Antidepressant-like effects of agmatine and NOS inhibitors in chronic unpredictable mild stress model of depression in rats: The involvement of NLRP inflammasomes.

Innate immunity activation in the central nervous system (CNS) is known to contribute to the development of depression through NOD-like receptors containing pyrin domain 3 (NLRP3) inflammasome assembly. Furthermore, administration of agmatine (AGM), a nitric oxide synthase (NOS) inhibitor, reverses stress-induced NLRP3 inflammasome activation in rats. We examined the effects of chronically-administered nitric oxide (NO) pathway modulating drugs on NLRP1/3-mediated neuroinflammatory responses and depressive-like behaviors in chronic unpredictable mild stress (CUMS) depression model of rats. CUMS model was applied to the adult male Sprague-Dawley rats for 6 weeks and the treatments were daily administered via intraperitoneal route in the last 3 weeks of CUMS procedure. Depressive-like behaviors were assessed by sucrose preference and forced swimming tests. The levels of NLRP inflammasome components (NLRP1, NLRP3, ASC, caspase-1 and IL-1ß) were investigated in the prefrontal cortex by real time PCR and western blot methods. CUMS-induced depressive-like behaviors were coupled with the overactivation of NLRP1 and NLRP3 inflammasome sensors and increased levels of IL-1ß. Depressive-like behaviors were ameliorated by chronic AGM and NOS inhibitor treatments. AGM and other NOS inhibitor treatments were found to be more effective in suppressing NLRP3 and NLRP1, respectively. All inhibitor reagents downregulated inflammasome components and IL-1ß. These results suggest that both neuronal NLRP1 and microglial NLRP3 inflammasomes are involved in chronic stress-induced depressive-like behaviors. The antidepressant effects of AGM, iNOS and nNOS inhibitors are associated with the downregulation of CNS inflammasome expression levels. NO-pathway modulating drugs might provide novel therapeutic strategies for depression.

Effect of betaine treatment on the regression of existing hepatic triglyceride accumulation and oxidative stress in rats fed on high fructose diet.

We investigated whether betaine has any regressive effect on existing high fructose diet (HFrD)-induced insulin resistance, dyslipidemia, inflammation as well as hepatic steatosis and oxidative stress. Rats were fed a HFrD containing 60% fructose for 8 weeks. After 8 weeks, rats were divided into two groups and fed a control diet for an additional 4-week period (regression groups). One of the regression groups received drinking water containing betaine (1%; w/v), having antioxidant and anti-inflammatory actions. HFrD feeding caused insulin resistance, elevated triglyceride (TG) and tumor necrosis factor-alfa (TNF-α) levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) activities in serum. This diet increased hepatic TG, thiobarbituric acid reactive substances (TBARS) and diene conjugate (DC) levels, decreased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. Marked macro-vesicular steatosis were detected. Serum TNF-α and ALT, hepatic TG, TBARS and DC levels and steatosis scores decreased in regression period of HFrD-fed rats. Additionally, serum TNF-α, hepatic TG, TBARS and DC levels significantly lower in betaine-treated regressed rats than non-treated regressed group. Our results indicate that betaine treatment may accelerate regression of HFrD-induced hepatic TG accumulation and oxidative stress in rats.

Reduced Peripheral Blood Mononuclear Cell ROCK1 and ROCK2 Levels in Obstructive Sleep Apnea Syndrome.

BACKGROUND/AIM: Obstructive sleep apnea syndrome (OSAS) is associated with intermittent episodes of hypoxia, endothelial dysfunction and associated cardiovascular problems. Our aim was to investigate whether OSAS-related hypoxia alters the expression of rho-associated protein kinase (ROCK), a marker of chronic hypoxia and endothelial dysfunction. MATERIALS AND METHODS: ROCK1 and ROCK2 levels were measured by immunoblotting in peripheral blood mononuclear cells (PBMC) of 47 OSAS patients and 17 healthy controls. RESULTS: OSAS patients showed significantly lower PBMC ROCK1 and ROCK2 levels than healthy controls in the morning, but not in the evening. ROCK1/2 levels were correlated with blood triglyceride, visceral adiposity index, minimum oxygen saturation, C-reactive protein concentration, lymphocyte levels and sleep efficiency. CONCLUSION: Intermittent hypoxia induced by OSAS does not permanently alter ROCK protein expression levels. OSAS appears to be associated with endothelial dysfunction through inflammation and lipid metabolism pathways.

