Copy number variations of satellite III (1q12) and ribosomal repeats in health and schizophrenia.

OBJECTIVE: Earlier we studied the copy number variations (CNVs) of ribosomal repeat (rDNA) and the satellite III fragment (1q12) (f-SatIII) in the cells of schizophrenia patients (SZ) and healthy controls (HC). In the present study we pursued two main objectives: (1) to confirm the increased rDNA and decreased f-SatIII content in the genomes of enlarged SZ and HC samples and (2) to compare the rDNA and f-SatIII content in the same DNA samples of SZ and HC individuals. METHODS: We determined the rDNA CN and f-SatIII content in the genomes of leukocytes of 1770 subjects [HC (N = 814) and SZ (N = 956)]. Non-radioactive quantitative hybridization method (NQH) was applied for analysis of the various combinations of the two repeats sizes in SZ and HC groups. RESULTS: f-SatIII in human leukocytes (N = 1556) varies between 5.7 and 44.7 pg/ng DNA. RDNA CN varies between 200 and 896 (N = 1770). SZ group significantly differ from the HC group by lower f-SatIII content and by rDNA abundance. The f-SatIII and rDNA CN are not randomly combined in the genome. Higher rDNA CN values are associated with higher f-SatIII index values in SZ and HC. The f-SatIII variation interval in SZ group increases significantly in the subgroup with the high rDNA CN index values (>300 copies). CONCLUSION: Schizophrenia patients' genomes contain low number of f-SatIII copies corresponding with a large ribosomal repeats CN. A scheme is proposed to explain the low f-SatIII content in SZ group against the background of high rDNA CN.

Accumulation of Circulating Cell-Free CpG-Enriched Ribosomal DNA Fragments on the Background of High Endonuclease Activity of Blood Plasma in Schizophrenic Patients.

INTRODUCTION: Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia? Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N = 100) and in the male healthy control group (HC) (N = 96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated. RESULTS: In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group. CONCLUSION: Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients' cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.

[Immune dysfunction and cognitive deficit in stress and physiological aging. Part II: New approaches to cognitive disorder prevention and treatment ].

Long-term stress as well as physiological aging result in similar immunological and hormonal disturbances including hypothalamic-pituitary-adrenal) axis depletion, aberrant immune response (regulatory T-cells, Tregs, and T(h17)-lymphocyte accumulation) and decreased dehydroepian-drosterone synthesis both in the brain and in the adrenal glands. Since the main mechanisms of inflammation control, "prompt" (stress hormones) and "delayed" (Tregs), are broken, serum cytokine levels increase and become sufficient for blood-brain-barrier disruption. As a result peripheral cytokines penetrate into the brain where they begin to perform new functions. Structural and functional alterations of blood-brain-barrier as well as stress- (or age-) induced neuroinflammation promote influx of bone marrow derived dendritic cells and lymphocyte effectors into the brain parenchyma. Thereafter, mass intrusion ofpro-inflammatory mediators and immune cells having a lot of specific targets alters the brain work that we can observe both in humans and in animal experiments. The concept of stressful cognitive dysfunction, which is under consideration in this review, allows picking out several therapeutic targets: 1) reduction of excessive Treg accumulation; 2) supporting hypothalamic-pituitary-adrenal axis and inflammatory reaction attenuation; 3) recovery of dehydroepiandrosterone level; 4) improvement of blood-brain-barrier function.

[Immune dysfunction and cognitive deficit in stress and physiological aging (Part I): Pathogenesis and risk factors].

The concept of stressful cognitive dysfunction, which is under consideration in this review, allows picking out several therapeutic targets. The brain, immune and endocrine systems being the principal adaptive systems in the body permanently share information both in the form of neural impulses and soluble mediators. The CNS differs from other organs due to several peculiarities that affect local immune surveillance. The brain cells secluded from the blood flow by a specialized blood-brain-barrier (BBB) can endogenously express pro- and anti-inflammatory cytokines without the intervention of the immune system. In normal brain the cytokine signaling rather contributes to exclusive brain function (e.g. long-term potentiation, synaptic plasticity, adult neurogenesis) than serves as immune communicator. The stress of different origin increases the serum cytokine levels and disrupts BBB. As a result peripheral cytokines penetrate into the brain where they begin to perform new functions. Mass intrusion of biologically active peptides having a lot of specific targets alters the brain work that we can observe both in humans and in animal experiments. In addition owing to BBB disruption dendritic cells and T cells also penetrate into the brain where they take up a perivascular position. The changes observed in stressed subject may accumulate during repeated episodes of stress forming a picture typical of the aging brain. Moreover long-term stress as well as physiological aging result in hormonal and immunological disturbances including hypothalamic-pituitary-adrenal axis depletion, regulatory T-cell accumulation and dehydroepiandrosterone decrease.

