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Investigation of the relationship between inflammation and energy metabolism is important for understanding biology of chronic noncommunicable diseases. Use of metformin, a drug for treatment of diabetes, is considered as a promising direction for treatment of neurodegenerative diseases and other neuropathologies with an inflammatory component. Astrocytes play an important role in the regulation of energy metabolism and neuroinflammation; therefore, we studied the effect of metformin on the cellular responses of primary rat astrocytes cultured in a medium with high glucose concentration (22.5 mM, 48-h incubation). Lipopolysaccharide (LPS) was used to stimulate inflammation. The effects of metformin were assessed by monitoring changes in the expression of proinflammatory cytokines and synthesis of oxylipins, assayed with ultra-high-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). Changes at the intracellular level were assessed by analyzing phosphorylation of ERK kinase and transcription factor STAT3, as well as enzymes mediating oxylipin synthesis, cyclooxygenase 1 and 2 (COX). It was found that, independent on glucose concentration, metformin reduced the LPS-stimulated release of cytokines IL-1β and IL-6, decreased activity of the transcription factor STAT3, ERK kinase, synthesis of the derivatives of the cyclooxygenase branch of metabolism of oxylipins and anandamide, and did not affect formation of ROS. The study of energy phenotype of the cells showed that metformin activated glycolysis and inhibited mitochondrial respiration and oxidative phosphorylation, independent on LPS stimulation and cell cultivation at high glucose concentration. Thus, it has been shown that metformin exhibits anti-inflammatory effects, and its effect on the synthesis of cytokines, prostaglandins, and other lipid mediators could determine beneficial effects of metformin in models of neuropathology.

Supplementary Figure 5 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Discussion from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Figure 2 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Figure Legends 1-5,Table Legends 1-8 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Figure 3 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Figure 4 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Figure 1 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 4 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Methods from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 6 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 8 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 2 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 1 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Supplementary Table 5 from Coexpression Network Analysis Identifies Transcriptional Modules Related to Proastrocytic Differentiation and Sprouty Signaling in Glioma

Using the previously developed rabbit model of perioperative dry eye syndrome (PDES) and quantitative mass spectrometric technique it is shown that the development of corneal erosion under conditions of general anesthesia is associated with changes in the content of inflammatory metabolites, namely, derivatives of linoleic (LA), alpha-linolenic (ALA), and arachidonic (AA) acids in tear fluid. The increase in the content of the metabolites of LA and ALA is found to be the most significant, while the content of AA derivatives (with the exception of 12-HETE) remains almost unchanged, indicating the key roles of the LA and ALA cascades in the inflammatory response in PDES. The increase in the concentration of oxylipins that can be formed by nonenzymatic oxidation of LA (9-KODE) or its processing by cytochromes (12,13-EpOME) under oxidative conditions indicates a significant contribution of oxidative stress to the development of PDES. The majority of metabolites of LA (13-HODE and 9-HODE), ALA (9-HOTrE and 13-HOTrE) and AA (12-HETE), the tear content of which was changed in PDES, are generated by the enzymes of lipoxygenase family. By contrast, the concentration of cyclooxygenase products does not exhibit any significant fluctuations. These data suggest a low therapeutic potential of cyclooxygenase inhibitors (such as nonsteroidal anti-inflammatory drugs) and a high therapeutic potential of antioxidants and lipoxygenase inhibitors in PDES, which should be taken into account upon developing a complex therapy for this disease.

Hyaluronic acid (HA) is one of the key polymer components of the extracellular matrix; it can exert different effects on intracellular signaling depending on its molecular weight. We compared the effects of low (LMW) and high molecular weight (HMW) HA polymers on the ability of primary astrocytes, glial brain cells, to release into the extracellular medium omega-3 unsaturated fatty acids, docosahexaenoic (DHA) and eicosapentaenoic (EPA), as well as omega-6 unsaturated arachidonic acid (AA), which were determined by HPLC-MS/MS. It was shown that upon the addition of LMW HA for 4.5 h the concentration of AA reduced. HMW HA did not affect the release of AA but increased the release of DHA and EPA. Adaptation of the cells for 48 h to the action of the HA polymers of different molecular weights led to a decrease in the release of omega-3 and omega-6 unsaturated fatty acids by astrocytes. A comparison of the ability of HA to modulate astrocyte responses to stimulation by Toll-like receptor (TLR) agonists showed that HA affected TLR4-stimulated induction of IL-1β proinflammatory marker gene after 0.5-h exposure to LMW HA and HMW HA followed by further stimulation with TLR4 agonist for 4 h. Thus, the release of polyunsaturated fatty acids in astrocytes was sensitive to HA and depended on the molecular weight of the polymers; long (48 h) exposure to HA led to adaptation of astrocytes; HA differently affected the release of omega-6 (AA) and omega-3 (DHA) unsaturated fatty acids, indicating a difference in the mechanisms of their release. As DHA is believed to perform anti-inflammatory and protective functions in the brain, these findings open up prospects for appliance of HA polymers as modulators of inflammatory responses of astrocytes.