Impact of Neuro-Behçet Disease Immunoglobulin G on Neuronal Apoptosis.

INTRODUCTION: Parenchymal neuro-Behçet disease (NBD) is encountered in 5%-15% of Behçet disease (BD) patients and is characterized by inflammation of the brainstem and diencephalon structures. Neuronal apoptosis has been shown to participate in neuronal cell loss. Anti-neuronal antibodies have been identified in NBD patients. However, the pathogenic properties of these antibodies have not been studied. METHODS: To delineate the potential pathogenic activity of serum antibodies on neurons, pooled sera from seven NBD patients and seven healthy controls were divided into purified immunoglobulin G (IgG) and IgG-depleted serum fractions, and each fraction was administered to cultured SH-SY5Y neuroblastoma cells. Cell death was evaluated with a toxicity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Moreover, expression levels of several apoptosis markers were evaluated with real time polymerase chain reaction (PCR). RESULTS: Administration of NBD IgG to cultured SH-SY5Y cells induced significantly increased cell death and apoptosis compared with other treatments. NBD IgG also enhanced the mRNA expression levels of major apoptosis and cell survival pathway factors. CONCLUSION: Our results suggest that IgGs isolated from the sera of NBD patients have a neurotoxic activity that is presumably mediated by apoptotic mechanisms.

Elevated IL-4 and IFN-γ Levels in Muscle Tissue of Patients with Dermatomyositis.

BACKGROUND/AIM: To investigate the contribution of muscle tissue-derived cytokines in dermatomyositis (DM). MATERIALS AND METHODS: Muscle homogenates were prepared from deltoid muscle biopsy specimens of 10 patients with DM and eight controls with no pathological signs of myopathy. Interleukin (IL)-4, interferon (IFN)-γ and IL-17 levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis. Muscle strength grades were recorded. RESULTS: Patients with DM showed significantly elevated muscle tissue IL-4 and IFN-γ levels, whereas IL-17 levels were comparable between patients with DM and controls. Immunoblotting studies confirmed ELISA results. In DM muscle specimens, IL-4 and IFN-γ levels were positively correlated, while no correlation was observed between IL-17 and the other two cytokines. Moreover, IL-4 and IFN-γ levels were significantly negative correlated with muscle strength grades for the deltoid muscle. CONCLUSION: Our results confirm the involvement of T helper (Th) 1-type and Th2-type immunity in DM pathogenesis. Muscle tissue appears to contribute to muscle weakness in DM by producing inflammatory cytokines.

Vascular function and arginine and dimethylarginines in gentamicin-induced renal failure: a possible effect of heme oxygenase 1 inducer hemin.

Increased oxidative stress and disturbance in nitric oxide bioavailability lead to endothelial dysfunction and cardiovascular complication in renal disease. Gentamicin (GM), a commonly used antibiotic, exhibits a toxic effect on renal proximal tubules. Prevention of its nephrotoxicity is important. Therefore, we investigated whether heme oxygenase 1 HO-1) induction influenced kidney and vascular function in GM-administered rats. GM (100 mg·kg-1·day-1; i.p.) was given to rats alone or together with hemin (20 mg·kg-1 on alternate days; i.p.) for 14 days. Plasma and kidney l-arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) as well as kidney 4-hydroxynonenal (HNE) levels and myeloperoxidase (MPO) activity were measured. Histopathological examinations of kidney and relaxation and contraction responses of aorta were also examined. GM increased serum SDMA, urea nitrogen (BUN), and creatinine levels and caused histopathological alterations in the kidney. GM elevated HO-1 protein and mRNA expressions, 4-HNE level, and MPO activity and decreased antioxidant enzyme activities and l-arginine levels in the kidney. Decreased relaxation and contraction were detected in the aorta. Hemin restored renal oxidative stress and inflammatory changes together with vascular dysfunction, but did not affect SDMA, BUN, or creatinine levels. We conclude that HO-1 induction may be effective in improving renal oxidative stress, inflammation, and vascular dysfunction mediated by GM.