[Regulatory T-cells: modern approaches to optimization of their numbers].

Regulatory T-cells (Tregs) are important components of the complex adaptive system of the body responsive to environmental challenges. Tregs ensure peripheral tolerance and play an important role in control of inflammatory reactions. Several subsets of Tregs have been described. Naturally occurring CD4+CD25+ Tregs are recognized as a major subset of immune cells responsible for peripheral immune self-tolerance. Another subtype of Tregs is inducible. Such Tregs are generated in the periphery and realize their suppressive potential largely in the form of anti-inflammatory activity. The latter plays an important role in cooperation of three principal anti-inflammatory mechanisms that developed in the course of evolution: macrophages possessed of suppressive activity, Tregs, and stress hormones. Normally, all the three mechanisms of inflammation control are in equilibrium. However, the balance may be disturbed with ageing due to repeated episodes of stress and HPA axis activation. As a result, secretion of stress hormones coupled to antigen overload leads to Treg accumulation. In the course of time activation of the HPA axis is replaced by its inhibition manifested both as a decrease of the baseline cortisol level and a reduction of stress-induced cortisol response. Cortisol present in blood at low concentrations is no longer capable of controlling inflammation and Tregs become a principal mechanism of anti-inflammatory machinery. Superfluous Treg accumulation results in the development of functional somatic syndromes, such as chronic fatigue syndrome, and (in some patients) in the growth of tumours resulting from the suppression of anticancer immunity. On the other hand, the lack of adequate antigen loading in the childhood may delay Treg maturation. Allergy and asthma manifestations may be a consequence of such Treg insufficiency. Thus, both excess and deficiency of Tregs may be at the bottom of morbid conditions. The advances in modern pharmacology open up opportunities for developing new methods to control the Treg level.

[Effect of steroid therapy on the clinical course of bronchial asthma].

Although only some 5% of the asthmatic population develop severe disease, such cases eat up more than half of the spendings for the management of asthma and they are poorly controlled by the currently available therapies. Most of these patients undergo long-term treatment with various steroid formulations that exert selective action in the airways eliminating one cell type and supporting the growth of another. For this reason, inflammation patterns in severe, as opposed to mild, asthma resemble those in COPD, with a high neutrophil count in the sputum, increased oxidative stress, and poor response to corticosteroids. Such differences in asthma pathogenesis may be due to the accumulation of two CD4+ cell subsets (regulatory T cells and Th17 cells) in the lungs. It is believed that the use of alkylating drugs in low (non-cytotoxic) doses selectively inactivating Tregs and Th17 cells is a promising method for the treatment of severe asthma.

HPA axis exhaustion and regulatory T cell accumulation in patients with a functional somatic syndrome: recent view on the problem of Gulf War veterans.

The authors proceeded from the assumption that physical and mental symptoms of functional somatic syndromes (including those observed in Gulf War veterans) are based on both underactivity of hypothalamic-pituitary-adrenal (HPA) axis and excessive accumulation of regulatory T cells (Tregs). Permanent psychogenic stress coupled with high antigen loading leads to gradual depletion of HPA axis, which is manifested by the reduction of stress-induced cortisol response. Under stress hormone deficiency, Tregs begin to play a principal role in anti-inflammatory mechanisms and each new pro-inflammatory stimulus increases their number. Superfluous accumulation of active Tregs results in malfunction of Th1 cells in the brain that leads to the appearance of neurodegeneration foci, which seems to be an anatomic substance for various cognitive and psychological symptoms. New approaches to the treatment of such conditions are also discussed.

[Inflammatory reactions in patients suffering from coronary artery disease with concomitant obesity and type 2 diabetes mellitus].

Of late, inflammatory reactions have been considered to play an important part in the development of atherosclerosis. Acute-phase inflammatory reaction, being initially a protective response directed within the homeostasis system towards lesion repair, may by itself due to various factors favor the development of pathological processes. Considering the role played by inflammation in the development of atherosclerosis, and inflammatory activity in obesity and diabetes mellitus (DM), a range of common and interrelated elements of these processes may be marked out. These are acute phase proteins and cytokines. Insulinoresistance, being the common precursor of obesity and DM, plays the key role in vascular lesion. The use of cytokine activity index seems to be a promising method of revealing patients with high risk of atherosclerosis.

Alkylating drugs applied in non-cytotoxic doses as a novel compounds targeting inflammatory signal pathway.

