Primary and Secondary micro-RNA Modulation the Extrinsic Pathway of Apoptosis in Hepatocellular Carcinoma.

Abstract-One of the most common malignant liver diseases is hepatocellular carcinoma, which has a high recurrence rate and a low five-year survival rate. It is very heterogeneous both in structure and between patients, which complicates the diagnosis, prognosis and response to treatment. In this regard, an individualized, patient-centered approach becomes important, in which the use of mimetics and hsa-miRNA inhibitors involved in the pathogenesis of the disease may be determinative. From this point of view hsa-miRNAs are of interest, their aberrant expression is associated with poor prognosis for patients and is associated with tumor progression due to dysregulation of programmed cell death (apoptosis). However, the effect of hsa-miRNA on tumor development depends not only on its direct effect on expression of genes, the primary targets, but also on secondary targets mediated by regulatory pathways. While the former are actively studied, the role of secondary targets of these hsa-miRNAs in modulating apoptosis is still unclear. The present work summarizes data on hsa-miRNAs whose primary targets are key genes of the extrinsic pathway of apoptosis. Their aberrant expression is associated with early disease relapse and poor patient outcome. For these hsa-miRNAs, using the software package ANDSystem, we reconstructed the regulation of the expression of secondary targets and analyzed their impact on the activity of the extrinsic pathway of apoptosis. The potential effect of hsa-miRNAs mediated by action on secondary targets is shown to negatively correlate with the number of primary targets. It is also shown that hsa-miR-373, hsa-miR-106b and hsa-miR-96 have the highest priority as markers of hepatocellular carcinoma, whose action on secondary targets enhances their anti-apoptotic effect.

[Primary and Secondary micro-RNA Modulation the Extrinsic Pathway of Apoptosis in Hepatocellular Carcinoma].

One of the most common malignant liver diseases is hepatocellular carcinoma, which has a high recurrence rate and a low five-year survival rate. It is very heterogeneous both in structure and between patients, which complicates the diagnosis, prognosis and response to treatment. In this regard, an individualized, patient-centered approach becomes important, in which the use of mimetics and hsa-miRNA inhibitors involved in the pathogenesis of the disease may be determinative. From this point of view hsa-miRNAs are of interest, their aberrant expression is associated with poor prognosis for patients and is associated with tumor progression due to dysregulation of programmed cell death (apoptosis). However, the effect of hsa-miRNA on tumor development depends not only on its direct effect on expression of genes, the primary targets, but also on secondary targets mediated by regulatory pathways. While the former are actively studied, the role of secondary targets of these hsa-miRNAs in modulating apoptosis is still unclear. The present work summarizes data on hsa-miRNAs whose primary targets are key genes of the extrinsic pathway of apoptosis. Their aberrant expression is associated with early disease relapse and poor patient outcome. For these hsa-miRNAs, using the software package ANDSystem, we reconstructed the regulation of the expression of secondary targets and analyzed their impact on the activity of the extrinsic pathway of apoptosis. The potential effect of hsa-miRNAs mediated by action on secondary targets is shown to negatively correlate with the number of primary targets. It is also shown that hsa-miR-373, hsa-miR-106b and hsa-miR-96 have the highest priority as markers of hepatocellular carcinoma, whose action on secondary targets enhances their anti-apoptotic effect.

Prioritization of potential pharmacological targets for the development of anti-hepatocarcinoma drugs modulating the extrinsic apoptosis pathway: the reconstruction and analysis of associative gene networks help.

Hepatocellular carcinoma (HCC) is a common severe type of liver cancer characterized by an extremely aggressive course and low survival rates. It is known that disruptions in the regulation of apoptosis activation are some of the key features inherent in most cancer cells, which determines the pharmacological induction of apoptosis as an important strategy for cancer therapy. The computer design of chemical compounds capable of specifically regulating the external signaling pathway of apoptosis induction represents a promising approach for creating new effective ways of therapy for liver cancer and other oncological diseases. However, at present, most of the studies are devoted to pharmacological effects on the internal (mitochondrial) apoptosis pathway. In contrast, the external pathway induced via cell death receptors remains out of focus. Aberrant gene methylation, along with hepatitis C virus (HCV) infection, are important risk factors for the development of hepatocellular carcinoma. The reconstruction of gene networks describing the molecular mechanisms of interaction of aberrantly methylated genes with key participants of the extrinsic apoptosis pathway and their regulation by HCV proteins can provide important information when searching for pharmacological targets. In the present study, 13 criteria were proposed for prioritizing potential pharmacological targets for developing anti-hepatocarcinoma drugs modulating the extrinsic apoptosis pathway. The criteria are based on indicators of the structural and functional organization of reconstructed gene networks of hepatocarcinoma, the extrinsic apoptosis pathway, and regulatory pathways of virus-extrinsic apoptosis pathway interaction and aberrant gene methylation-extrinsic apoptosis pathway interaction using ANDSystem. The list of the top 100 gene targets ranked according to the prioritization rating was statistically significantly (p-value = 0.0002) enriched for known pharmacological targets approved by the FDA, indicating the correctness of the prioritization method. Among the promising potential pharmacological targets, six highly ranked genes (JUN, IL10, STAT3, MYC, TLR4, and KHDRBS1) are likely to deserve close attention.