Viability of SH-SY5Y cells is associated with purinergic P2 receptor expression alterations.

To investigate the role of metabotrophic purinergic P2Y receptors in neuroblastoma cell survival, expression of P2 receptors by normal mouse (C57BL/6) brain and human neuroblastoma SH-SY5Y cells was investigated by Western blot and real time PCR studies. Viability of SH-SY5Y cells treated with purinergic receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) was evaluated by MTT assay and flow cytometry. In the brain samples of C57BL/6 mice, expressions of P2Y4 and P2X7 were significantly reduced, whereas that of P2Y1 was significantly elevated in an age-dependent manner. SH-SY5Y cell viability was significantly reduced and necrotic cell rates were mildly increased by 400 µM suramin and 100 µM PPADS treatment. Antagonist treatment downregulated P2Y1, P2Y2 and P2Y4 and upregulated P2Y6, P2Y12 and P2X7 mRNA levels in SH-SY5Y cells on the 24th hour. These alterations were abolished for all P2 receptors except P2Y1 in the 48th hour. P2Y receptors are expressed by both normal mouse brain and human neuroblastoma cells. Purinergic receptor antagonism interferes with neuroblastoma viability through elevation of necrotic cell death and modulation of P2 receptor expression. P2Y receptors might thus be useful targets for future anti-tumor treatment trials.

Neuroprotective effects of intravenous immunoglobulin are mediated through inhibition of complement activation and apoptosis in a rat model of sepsis.

BACKGROUND: Intravenous (IV) immunoglobulin (Ig) treatment is known to alleviate behavioral deficits and increase survival in the experimentally induced model of sepsis. To delineate the mechanisms by which IVIg treatment prevents neuronal dysfunction, an array of immunological and apoptosis markers was investigated. METHODS: Sepsis was induced by cecal ligation perforation (CLP) in rats. The animals were divided into five groups: sham, control, CLP + saline, CLP + immunoglobulin G (IgG) (250 mg/kg, iv), and CLP + immunoglobulins enriched with immunoglobulin M (IgGAM) (250 mg/kg, iv). Blood and brain samples were taken in two sets of experiments to see the early (24 h) and late (10 days) effects of treatment. Total complement activity, complement 3 (C3), and soluble complement C5b-9 levels were measured in the sera of rats using ELISA-based methods. Cerebral complement, complement receptor, NF-κB, Bax, and Bcl-2 expressions were analyzed by western blot and/or RT-PCR methods. Immune cell infiltration and gliosis were examined by immunohistochemistry using CD3, CD4, CD8, CD11b, CD19, and glial fibrillary acidic protein antibodies. Apoptotic neuronal death was investigated by TUNEL staining. RESULTS: IVIgG and IgGAM administration significantly reduced systemic complement activity and cerebral C5a and C5a receptor expression. Likewise, both treatment methods reduced proapoptotic NF-κB and Bax expressions in the brain. IVIgG and IgGAM treatment induced considerable amelioration in glial cell proliferation and neuronal apoptosis which were increased in non-treated septic rats. CONCLUSIONS: We suggest that IVIgG and IgGAM administration ameliorates neuronal dysfunction and behavioral deficits by reducing apoptotic cell death and glial cell proliferation. In both treatment methods, these beneficial effects might be mediated through reduction of anaphylatoxic C5a activity and subsequent inhibition of inflammation and apoptosis pathways.

Dynamic collagen changes in cervix during the first trimester and decreased collagen content in cervical insufficiency.

OBJECTIVE: To determine the changes in cervical collagen during the first trimester of pregnancy and to evaluate the collagen deficit in cases with a previous diagnosis of cervical insufficiency (CI). MATERIALS AND METHODS: Cervical punch biopsies were obtained from 66 patients divided into three groups: patients with recurrent abortions due to CI (CI group; n = 8); first-trimester abortion group (study group; n = 37), subdivided into three groups according their gestational week (<7, 7-9 and 9-12 weeks), and patients with cervical biopsy due to gynecologic reasons (control group; n = 12). Collagen quantity was determined by a biochemical method that measured the levels of hydroxyproline (HOP) in dry cervix tissue. RESULTS: The HOP concentrations were significantly higher at lower gestational ages (p = 0.001). Collagen quantity was lowest in the CI group compared with other groups (p < 0.001). CONCLUSION: This study shows collagen component of cervix decreases as pregnancy advances through the first trimester. Cervical collagen concentration is lower in women with a history of CI compared to controls who has not a history of CI.