Alkylating drugs (ADs) belonging to the nitrogen mustard family are commonly used as cytostatic and immunosuppressive agents. Our previous in vitro studies demonstrated that in the case of gradual dose decrease, the number of targets for alkylation in the cell is also reduced and the drug switches from brutal cytostatic to cell growth modifier. At doses of 0.3 microg/ml and lower, the effects of ADs are no longer associated with DNA damage or stress/MAPK pathways activation. Instead, the disruption of signal transduction by the IL-2beta and/or TNFalpha cell surface receptors is observed. As a result, ADs in the doses 100-fold lower than cytostatic ones are capable to modify lymphocyte activity including the activity of regulatory T cells. We hypothesized that ADs may have a beneficial effect in the treatment of inflammatory diseases. Indeed, the application of non-cytotoxic doses of an AD melphalan reduces the severity of murine experimental colitis. Daily administration of melphalan (25 microg/kg body weight) markedly reduced the severity of DSS-colitis as determined by clinical and histological criteria. Moreover, the beneficial effect of melphalan was also shown in asthmatic patients. In 60% of these patients histological and ultrastructural signs of bronchial epithelium regeneration were also revealed. Thus, ADs at non-cytotoxic concentrations exert beneficial effect both in acute and chronic inflammatory diseases. Such anti-inflammatory activity is thought to be due to blocking of signal transduction through various cell surface receptor including IL-2R and TNFR. Consequently different steps of inflammatory cascade turn out to be inhibited.

Melphalan in ultralow doses decreases the severity of experimental colitis in mice.

We studied the effect of melphalan in ultralow doses on mice with experimental colitis induced by substitution of drinking water for 5% dextran sulfate. Daily treatment with melphalan in a dose of 0.025 mg/kg improved the general state of animals. The influence of melphalan was evaluated by quantitative clinical, pathomorphological, and laboratory parameters. Melphalan had a local and systemic antiinflammatory effect.

Preventive and therapeutic effects of alpha-acid glycoprotein in mice infected with B. anthracis.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

[Dynamics of markers of blood inflammation against the background of antibacterial therapy in children with mucoviscidosis, with the various regimens of intake of pancreatic enzymes]. / Dinamika markerov vospaleniia krovi na fone antibakterial'noi terapii u detei, bol'nykh mukovistsidozom, s razlichnym rezhimom priema pankreaticheskikh fermentov.

The results of the studies show that patients with mucoviscidosis diagnosed for the first time who do not receive any adequate replacement pancreatic therapy should not be prescribed high doses of pancreatic enzymes along with antibacterial therapy when they are hospitalized on account of exacerbation of the bronchopulmonary process. Sharply increased lipid absorption can cause an aggravation of oxidative stress and impair the patient's state instead of improving it. In our opinion, such patients should be prescribed delayed adequate pancreatic therapy with immediate introduction of high doses of antioxidants (vitamins E, A, C, beta-carotene, etc.). There is no doubt that it is necessary to conduct further research into the problem for a greater number of patients.

Tumor necrosis factor-alpha/interleukin-10 balance in normal and cystic fibrosis children.

BACKGROUND: The balance between tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) is important for immune homeostasis maintenance. Exuberant production of TNF-alpha contributes to overwhelming inflammatory response and tissue damage. But, commonly, increase in TNF-alpha is counterbalanced by simultaneous synthesis of an anti-inflammatory cytokine IL-10, which suppresses production of many activating and regulatory mediators. AIMS: In the present study, the relationships between TNF-alpha and IL-10 in the plasma of healthy school-children and cystic fibrosis (CF) patients have been investigated. METHODS: Blood samples were obtained from 12 CF patients with chronic pulmonary disease and 18 healthy schoolchildren vaccinated with live attenuated rubella vaccine. IL-10 and TNF-alpha were determined in the plasma samples using commercially available enzyme-linked immunosorbent assay kits. RESULTS: Before vaccination, most healthy children (13 of 18) demonstrated superiority of pro-inflammatory TNF-alpha over anti-inflammatory IL-10 (TNF-alpha/IL-10 > 1). In these subjects, a significant positive linear association between the cytokine values has been found. Vaccine challenge resulted in a marked reduction of TNF-alpha/IL-10 ratios. In addition, a disappearance of correlation between the cytokine values was observed. Such disturbance was related to exuberant elevation of the IL-10 levels after inoculation. On the contrary, in CF individuals, plasma cytokine values remained in strong linear association independently of TNF-alpha or IL-10 predominance. No spikes in the plasma levels of IL-10 in CF patients during a 6-month observation period have been revealed. CONCLUSIONS: There were no fundamental differences between CF and healthy children in the regulation of TNF-alpha and IL-10 secretion. Thus, immune quiescence seemed to be associated with the predominance of TNF-alpha, whereas immune disturbance was characterized by IL-10 superiority. The only abnormality that was found in CF patients consisted of their inability to produce unlimitedly IL-10 in response to antigen stimuli.

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities.

We studied the effects of alpha1-acid glycoprotein on tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to alpha1-acid glycoprotein: first, stimulation of TNF-alpha and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-alpha/IL-10 ratio remained unchanged after addition of native alpha1-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of alpha1-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.

