles. Protein levels of the antioxidant enzymes Mn-SOD, catalase, and HO-1 were not significantly modified in the rat cachectic muscles compared to controls. The inefficiency of the antioxidant enzymes in neutralizing excessive ROS production may account for elevated markers of protein oxidation and be responsible for the development of both oxidative and nitrosative stress in cancer-induced cachexia.     

Effects of oxidative and nitrosative stress on Tetrahymena pyriformis glyceraldehyde-3-phosphate dehydrogenase

Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H(2)O(2) and NO, mainly due to a decrease in its V(max) without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H(2)O(2)-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.     

Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

Exposure of mitochondria to oxidative stress and elevated Ca²⁺ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 h later. Respiring mitochondria actively accumulated added Ca²⁺, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca²⁺ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.     

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.     

Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli

The Vitreoscilla hemoglobin gene (vhb) is expressed under oxygen-limited conditions via an FNR-dependent mechanism. Furthermore, cAMP-CRP has been implicated in its regulation. Recently, VHb protein has been reported to protect a heterologous host from nitrosative stress. In this study we analyzed the regulation of the Vitreoscilla hemoglobin promoter (Pvhb) in Escherichia coli under nitrosative and oxidative stress conditions. Our results show unambiguously that expression of neither VHb nor chloramphenicol acetyltransferase under the control of Pvhb is induced under the experimental conditions used. Thus, a clear discrepancy between in vivo function, i.e. protection against nitrosative stress, and regulation of gene expression is obvious. The regulation of Pvhb reported here is in clear contrast to the expression pattern of flavohemoglobins from various microorganisms, which are generally induced by nitrosative stress. However, the length of Pvhb is only 146 bp and therefore, we cannot rule out that additional regulatory sequences may be located in the upstream region of Pvhb.     

Selected contribution: Hyperthermia-induced intestinal permeability and the role of oxidative and nitrosative stress.

The purpose of this study was to characterize intestinal permeability changes over a range of physiologically relevant body temperatures in vivo and in vitro. Initially, FITC-dextran (4,000 Da), a large fluorescent molecule, was loaded into the small intestine of anesthetized rats. The rats were then maintained at approximately 37 degrees C or heated over 90 min to a core body temperature of approximately 41, approximately 41.5, or approximately 42.5 degrees C. Permeability was greater in the 42.5 degrees C group compared with the 37, 41, or 41.5 degrees C groups. Histological analysis revealed intestinal epithelial damage in heated groups. Everted intestinal sacs were then used to further characterize hyperthermia-induced intestinal permeability and to study the potential role of oxidative and nitrosative stress. Increased permeability to 4,000-Da FITC-dextran in both small intestinal and colonic sacs was observed at a temperature of 41.5-42 degrees C compared with 37 degrees C, along with widespread intestinal epithelial damage. Administration of antioxidant enzyme mimics or a nitric oxide synthase inhibitor did not reduce permeability due to heat stress, and tissue concentrations of a lipid peroxidation product were not altered by heat stress, suggesting that oxidative and nitrosative stress were not likely mediators of this phenomenon in vitro. In conclusion, hyperthermia produced increased permeability and marked intestinal epithelial damage both in vivo and in vitro, suggesting that thermal disruption of epithelial membranes contributes to the intestinal barrier dysfunction manifested with heat stress.     

alpha-Glycerylphosphorylethanolamine rescues astrocytes from mitochondrial impairment and oxidative stress induced by amyloid beta-peptides

The present work shows that alpha-glycerylphosphorylethanolamine (alpha-GPE) is effective in recovering astrocytes from mitochondrial membrane integrity and potential derangement and cellular oxidative stress that occur under amyloid beta-peptides-induced reactive gliosis.alpha-Glycerylphosphorylethanolamine (alpha-GPE), a new compound with nootropic properties, known to improve in vivo the learning and memory processes, has been tested for its protective properties on an in vitro model of degeneration. Rat primary astrocytic cultures treated with two amyloid-derived peptides, Abeta((1-40)) and Abeta(3(pE)-42), showed a marked reduction of the mitochondrial redox activity and membrane potential, together with an increase of oxidative species production. Plasma membrane lipid peroxidation (LPO) as well as generation of peroxides is greatly increased under Abeta-peptides toxicity. These features, typical of the reactive gliosis that accompanies neuronal degeneration, were readily recovered by pretreatment with alpha-GPE. alpha-GPE, likely improving the fluidity of cell membrane, has the potential to recover astrocytes from the general redox derangement induced by different amyloid fragments and possibly to protect from inflammation, gliosis and neurodegeneration. This is the first evidence of an antioxidant effect of the ethanolamine derivative on a rat model of chronic gliosis.