Serum Prolactin Levels in Multiple Sclerosis, Neuromyelitis Optica, and Clinically Isolated Syndrome Patients.

INTRODUCTION: Prolactin has been discussed as a factor likely to play a mediating role in multiple sclerosis (MS). Our aim was to investigate the possible association between prolactin production and clinical features of autoimmune demyelinating central nervous system disorders. METHODS: Serum prolactin levels of 255 MS patients, 19 neuromyelitis optica (NMO) patients, 15 clinically isolated syndrome (CIS) patients, and 240 healthy controls were measured by a heterogeneous sandwich magnetic separation assay. RESULTS: MS and NMO cohorts had a significantly higher number of patients with hyperprolactinemia than healthy controls. Sera obtained during attacks of both MS and NMO patients displayed higher prolactin levels than those collected during remission. Prolactin level elevations were found to be more prominent in myelitis attacks in MS. No significant correlation was found between prolactin levels and age, disease duration, disability status, number of attacks, and oligoclonal band positivity. CIS patients who converted to MS had higher prolactin levels than those who did not. CONCLUSION: Our findings support the possible mediating role of prolactin in the immunopathogenesis of MS, NMO, and conversion from CIS to MS.

Effect of olive leaf extract treatment on doxorubicin-induced cardiac, hepatic and renal toxicity in rats.

Doxorubicin (DOX) is known to increase in oxidative stress in several organs. Olive leaf extract (OLE) has potent antioxidant effects; therefore, we evaluated the ability of OLE to reduce DOX-induced toxicity in the heart, liver, and kidneys of rats. DOX (30mg/kg; i.p.) was administered to rats, which were sacrificed 4 days after DOX. The rats received OLE (6 and 12mL/L in drinking water) for 12 days. Serum cardiac troponin I (cTnI) levels, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities, urea and creatinine levels, as well as prooxidant and antioxidant status in organs were measured. DOX was found to increase serum markers that indicate tissue injury, malondialdehyde (MDA), diene conjugate (DC), and protein carbonyl (PC) levels, and to decrease glutathione (GSH) levels in organs. Histopathologic changes were also evaluated. OLE, especially OLE 1000, led to decreases in serum cTnI and urea levels, ALT and AST activities, and amelioration in histopathologic findings. Decreases in MDA, DC, and PC, and increases in GSH levels were observed in organs of DOX-treated rats due to OLE. We conclude that OLE treatment may be effective in decreasing DOX-induced cardiac, hepatic and renal oxidative stress and injury.

A case of hyperkinetic movement disorder associated with LGI1 antibodies.

Encephalitis associated with leucine-rich glioma inactivated 1 (LGI1) antibodies is often encountered in elderly male patients and may infrequently present with isolated syndromes. A 6-year-old boy was admitted with acute onset severe oral and facial stereotypic and choreiform movements. On his neurologic examination, he had repetitive and rhythmic movements in orolingual muscles including tongue protrusion, limb chorea and minimal facial stereotypic movements. Anti-streptolysin O (ASO) titers were found severely elevated in several measurements. Well-characterized antibodies against ion channels and synapse proteins were negative whereas LGI1 antibody was positive in both serum and CSF. Marked clinical improvement was observed after immunotherapy. Here, we present the first pediatric case with LGI1 antibody associated hyperkinetic movement disorders and emphasize the importance of investigating neuronal autoantibodies in patients with isolated and treatment resistant movement disorders.

Effect of carnosine alone or combined with α-tocopherol on hepatic steatosis and oxidative stress in fructose-induced insulin-resistant rats.