Polyunsaturated fatty acid (PUFA) metabolism is currently a focus in cancer research due to PUFAs functioning as structural components of the membrane matrix, as fuel sources for energy production, and as sources of secondary messengers, so called oxylipins, important players of inflammatory processes. Although breast cancer (BC) is the leading cause of cancer death among women worldwide, no systematic study of PUFA metabolism as a system of interrelated processes in this disease has been carried out. Here, we implemented a Boruta-based feature selection algorithm to determine the list of most important PUFA metabolism genes altered in breast cancer tissues compared with in normal tissues. A rank-based Random Forest (RF) model was built on the selected gene list (33 genes) and applied to predict the cancer phenotype to ascertain the PUFA genes involved in cancerogenesis. It showed high-performance of dichotomic classification (balanced accuracy of 0.94, ROC AUC 0.99) We also retrieved a list of the important PUFA genes (46 genes) that differed between molecular subtypes at the level of breast cancer molecular subtypes. The balanced accuracy of the classification model built on the specified genes was 0.82, while the ROC AUC for the sensitivity analysis was 0.85. Specific patterns of PUFA metabolic changes were obtained for each molecular subtype of breast cancer. These results show evidence that (1) PUFA metabolism genes are critical for the pathogenesis of breast cancer; (2) BC subtypes differ in PUFA metabolism genes expression; and (3) the lists of genes selected in the models are enriched with genes involved in the metabolism of signaling lipids.

The involvement of oxylipins, metabolites of polyunsaturated fatty acids, in cancer pathogenesis was known long ago, but only the development of the high-throughput methods get the opportunity to study oxylipins on a system level. The study aimed to elucidate alterations in oxylipin metabolism as characteristics of breast cancer patients. We compared the ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) oxylipin profile signatures in the blood plasma of 152 healthy volunteers (HC) and 169 patients with different stages of breast cancer (BC). To integrate lipidomics, transcriptomics, and genomics data, we analyzed a transcriptome of 10 open database datasets obtained from tissues and blood cells of BC patients and SNP data for 33 genes related to oxylipin metabolism. We identified 18 oxylipins, metabolites of omega-3 or omega-6 polyunsaturated fatty acids, that were differentially expressed between BCvsHC patients, including anandamide, prostaglandins and hydroxydocosahexaenoic acids. DEGs analysis of tissue and blood samples from BC patients revealed that 19 genes for oxylipin biosynthesis change their expression level, with CYP2C19, PTGS2, HPGD, and FAAH included in the list of DEGs in the analysis of transcriptomes and the list of SNPs associated with BC. Results allow us to suppose that oxylipin signatures reflect the organism’s level of response to the disease. Our data regarding changes in oxylipins at the system level show that oxylipin profiles can be used to evaluate the early stages of breast cancer.

An increased interest in neuroinflammation is conditioned by its involvement in various pathological processes in the brain. Astrocytes play an important role in neuroinflammation, participating in its regulation, throwing out a large number of signaling molecules. Steroid compounds, actively produced by astrocytes, are of interest with regards to the regulation of inflammatory processes in the central nervous system. In the present work the effect of dehydroepiandrosterone (DHEA) on astroglial cells (cultured primary rat astrocytes) in a model of inflammation was studied. The inflammatory response was stimulated with lipopolysaccharide (LPS). Expression levels of pro-inflammatory factor TNFα, antinflammatory interleukin IL-10, and both pro- and antiinflammatory protein COX-2 were measured. The expression of IL-10, COX-2, and TNFα mRNA was determined by real-time PCR, COX-2 protein level by immunoblotting method, TNFα and IL-10 release by enzyme immunoassay. The effect of short-term (30 min) and long-term (24 h) exposure to DHEA was evaluated. It was shown that DHEA potentiates LPS-stimulated (1) increase in the IL-10 mRNA level; (2) IL-10 release; (3) does not affect TNFα level, and (4) exerts a weak pulsating bidirectional effect on COX-2. Using trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, a key enzyme of DHEA metabolism, it was shown that DHEA metabolites make the main contribution to its effect. Thus, DHEA is of interest as a stimulant of anti-inflammatory processes in the brain.

Ocular inflammation is a key pathogenic factor in most blindness-causing visual disorders. It can manifest in the aqueous humor (AH) and tear fluid (TF) as alterations in polyunsaturated fatty acids (PUFAs) and their metabolites, oxylipins, lipid mediators, which are biosynthesized via enzymatic pathways involving lipoxygenase, cyclooxygenase or cytochrome P450 monooxygenase and specifically regulate inflammation and resolution pathways. This study aimed to establish the baseline patterns of PUFAs and oxylipins in AH and TF by their comprehensive lipidomic identification and profiling in humans in the absence of ocular inflammation and comparatively analyze these compounds in the eye liquids of rabbits, the species often employed in investigative ophthalmology. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for qualitative and quantitative characterization of lipid compounds in the analyzed samples. A total of 28 lipid compounds were identified, including phospholipid derivatives and PUFAs, as well as 22 oxylipins. Whereas the PUFAs included arachidonic, docosahexaenoic and eicosapentaenoic acids, the oxylipins were derived mainly from arachidonic, linoleic and α-linolenic acids. Remarkably, although the concentration of oxylipins in AH was lower compared to TF, these liquids showed pronounced similarity in their lipid profiles, which additionally exhibited noticeable interspecies concordance. The revealed correlations confirm the feasibility of rabbit models for investigating pathogenesis and trialing therapies of human eye disorders. The identified metabolite patterns suggest enzymatic mechanisms of oxylipin generation in AH and TF and might be used as a reference in ocular inflammation studies.