Improvement of nutrient absorption may enhance systemic oxidative stress in cystic fibrosis patients.

BACKGROUND: The life expectancy of patients with cystic fibrosis (CF) is largely dependent on the pulmonary disease severity and progress. Malnutrition may be an important complicating factor in active and chronic lung disease. AIMS: The focus of this study was to investigate several inflammatory markers in pancreatic-insufficient CF patients with different enzyme treatment regimens. METHODS: CF patients with pancreatic insufficiency were examined at a time of symptomatic exacerbation of their lung disease. Group A (n = 11) regularly received microspheric enzymes. Group B (n = 8) were treated with enzymes during the hospitalization period only and demonstrated the presence of malnutrition. Inflammatory markers in the sputa (neutrophil elastase activity, interleukin-8 and tumour necrosis factor-alpha levels) and in the peripheral blood (plasma malondialdehyde (MDA), lymphocyte response to PHA, and the cell sensitivity to steroid suppression) have been investigated. RESULTS: During acute lung exacerbation, group B demonstrated reduced levels of lymphocyte proliferation. This parameter was normalized after combined antibiotic and pancreatic enzyme therapy. Simultaneously, plasma MDA in group B markedly increased following treatment. For this group, a significant positive linear association between values of plasma MDA and lymphocyte proliferation has been observed. For group A, neither the same correlation nor changes in MDA levels and lymphocyte proliferation have been found. CONCLUSIONS: Our data indicate that acute lung exacerbation in malnourished CF patients may be associated with alteration in T-lymphocyte activity. Adequate therapy normalizes lymphocyte function but results in systemic oxidative stress.

Stimulatory and protective effects of alkylating agents applied in ultra-low concentrations.

Alkylating drugs belonging to the nitrogen mustard family are known as cytostatic and immunosuppressive agents. Ultra-low doses of these drugs may demonstrate pharmacological effects unlike this category of drugs. In the case of a gradual dose decrease, the number of targets for alkylation is also reduced and the drug switches from cytostatic to cell growth modifier. We postulate that application of ultra-low doses of alkylating drugs may result in a beneficial effect in the therapy of diseases associated with chronic inflammation of the mucosa, especially with the signs of epithelial atrophy.

Effect of semisynthetic analog of alpha(1)-acid glycoprotein on immunomodulatory and antiinflammatory activity of natural glycoprotein.

Pseudo-alpha(1)-acid glycoprotein with carbohydrate chain ratio typical of native form was synthesized by a previously developed original technique of quantitative transfer of alpha(1)-acid glycoprotein carbohydrate chains to other polymeric carrier. Similarly to native glycoprotein, the semisynthetic analog inhibited lymphocyte proliferation and stimulated the production of antiinflammatory cytokines by peripheral blood mononuclear leukocytes. However, it possessed no antioxidant activity and did not inhibit complement activation by the alternative pathway. The role of carbohydrate and protein components of alpha(1)-acid glycoprotein molecule in the realization of its biological effects is discussed.

[Immunologic monitoring of patients with cystic fibrosis: value of different laboratory findings]. / Immunologicheskii monitoring bol'nykh mukovistsidozom, znachenie razlichnykh laboratornykh pokazatelei.

Cystic fibrosis (CF) is a common, serious, and frequently fatal autosomal recessive genetic disorder associated with the poor function of chloride channels. Chronic endobronchial inflammation and bacterial infection are main causes of morbidity and mortality due to CF. The study dealt with a relationship between progression and inflammation markers. Twenty one CF children with acute pulmonary exacerbation were examined. The signs of peripheral blood inflammation (responses of lymphocytes to PHA and their sensitivity to the antiproliferative effect of glucocorticoids) and in situ inflammation markers (sputum elastase activity, IL-8 and TNF-alpha, and protein concentrations in the same sputum specimens). These laboratory findings were used to calculate a "laboratory index" (LI). The clinical status of each patient was evaluated with a "clinical index" (CI), a parameter that includes respiratory secretion cultures, pulmonary function test results, nutritional status, and the presence of disease-related complications. There was a positive linear correlation between LI and CI. The presence of P. aeruginosa was strongly associated with the changes of inflammatory markers. CF patients with prolonged P. aeruginosa infection demonstrated extremely enhanced elastase activity and elevated amounts of sputum IL-8 and TNF-alpha as compared to uninfected subjects. The lung elastase activities, sputum protein contents, and TNF-alpha levels in individuals with short-term colonization were at or below those without P. aeruginosa infection. In patients with or without short-term colonization, the normalization of laboratory parameters was strongly related to evident clinical improvement. At the same time, antibiotic treatment failed to suppress an excessive inflammatory response in the lungs of patients with prolonged P. aeruginosa infection. The importance of individual inflammation markers is discussed in the paper.