A diet high in fructose (HFr) induces insulin resistance in animals. Free radicals are involved in the pathogenesis of HFr-induced insulin resistance. Carnosine (CAR) is a dipeptide with antioxidant properties. We investigated the effect of CAR alone or in combination with α-tocopherol (CAR + TOC) on HFr-induced insulin-resistant rats. Rats fed with HFr containing 60% fructose received CAR (2 g/L in drinking water) with/without TOC (200 mg/kg, i.m. twice a week) for 8 weeks. Insulin resistance, serum lipids, inflammation markers, hepatic lipids, lipid peroxides, and glutathione (GSH) levels together with glutathione peroxidase (GSH-Px) and superoxide dismutase 1 (CuZnSOD; SOD1) activities and their protein expressions were measured. Hepatic histopathological examinations were performed. HFr was observed to cause insulin resistance, inflammation and hypertriglyceridemia, and increased triglyceride and lipid peroxide levels in the liver. GSH-Px activity and expression decreased, but GSH levels and SOD1 activity and expression did not alter in HFr rats. Hepatic marker enzyme activities in serum increased and marked macro- and microvesicular steatosis were seen in the liver. CAR treatment did not alter insulin resistance and hypertriglyceridemia, but it decreased steatosis and lipid peroxidation without any change in the antioxidant system of the liver. However, CAR + TOC treatment decreased insulin resistance, inflammation, hepatic steatosis, and lipid peroxidation and increased GSH-Px activity and expression in the liver. Our results may indicate that CAR + TOC treatment is more effective to decrease HFr-induced insulin resistance, inflammation, hepatic steatosis, and dysfunction and pro-oxidant status in rats than CAR alone.

Serum anti-neuronal antibodies in amyotrophic lateral sclerosis.

We aimed to investigate various anti-neuronal antibodies in sera of amyotrophic lateral sclerosis (ALS) patients to detect possible autoimmune encephalitis patients imitating ALS findings and to delineate the validity of routine screening of well-characterized anti-neuronal antibodies in ALS. The patients fulfilling the revised El Escorial diagnostic criteria for definite ALS were included. Their serum samples were investigated for antiganglioside (IgM/IgG) and onconeural (IgG) antibodies by immunoblotting, for ion channel antibodies (IgG) by a cell-based assay and for IgG binding patterns to the rat brain by indirect immunohistochemistry. Thirty-five patients with definite ALS and 30 healthy individuals were included. Ganglioside antibodies were detected in 2 out of 35 (5.7%) patients with ALS. The onconeural and ion channel antibodies were negative in our series. Varied serum IgG binding patterns were identified in eight (22.9%) patients. Although autoimmune encephalitis patients may occasionally present with atypical motor neuron disease findings, definite ALS patients do not appear to exhibit onconeural or ion channel antibodies, suggesting that routine analysis of these antibodies in typical ALS is not mandatory. By contrast, some ALS patients display anti-neuronal antibodies against undetermined target antigens, prompting investigation of these novel antibodies with more advanced methods.

Response of liver to lipopolysaccharide treatment in male and female rats.

Gender is considered to be an important factor in endotoxin-induced tissue damage. Our aim was to examine the role of sex on the prooxidant-antioxidant status, necrotic and apoptotic events in the liver of lipopolysaccharide (LPS)-treated rats. We determined levels of lipid peroxides, non-enzymatic and enzymatic antioxidants, and expressions of apoptosis-related proteins, antiapoptotic B cell lymphoma-2 (Bcl-2) and proapoptotic Bax, caspase-3 activity and apoptotic cell numbers in the liver. Hepatic histopathology and serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were also investigated. Male and female Wistar rats (180-200 g) were injected with LPS (10 mg/kg, i.p.) and examinations were performed 6 h after the injection. Significant increases in hepatic thiobarbituric acid reactive substances and diene conjugate levels were observed in male and female rats following LPS treatment. However, there were no changes in hepatic glutathione, vitamin E and vitamin C levels together with superoxide dismutase, glutathione peroxidase and glutathione transferase activities. LPS treatment caused significant increases in serum ALT and AST activities and lymphocyte infiltration and necrotic changes in the livers. Bcl-2 and Bax expressions, caspase-3 activity and apoptotic cell numbers were also found to be increased in both groups. In conclusion, no sex-dependent difference was observed in the changed hepatic prooxidant-antioxidant status of rats following LPS treatment. Besides, the process leading to apoptosis and necrosis in the liver showed a similar pattern in both gender of rats.