Astrocytes play a vital role in regulating central nervous system inflammation, energy metabolism and brain homeostasis. Unlike macrophages and microglia, which are cells of myeloid ancestry, astrocytes are of ectodermal origin. However, regulatory specificities of signaling pathways connecting inflammatory and metabolic processes are still largely unknown. We analyzed firstly cellular responses to toll-like receptor (TLR) agonists and secondly, modulation of the mRNA of the three isoforms of the transcription factors PPARs (peroxisome proliferator-activated receptors) in primary rat astrocytes exposed to normal glucose (5.5 mM) and high glucose (25 mM). Cell culturing of rat brain astrocytes for 2 days in high glucose did not alter cellular morphology, but i) enhanced the release of TNFα that was induced by TLR4 agonist LPS or TLR3 agonist PIC and the synthesis of prostaglandin E2 (PGE2), ii) changed the signaling pathways of TLR4/MAPK (increase in p38 MAPK, and decrease in JNK activities at early stages of TLR activation) and iii) modulated mRNA expression of PPARs. High glucose cultivation reduced PPARα and PPARβ mRNA levels, without altering PPARγ mRNA level and changed the sensitivity of expressions to agonists of TLR1/2 (PGN), TLR4 (LPS), TLR3 (PIC), and TLR5 (FGN). Differences between low and high glucose-adapted cells were obtained for agonists of TLR1/2 (PPARα, PPARβ), TLR4 (PPAR β), TLR3 (PPARα). In the TLR4/p38/PPARβ signaling pathway, there was a stimulatory connection in normal glucose but an inhibitory connection in high glucose. TLR4/JNK/activated PPARβ, TLR4/JNK/inhibited PPARγ both in cells adapted to normal or high glucose, but PPARα expression was not affected. As PPARs in astrocytes are involved in inflammatory processes in the form of the recently published PPAR triad, the changes in expression revealed here are most likely resulting in implications of high glucose in inflammatory processes. Our data underline the complexity of multiple regulatory interactions between inflammatory responses and energy metabolism in astrocytes.

The ultraviolet (UV) B-induced damage of the eye surface of experimental animals (rabbits) includes loss of corneal epithelium, apoptosis of keratocytes and stromal edema. These changes are accompanied by clinically and histologically manifested corneal inflammation, neutrophil infiltration, and exudation of the anterior chamber of the eye. According to mass spectrometric analysis, UV-induced corneal damage is associated with pronounced changes in the lipid composition of tears, including a decrease in the amount of arachidonic acid and prostaglandin E2 and an increase in the concentrations of prostaglandin D2 and its derivative 15d-PGJ2. In addition, it is accompanied by an alteration in the levels of hydroxyeicosate tetraenic acid derivatives, namely upregulation of 12-HETE and downregulation of 5-HETE. The revealed changes indicate the activation of metabolic pathways involving 5-lipoxygenase, 12-lipoxygenase, cyclooxygenase 1 and 2, and prostaglandin-D-synthase. These findings contribute to understanding mechanisms of UV-induced keratitis and point on feasibility of selective anti-inflammatory therapy for improving corneal regeneration after iatrogenic UV damage.Povrezhdeniia poverkhnosti glaza éksperimental'nykh zhivotnykh (krolikov), indutsirovannye ul'trafioletovym (UF) izlucheniem (ul'trafiolet V), vkliuchaiut polnuiu deépitelizatsiiu rogovitsy, apoptoz keratotsitov i otek stromy étoĭ tkani. Ukazannye izmeneniia soprovozhdaiutsia razvitiem klinicheski i gistologicheski detektiruemoĭ vospalitel'noĭ reaktsii v rogovitse, a takzhe neĭtrofil'noĭ infil'tratsieĭ i ékssudatsieĭ peredneĭ kamery glaza. Po dannym mass-spektrometricheskogo analiza, UF-indutsirovannye povrezhdeniia rogovitsy assotsiirovany s vyrazhennymi izmeneniiami lipidnogo sostava slezy, vkliuchaiushchimi snizhenie kolichestva arakhidonovoĭ kisloty i prostaglandina E2, uvelichenie kontsentratsiĭ prostaglandina D2 i ego proizvodnogo 15d-PGJ2, a takzhe izmenenie urovneĭ gidroksiéĭkozatetraenovykh kislot 12-HETE (snizhenie) i 5-HETE (povyshenie). Éto ukazyvaet na aktivatsiiu metabolicheskikh puteĭ s uchastiem 5-lipoksigenazy, 12-lipoksigenazy, tsiklooksigenaz 1 i 2, i prostaglandin-D-sintazy. Rezul'taty raboty rasshiriaiut ponimanie mekhanizmov razvitiia UF-indutsirovannogo keratita, a takzhe svidetel'stvuiut o perspektivnosti ispol'zovaniia selektivnoĭ protivovospalitel'noĭ terapii dlia uskoreniia regeneratsii rogovitsy posle iatrogennykh povrezhdeniĭ, vyzvannykh UF-izlucheniem.

The research is relevant as educational computer games are included in students’ mathematical activity and form additional opportunities to improve the quality of teaching mathematics in a digital school, to support the formation of the demanded professional competence - computational thinking. To form the appropriate skills that determine the essence of special computational thinking, the authors propose to include game educational spaces based on digital gamification resources into students’ mathematical activities. The research aims to resolve the contradiction between requirements of the modern economy for specialists’ high level of computational thinking and an insufficiently developed methodological base for training graduates that meets these requirements. The purpose of this research is to study the features of using gamification technologies in teaching mathematics to form the skills and abilities that make up the essence of computational thinking. The article describes directions of educational and cognitive mathematical activity based on the principles of gamification. The authors clarify the concept of “computational thinking”, which includes a system of actions for activating patterns, connections between them from human memory, and compile an effective algorithm for solving them: to obtain relevant information on advanced technological developments; to state the problem and model; to use a software product with mathematical content.

Nanoparticles of the steroidal hormone dihydroepiandrosterone (DHEA) were prepared by cryosynthesis technology, which allowed starting DHEA particles of size (100 ± 50) μm to be converted to nanoparticles of size (100 ± 20) nm. The particle sizes were determined by optical, electron transmission, and scanning atomic-force microscopy. HPLC with mass-spectrometric detection of starting and modified DHEA samples showed that the hormone molecular structure did not change during cryosynthesis of the nanoparticles. A comparison of the cytotoxicities of the samples on C6 glial cell culture showed that modified DHEA was less toxic. The results indicated that cryosynthesis technology could be used effectively to prepare nanoparticles of steroidal hormones.

The speed of modern changes in the system of teaching reflects an unprecedented accelerated renewal of means, forms and methods of teaching. Today, it is very important to test new learning solutions that reduce teachers’ time on organization of students’ educational activities. The idea of solving this problem is to combine the theory and practice of taking managerial actions and pedagogy in order to identify the type of learning solutions that reduce teachers’ time, in particular teachers of mathematics, to prepare for classes. Thus, the purpose of the article is to justify full packaged learning solutions as an effective means of reducing the time spent on organizing the educational activities of schoolchildren. The authors of the article have determined the full packaged product as a package of program-methodical and subject-developing support that can be used by consumers of educational services (children, parents, teachers, administrators, employers) for independent use (a turn-key project). The leading methods of research are monitoring the organizational activities of teachers during math lessons, talking to teachers, analyzing methodical work and teachers’ profiles, modeling and statistical processing of research results. As a result of the 2016-2017 experiment, where 21 teachers of mathematics took part, the authors of the article have defined types of learning solutions for mathematics teachers (adjustable, integrated and packaged); have described the stages of development and phases of creating a full packaged learning solution. Evaluation of the effectiveness of using full packaged product allowed to make a conclusion about an average decrease of time costs by 22% while preparing for classes. The theoretical significance of the article is due to the contribution to the development of scientific ideas about the means of methodical support for teachers of mathematics. The practical use of the proposed methods allows to organize a step-by-step transition from the development of adjustable solutions to full packaged learning solutions for studying school mathematics that contribute to reducing teachers’ time spent on the organization of educational activities of students. The value of the full packaged product is justified with the help of a “project triangle”, which connects key parameters for assessing the effectiveness of providing methodical support to mathematics teachers: the amount of work, time and costs. Changing the value of one parameter leads to changes of the values of others. Full packaged product allows to balance these parameters and achieve the planned educational result.

Correction on Formation of Academic Mobility of Future Foreign Language Teachers by Means of Media Education Technologies

Relationship between mood disorders and inflammation is now well-documented, although molecular mechanisms are not understood. Previously mostly pro-inflammatory cytokines of immune system (IL-6, TNF, etc.) were taken into account. However, recent understanding of resolution of inflammation as an active process drew attention to mediators of resolution, which include both proteins and ω-3 and ω-6 polyunsaturated fatty acids derivatives (resolvins, cyclopentenone prostaglandins, etc.). This review takes into account new data on resolution of inflammation and action of mediators of resolution in models of depression. New facts and ideas about mechanisms of chronic inflammation onset are considered in relation to mood disorders. Basic control mechanisms of inflammation at the cellular level and the role of resolution substances in regulation of depression and other mood disorders are discussed. Signaling systems of innate immunity located in non-immune cells and their ability to generate substances that affect an onset of depression are reviewed. A novel hypothesis of depression as a type of abnormal resolution is proposed.

Recently, the modulation of cellular inflammatory responses via endogenous regulators became a major focus of medically relevant investigations. Prostaglandins (PGs) are attractive regulatory molecules, but their synthesis and mechanisms of action in brain cells are still unclear. Astrocytes are involved in manifestation of neuropathology and their proliferation is an important part of astrogliosis, a cellular neuroinflammatory response. The aims of our study were to measure synthesis of PGs by astrocytes, and evaluate their influence on proliferation in combination with addition of inflammatory pathway inhibitors. With UPLC-MS/MS analysis we detected primary PGs (1410 ± 36 pg/mg PGE2, 344 ± 24 PGD2) and cyclopentenone PGs (cyPGs) (87 ± 17 15d-PGJ2, 308 ± 23 PGA2) in the extracellular medium after 24-h lipopolysaccharide (LPS) stimulation of astrocytes. PGs reduced astrocytic proliferation with the following order of potencies (measured as inhibition at 20 μM): most potent 15d-PGJ2 (90%) and PGA2 (80%), > PGD2 (40%) > 15d-PGA2 (20%) > PGE2 (5%), the least potent. However, PGF2α and 2-cyclopenten-1-one, and ciglitazone and rosiglitazone (synthetic agonists of PPARγ) had no effect. Combinations of cyPGs with SC-560 or NS-398 (specific anti-inflammatory inhibitors of cyclooxygenase-1 and -2, respectively) were not effective; while GW9662 (PPARγ antagonist) or MK-741 (inhibitor of multidrug resistance protein-1, MRP1, and CysLT1 receptors) amplified the inhibitory effect of PGA2 and 15d-PGJ2. Although concentrations of individual PGs and cyPGs are low, all of them, as well as primary PGs suppress proliferation. Thus, the effects are potentially additive, and activated PGs synthesis suppresses proliferation in astrocytes.