Switch-associated protein 70 antibodies in multiple sclerosis: relationship between increased serum levels and clinical relapse.

OBJECTIVE: To identify an antibody biomarker for multiple sclerosis (MS) that can be used as a predictor of MS relapses. METHODS: MS patients' sera were screened by a protein macroarray derived from human fetal brain cDNA library (hEX1). Sera of 90 consecutive relapsing remitting MS (RRMS) patients and age-matched 145 Behçet's disease (BD) patients, 40 infectious meningoencephalitis patients, and 70 healthy controls were screened by ELISA for serum antibodies against the selected clone. RESULTS: Sequencing of the clone with the highest signal intensity revealed switch-associated protein 70 (SWAP70) as a potential target autoantigen in RRMS. ELISA studies showed high-titer SWAP70-antibodies in 21 (23.3 %) RRMS and 7 (4.8 %) BD patients. SWAP70 antibodies were more likely to be found positive in sera obtained during or shortly after a relapse. CONCLUSION: Detection of SWAP70 antibodies during the attack period might suggest that SWAP70 is involved in MS relapse pathogenesis. Whether serum SWAP70 antibody detection may be utilized as an MS relapse predictor should be tested in prospective studies.

Dual inhibition of wound healing and oxidative process by montelukast in experimental colon anastomoses.

BACKGROUND: The authors aimed to investigate the effects of montelukast (ML) on the experimental rat colon anastomosis. METHODS: A total of 80 Wistar albino rats were divided into 4 groups: sham-operated, colon anastomosis, and colon anastomosis with oral administration (OAML) and rectal administration of 10 mg/kg/d ML (RAML). Anastomotic bursting pressure, anastomotic hydroxyproline contents, malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GPX), and superoxide dismutase (SOD) levels, and the expressions of Ki-67, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) with immunohistochemistry were assessed on postoperative day 5. RESULTS: Anastomotic bursting pressures and bFGF expressions were not changed, whereas tissue hydroxyproline concentrations and MDA levels and the expressions of Ki-67 and VEGF were significantly decreased, and GSH, GPX, and SOD levels were significantly increased in the OAML and RAML groups. CONCLUSION: ML causes impairment of wound healing without altering the anastomosis bursting pressure and reverses the oxidative damage of the colon anastomoses in rats.

Heme oxygenase-1 prevents hyperthyroidism induced hepatic damage via an antioxidant and antiapoptotic pathway.

BACKGROUND: The exact pathogenesis of hepatic dysfunction in hyperthyroidism is still unknown. We aimed to investigate the pathogenesis of liver dysfunction caused by hyperthyroidism through inducing heme oxygenase-1 (HO-1) expression, which has antioxidant and anti-apoptotic properties. METHODS: Rats were divided into six groups: untreated (group 1), treated with zinc protoporphyrin (ZnPP) (group 2), treated with hemin (group 3), treated with tri-iodothyronine (T3) (group 4), treated with T3 and ZnPP (group 5), and treated with T3 and hemin (group 6). After 22 d, oxidative stress and antioxidant enzymes and the expression of HO-1, mitochondrial permeability transition, cytochrome c, Bax, Bcl-2, caspase-3, caspase-8, and caspase-3 activity, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were examined. RESULTS: Hyperthyroidism induced oxidative stress of liver tissue was ameliorated by HO-1 induction. Administration of hemin (HO-1 inducer) increased Bcl-2 expression. Decreased expression of cytochrome c was accompanied by a decrease in caspase-3, caspase-8, Bax expression, and caspase-3 activity. The apoptotic activity and oxidative damage were found to be increased by the administration of ZnPP (HO-1 inhibitor). Immunohistochemistry findings supported these results. CONCLUSION: HO-1 induction plays a protective role in the pathogenesis of the liver dysfunction in hyperthyroidism. This effect is dependent on modulation of the antiapoptotic and antioxidative pathways by HO-1 expression.