Endotoxin tolerance (ET) represents a state of an altered immune response induced by multiple stimulations of a cell, a tissue, or an organism with lipopolysaccharide. Characteristics of ET include downregulation of induction of proinflammatory genes (TNFα, IL6, and others) and enhancement of induction of antiinflammatory genes (IL10, TGFβ). ET generally has protective functions; nevertheless, it might result in a state of innate immune deficiency and cause negative outcomes. A current issue is the search for the mechanisms controlling the level of inflammation in the course of endotoxin tolerance. In this work, we investigated the change in cyclooxygenase 2 (Cox2) expression in the model of endotoxin tolerance in astrocytes and analyzed the possibility of regulating this process applying nuclear receptor PPAR agonists. Our results indicate that: 1) endotoxin tolerance can be induced in astrocytes and results in TNFα and Cox2 mRNA induction decrease upon secondary stimulation; 2) tolerance is revealed on the level of TNFα release and Cox2 protein expression; 3) PPAR agonists GW7647, L-165041, and rosiglitazone control Cox2 mRNA expression levels under conditions of endotoxin tolerance. In particular, rosiglitazone (a PPARγ agonist) induces Cox2 mRNA expression, while GW7647 (a PPARα agonist) and L-165041 (a PPARβ agonist) suppress the expression. Our results demonstrate that Cox2 can be up- and downregulated during endotoxin tolerance in astrocytes, and PPAR agonists might be effective for controlling this target under conditions of multiple proinflammatory stimulations of brain tissues with endotoxin.

It has recently become clear that regulation of mRNA stability plays an important role in the development of cell responses to stimulation of toll-like receptors during inflammation. This discovery motivated a search for low molecular weight substances modulating the stability of mRNA encoding proteins involved in inflammatory responses. In this work, regulation of the cyclooxygenase 2 (COX-2) expression by rosiglitazone–a promising drug for regulation of inflammation–was studied on rat glioma cell line C6. Inflammatory response was induced by lipopolysaccaride (LPS). The concentration of prostaglandin E2 (PGE2) was measured by ELISA, the level of COX-2 mRNA was determined by real-time PCR. It was shown that treatment with LPS caused a 6-fold increase in the PGE2 synthesis, which correlated with an increase in the COX-2 mRNA expression. Rosiglitazone induced a 2-fold decrease of the LPS-stimulated PGE2 release and reduced the levels of COX-2 transcripts. To explore the molecular mechanisms of the rosiglitazone effect, we estimated the stability of COX-2 mRNA. Cells were incubated in the presence of LPS for 1 h, and then de novo mRNA transcription was blocked with actinomycin D. The levels of mRNA were determined at various time points. Treatment with rosiglitazone was carried out for 30 min before the LPS addition. The COX-2 mRNA half-life in native cells was found to be 75 min. LPS stimulation slowed down the mRNA degradation so that its half-life time was 120 min, and the treatment with rosiglitazone restored this process back to the normal level. The results suggest that rosiglitazone regulates the stability of COX-2 mRNA. This opens up new perspectives for therapeutic applications of this drug.

Peroxisome proliferator-activated receptors (PPAR)-α and -γ in astrocytes play important roles in inflammatory brain pathologies. Understanding the regulation of both activity and expression levels of PPARs is an important neuroscience issue. Toll-like receptor (TLR) agonists are inflammatory stimuli that could modulate PPAR, but the mechanisms of their control in astrocytes are poorly understood. In the present study, we report that lipopolysaccharide, peptidoglycan, and flagellin, which are agonists of TLR4, TLR1/2, and TLR5, respectively, exert time- and nuclear factor kappa-light-chain-enhancer of activated B cells-dependent suppression of mRNA, protein and activity of PPARα and PPARγ. In naïve astrocytes, PPARα and PPARγ mRNA have short turnover time (half-life about 30 min for PPARα, 75 min for PPARγ) with a nearly two-fold stabilization after TLR-activation. p38 inhibition abolished TLR-induced stabilization. The levels of PPARα and PPARγ mRNA, and protein and DNA-binding activity could be modified using c-Jun N-terminal Kinase and p38 inhibitors. In addition, the expression levels of both PPARα and PPARγ isotypes were induced after inhibition of protein synthesis. This induction signifies participation of additional regulatory proteins with short life-time. They are p38-sensitive for PPARα and c-Jun N-terminal Kinase-sensitive for PPARγ. Thus, PPARα and PPARγ are regulated in astrocytes on mRNA and protein levels, mRNA stability, and DNA-binding activity during TLR-mediated responses. Astrocytes have the triad of PPARα, PPARβ/δ, and PPARγ in regulation of proinflammatory responses. Activation of Toll-like receptors (TLR) leads to PPARβ/δ overexpression, PPARα and PPARγ suppression via TLR/NF-κB pathway on mRNA, protein and activity levels. Mitogen-activated protein kinases (MAPK) p38 and JNK are involved in regulation of PPAR expression. p38 MAPK plays a special role in stabilization of PPAR mRNA.

The information society reveals the social impact of the dissemination of information technologies. The purpose of this article is to study the negative social, psychological, and pedagogical implications of the Internet on young people. The authors analyzed the internet addiction problem among students (14-19 years) from the standpoint of the social health of the individuals and society as a whole. In a pilot study, which involved more than 600 adolescent participants aged 14-19 years (secondary school, college and university students), the authors have defined internet addiction as a complex phenomenon. The prerequisites of its development identify and highlights its formation in stages among students (mild fascination, passion, addiction, attachment). At the ascertaining stage of the experiment, a screening study was carried out to examine the state of internet addiction in young students in social networks. The results showed the necessity to design and implement internet addiction prevention programs for young students, which include three main blocks (motivational and cognitive, practice-oriented, reflexive), and a systematic plan for its implementation in the educational space framework. The stages of the experiment provided evidence of the authors’ proposed effectiveness of methodologies for young people aged 14-19 years. This article may be useful to pedagogues, psychologists, and parents of students, social workers, and researchers working in the field of addiction prevention among young people.

The purpose of this research is to identify efficient forms and means of academic mobility of future foreign language teachers. The leading research method that tests hypothesis is pedagogical experiment (ascertaining, forming and control stages of experiment) and also the method of processing of quantitative results of the research. 270 future foreign language teachers took part in the experimental research where 138 respondents belonged to an experimental group and 132 future teachers belonged to a control group. the model of formation of academic mobility of future foreign language teachers was designed and scientifically grounded. Formation of academic mobility is implemented by means of media-education technologies, methodological principles which include systemic, competency-based, synergetic, student-oriented approach and the following principles such as humanization, viability, self-education, succession, continuity, dialogism. The proposed model was realized in three stages (motivational-cognitive, organizational- action-based, professional-communicative) and provided for gradual acquisition by future educators the motivation for academic migration and their ability to master their foreign language communicative competence. The purpose of the above mentioned model is to develop such personal qualities which will contribute to students’ readiness to get education in foreign educational environment. The result of the realization of the model is the formation of the academic mobility of foreign language teachers on a creative level.

Control of decay of mRNA containing the adenine-uridine rich elements (AREs) is an important post-transcriptional mechanism involved in the regulation of inflammatory gene expression. Two widely recognized proteins in this machinery are HuR (human antigen R) - a protein that stabilizes ARE-containing mRNA and TTP (tristetraprolin) - a protein that shortens half-lives of ARE-containing mRNA. Although HuR and TTP regulation mechanisms have been well studied in cells of hematopoietic origin, there are no respective data in astrocytes, cells of ectodermal origin which play an important role in neuroinflammation. Therefore we evaluated the existence of TTP and HuR in primary astrocytes and characterized the features of their regulation after stimulation by the proinflammatory stimuli thrombin, ATP, and agonists of TLR4, TLR2. All proinflammatory stimuli increased levels of TTP mRNA, but not HuR mRNA. Transcripts of both HuR and TTP underwent stabilization upon lipopolysaccharide (LPS) treatment, measured with the actinomycin D protocol. This effect was abolished by treatment with SB203580, an inhibitor of р38 МАРК. Both TTP and HuR transcripts were sensitive to modulation by anisomycin and cycloheximide. LPS induced translocation of HuR protein from nucleus to cytoplasm. TTP is localized in the cytosolic fraction and localization is not sensitive to LPS treatment. Our data for the first time reveal specificity of regulation of ARE-binding proteins in astrocytes. We propose possibilities to manipulate brain inflammatory processes via post-transcription regulatory steps in astrocytes.

Hyaluronic acid (HA), a major glycosaminoglycan of the extracellular matrix, has cell signaling functions that are dependent on its molecular weight. Anti-inflammatory effects for high-molecular-weight (HMW) HA and pro-inflammatory effects for low-molecular-weight (LMW) HA effects were found for various myeloid cells, including microglia. Astrocytes are cells of ectodermal origin that play a pivotal role in brain inflammation, but the link between HA with different molecular weights and an inflammatory response in these cells is not clear. We tested the effects of LMW and HMW HA in rat primary astrocytes, stimulated with Poly:IC (PIC, TLR3 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Oxylipin profiles were measured by the UPLC-MS/MS analysis and metabolites HDoHEs (from docosahexaenoic acid), -HETEs, prostaglandins (from arachidonic acid), DiHOMEs and HODEs (from linoleic acid) were detected. Both, HMW and LMW HA downregulated the cyclooxygenase-mediated polyunsaturated fatty acids metabolism, LMW also reduced lipoxygenase-mediated fatty acid metabolism. Taken together, the data show that both LMW and HMW (i) influence themselves on cytokines (TNF&alpha, IL-6, IL-10), enzymes iNOS, COX-2, and oxylipin levels in extracellular medium of cultured astrocytes, (ii) induced cellular adaptations in long-term applications, (iii) modulate TLR4- and TLR3-signaling pathways. The effects of HMW and LMW HA are predominantly revealed in TLR4&ndash, and TLR3- mediated responses, respectively.

Project-based approach is the way to overcome the gap between school education and modern science. This pathway allows students to be a part of modern scientific research and obtain valuable results. The best efficiency would lead to full collaboration between school and research centers.

Peroxisome proliferator-activated receptors (PPARα, -β/δ and -γ) are lipid-activated transcription factors. Synthetic PPARα and PPARγ ligands have neuroprotective properties. Recently, PPARβ/δ activation emerged as the focus of a novel approach for the treatment of a wide range of neurodegenerative diseases. To fill the gap of knowledge about the role of PPARβ/δ in brain, new hypotheses about PPARβ/δ involvement in neuropathological processes are requested. In this paper, we describe a novel hypothesis, claiming the existence of tight interactions between the three PPAR isotypes, which we designate the “PPAR triad”. We propose that PPARβ/δ has a central control of the PPAR triad. The majority of studies analyze the regulation only by one of the PPAR isotypes. A few reports describe the mutual regulation of expression levels of all three PPAR isotypes by PPAR agonists. Analysis of these studies where pairwise interactions of PPARs were described allows us to support the existence of the PPAR triad with central role for PPARβ/δ. In the present review, we propose the hypothesis that in a wide range of brain disorders, PPARβ/δ plays a central role between PPARα and PPARγ. Finally, we prove the advantages of the PPAR triad concept by describing hypotheses of PPARβ/δ involvement in the regulation of myelination, glutamate-induced neurotoxicity, and signaling pathways of reactive oxygen species/NO/Ca 2+ .

Epidemiological studies have shown that severe inflammatory responses occur in patients with hyperglycemia. The molecular nature of these changes is currently under intense investigations. A central role of nuclear receptors PPAR has been shown in the regulation of metabolic changes associated with hyperglycemia, a selective agonist of nuclear receptor PPARγ rosiglitazone is used as a hypoglycemic drug. Rosiglitazone is known to have anti-inflammatory effects, but its properties as an anti-inflammatory drug in hyperglycemic conditions have not been studied. This was an aim of our work. We used a human cell culture model of hyperglycemia: HeLa cells incubated in the conditions of 25 mM glucose for 3 days. Control cells were incubated with 5 mM glucose. The cells were stimulated with lipopolysaccharide (LPS) that is known to trigger innate immune response through activation of Toll-like receptor 4 and influence mRNA expression levels of three of PPAR (α, β/δ, γ) isotypes as well as cyclooxygenase (COX-1 and COX-2). We have shown that under hyperglycemic conditions expression levels of PPARα and PPARβ/δ decreased almost twofold, expression level of COX-2 also decreased, while expression levels of COX-1 and PPARγ remained unchanged compared to those under normal glucose concentration. LPS administration in control cells leads to a 1.5–2.5-fold stimulation of expression of COX-2 and PPAR isotypes. In contrast, under hyperglycemia, LPS exhibited no effect on expression of COX-2 and the PPAR isotypes, which indicates potential mechanisms of hyperglycemia-related alterations in innate immunity. Rosiglitazone, an agonist of PPARγ, decreased expression level of PPARβ/δ and abolished the effect of LPS under hyperglycemia. Rosiglitazone also reduced expression level of COX-1 and COX-2, which indicates on the agonist possible role as an anti-inflammatory agent under high glucose concentrations. These data broaden applicability of rosiglitazone as an anti-inflammatory agent in hyperglycemic conditions.

Despite the importance of cytosolic phospholipase A(2) type IVA (cPLA(2)) and secretory PLA(2) (sPLA(2)) in physiological and pathological responses of astrocytes in inflammatory conditions, the regulation of the expression of these genes is still unclear. Both genes have peroxisome proliferator-activated receptors (PPAR) binding sites in their promoters. The role of synthetic PPAR agonists in the regulation of gene expression in naive and lipopolysaccharide (LPS)-stimulated rat astrocytes in culture was investigated. Exposure to LPS resulted in a time-dependent, fourfold transient increase of sPLA(2) expression, with maximum at 4 h; cPLA(2) expression was notably increased after 16-h LPS stimulation. Using selective PPARα, PPARβ/δ, and PPARγ agonists, we found that expression of both cPLA(2) and sPLA(2) is under PPAR control, but with different isotypes sensitivity. In naive astrocytes, all three PPAR agonists significantly suppressed the expression of sPLA(2), while only PPARα and PPARγ activation suppressed cPLA(2) expression. Astonishingly, simultaneous addition of LPS with PPAR agonists evoked the opposite effect. All three PPAR agonists induced potentiation of cPLA(2) expression level. Potentiation of sPLA(2) expression was induced only by simultaneous addition of LPS with PPARγ agonist. By knockdown of PPARα, PPARβ/δ, and PPARγ, we confirmed the involvement of PPAR-dependent pathways. The important novelty of our findings is that both sPLA(2) and cPLA(2) are under dichotomous control of PPARs: suppression in naive control cells, but induction in LPS-stimulated astrocytes.

Peroxisome proliferator-activated receptor (PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (alpha, beta/delta, and gamma) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPARalpha [2-[4-[2-[4-cyclohexylbutyl (cyclohexylcarbamoyl)amino]ethyl]phenyl] sulfanyl-2-methylpropanoic acid (GW7647)], PPARbeta/delta [4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy)propyloxyphenoxy acetic acid, (L-165041)], and PPARgamma (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) synthesis in lipopolysaccharide (LPS)-stimulated cells. PPARalpha, PPARbeta/delta, and PPARgamma knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPS-induced COX-2 expression via PPARgamma-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPARgamma activation through PPARbeta/delta-dependent mechanism. On the contrary, PPARalpha activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPARbeta/delta overexpression model confirmed that PPARbeta/delta expression level is the point at which PPARgamma and PPARalpha pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPARgamma has a positive influence and PPARalpha has a negative influence on PPARbeta/delta expression and activity. A positive/negative-feedback loop is formed by PPARbeta/delta-dependent increase in PPARalpha expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPARalpha,-beta/delta, and -gamma isoforms on the level of their expression.

The identification of orthologs to a set of known genes is often the starting point for evolutionary studies focused on gene families of interest. To date, the existing orthology detection tools (COG, InParanoid, OrthoMCL, etc.) are aimed at genome-wide ortholog identification and lack flexibility for the purposes of case studies. We developed a program OrthoFocus, which employs an extended reciprocal best hit approach to quickly search for orthologs in a pair of genomes. A group of paralogs from the input genome is used as the start for the forward search and the criterion for the reverse search, which allows handling many-to-one and many-to-many relationships. By pairwise comparison of genomes with the input species genome, OrthoFocus enables quick identification of orthologs in multiple genomes and generates a multiple alignment of orthologs so that it can further be used in phylogenetic analysis. The program is available at http://www.lipidomics.ru/.

In the current study, we reveal that in astrocytes the VIB Ca2+-independent phospholipase A2 is the enzyme responsible for the release of docosahexaenoic acid (22:6n-3). After pharmacological inhibition and siRNA silencing of VIB Ca2+-independent phospholipase A2, docosahexaenoic acid release was strongly suppressed in astrocytes, which were acutely stimulated (30 min) with ATP and glutamate or after prolonged (6 h) stimulation with the endotoxin lipopolysaccharide. Docosahexaenoic acid release proceeds simultaneously with arachidonic acid (20:4n-6) release and prostaglandin liberation from astrocytes. We found that prostaglandin production is negatively controlled by endogenous docosahexaenoic acid, since pharmacological inhibition and siRNA silencing of VIB Ca2+-independent phospholipase A2 significantly amplified the prostaglandin release by astrocytes stimulated with ATP, glutamate, and lipopolysaccharide. Addition of exogenous docosahexaenoic acid inhibited prostaglandin synthesis, which suggests that the negative control of prostaglandin synthesis observed here is likely due to competitive inhibition of cyclooxygenase-1/2 by free docosahexaenoic acid. Additionally, treatment of astrocytes with docosahexaenoic acid leads to the reduction in cyclooxygenase-1 expression, which also contributes to reduced prostaglandin production observed in lipopolysaccharide-stimulated cells. Thus, we identify a regulatory mechanism important for the brain, in which docosahexaenoic acid released from astrocytes by VIB Ca2+-independent phospholipase A2 negatively controls prostaglandin production.

Arachidonic acid (AA) and its metabolites (eicosanoids) represent powerful mediators, used by organisms to induce and suppress inflammation as a part of the innate response to disturbances. Several cell types participate in the synthesis and release of AA metabolites, while many cell types represent the targets for eicosanoid action. Endothelial cells (EC), forming a semi-permeable barrier between the interior space of blood vessels and underlying tissues, are of particular importance for the development of inflammation, since endothelium controls such diverse processes as vascular tone, homeostasis, adhesion of platelets and leukocytes to the vascular wall, and permeability of the vascular wall for cells and fluids. Proliferation and migration of endothelial cells contribute significantly to new vessel development (angiogenesis). This review discusses endothelial-specific synthesis and action of arachidonic acid derivatives with a particular focus on the mechanisms of signal transduction and associated intracellular protein targets.

1. Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2. Primary astrocytes were cultured under standard conditions and prelabeled with [(14)C]DHA or with [(3)H]AA. Adenosine 5'-triphosphate (ATP) (20 micro M applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca(2+)-independent phospholipase A(2) (iPLA(2)), since it is blocked by the selective iPLA(2) inhibitor 4-bromoenol lactone (BEL, 5 micro M) and is not affected either by removal of Ca(2+) from extracellular medium or by suppression of intracellular Ca(2+) release through PLC inhibitor (U73122, 5 micro M). 3. AA release, on the other hand, which is stimulated by ATP, is attributed to Ca(2+)-dependent cytosolic PLA(2) (cPLA(2)). AA release is abolished by U73122 and, by removal of extracellular Ca(2+), is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 micro M), a general inhibitor of intracellular PLA(2) s. 4. Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA(2) and 40 kDa protein reactive to iPLA(2) antibodies. 5. The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 micro M), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mM). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 micro M) and inhibited by 34% by H89 (10 micro M), inhibitors of adenylyl cyclase and PKA, respectively. 6. Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7. Different sensitivities of iPLA(2) and cPLA(2) to Ca(2+) and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

ATP-elicited oscillations of the concentration of free intracellular Ca(2+) ([Ca(2+)](i)) in rat brain astrocytes were abolished by simultaneous arachidonic acid (AA) addition, whereas the tetraenoic analogue 5,8,11,14-eicosatetraynoic acid (ETYA) was ineffective. Inhibition of oscillations is due to suppression by AA of intracellular Ca(2+) store refilling. Short-term application of AA, but not ETYA, blocked Ca(2+) influx, which was evoked by depletion of stores with cyclopiazonic acid (CPA) or thapsigargin (Tg). Addition of AA after ATP blocked ongoing [Ca(2+)](i) oscillations. Prolonged AA application without or with agonist could evoke a delayed [Ca(2+)](i) increase. This AA-induced [Ca(2+)](i) rise developed slowly, reached a plateau after 5 min, could be reversed by addition of bovine serum albumin (BSA), that scavenges AA, and was blocked by 1 microM Gd(3+), indicative for the influx of extracellular Ca(2+). Specificity for AA as active agent was demonstrated by ineffectiveness of C16:0, C18:0, C20:0, C18:2, and ETYA. Moreover, the action of AA was not affected by inhibitors of oxidative metabolism of AA (ibuprofen, MK886, SKF525A). Thus, AA exerted a dual effect on astrocytic [Ca(2+)](i), firstly, a rapid reduction of capacitative Ca(2+) entry thereby suppressing [Ca(2+)](i) oscillations, and secondly inducing a delayed activation of Ca(2+) entry, also sensitive to low Gd(3+) concentration.