Rational metabolic engineering of Corynebacterium glutamicum to create a producer of L-valine.

L-Valine is one of the nine amino acids that cannot be synthesized de novo by higher organisms and must come from food. This amino acid not only serves as a building block for proteins, but also regulates protein and energy metabolism and participates in neurotransmission. L-Valine is used in the food and pharmaceutical industries, medicine and cosmetics, but primarily as an animal feed additive. Adding L-valine to feed, alone or mixed with other essential amino acids, allows for feeds with lower crude protein content, increases the quality and quantity of pig meat and broiler chicken meat, as well as improves reproductive functions of farm animals. Despite the fact that the market for L-valine is constantly growing, this amino acid is not yet produced in our country. In modern conditions, the creation of strains-producers and organization of L-valine production are especially relevant for Russia. One of the basic microorganisms most commonly used for the creation of amino acid producers, along with Escherichia coli, is the soil bacterium Corynebacterium glutamicum. This review is devoted to the analysis of the main strategies for the development of L- valine producers based on C. glutamicum. Various aspects of L-valine biosynthesis in C. glutamicum are reviewed: process biochemistry, stoichiometry and regulation, enzymes and their corresponding genes, export and import systems, and the relationship of L-valine biosynthesis with central cell metabolism. Key genetic elements for the creation of C. glutamicum-based strains-producers are identified. The use of metabolic engineering to enhance L-valine biosynthesis reactions and to reduce the formation of byproducts is described. The prospects for improving strains in terms of their productivity and technological characteristics are shown. The information presented in the review can be used in the production of producers of other amino acids with a branched side chain, namely L-leucine and L-isoleucine, as well as D-pantothenate.

Computer analysis of regulation of hepatocarcinoma marker genes hypermethylated by HCV proteins.

Hepatitis C virus (HCV) is a risk factor that leads to hepatocellular carcinoma (HCC) development. Epigenetic changes are known to play an important role in the molecular genetic mechanisms of virus-induced oncogenesis. Aberrant DNA methylation is a mediator of epigenetic changes that are closely associated with the HCC pathogenesis and considered a biomarker for its early diagnosis. The ANDSystem software package was used to reconstruct and evaluate the statistical significance of the pathways HCV could potentially use to regulate 32 hypermethylated genes in HCC, including both oncosuppressor and protumorigenic ones identified by genome-wide analysis of DNA methylation. The reconstructed pathways included those affecting protein-protein interactions (PPI), gene expression, protein activity, stability, and transport regulations, the expression regulation pathways being statistically significant. It has been shown that 8 out of 10 HCV proteins were involved in these pathways, the HCV NS3 protein being implicated in the largest number of regulatory pathways. NS3 was associated with the regulation of 5 tumor-suppressor genes, which may be the evidence of its central role in HCC pathogenesis. Analysis of the reconstructed pathways has demonstrated that following the transcription factor inhibition caused by binding to viral proteins, the expression of a number of oncosuppressors (WT1, MGMT, SOCS1, P53) was suppressed, while the expression of others (RASF1, RUNX3, WIF1, DAPK1) was activated. Thus, the performed gene-network reconstruction has shown that HCV proteins can influence not only the methylation status of oncosuppressor genes, but also their transcriptional regulation. The results obtained can be used in the search for pharmacological targets to develop new drugs against HCV-induced HCC.

The molecular view of mechanical stress of brain cells, local translation, and neurodegenerative diseases.

The assumption that chronic mechanical stress in brain cells stemming from intracranial hypertension, arterial hypertension, or mechanical injury is a risk factor for neurodegenerative diseases was put forward in the 1990s and has since been supported. However, the molecular mechanisms that underlie the way from cell exposure to mechanical stress to disturbances in synaptic plasticity followed by changes in behavior, cognition, and memory are still poorly understood. Here we review (1) the current knowledge of molecular mechanisms regulating local translation and the actin cytoskeleton state at an activated synapse, where they play a key role in the formation of various sorts of synaptic plasticity and long-term memory, and (2) possible pathways of mechanical stress intervention. The roles of the mTOR (mammalian target of rapamycin) signaling pathway; the RNA-binding FMRP protein; the CYFIP1 protein, interacting with FMRP; the family of small GTPases; and the WAVE regulatory complex in the regulation of translation initiation and actin cytoskeleton rearrangements in dendritic spines of the activated synapse are discussed. Evidence is provided that chronic mechanical stress may result in aberrant activation of mTOR signaling and the WAVE regulatory complex via the YAP/TAZ system, the key sensor of mechanical signals, and influence the associated pathways regulating the formation of F actin filaments and the dendritic spine structure. These consequences may be a risk factor for various neurological conditions, including autistic spectrum disorders and epileptic encephalopathy. In further consideration of the role of the local translation system in the development of neuropsychic and neurodegenerative diseases, an original hypothesis was put forward that one of the possible causes of synaptopathies is impaired proteome stability associated with mTOR hyperactivity and formation of complex dynamic modes of de novo protein synthesis in response to synapse-stimulating factors, including chronic mechanical stress.

Causes of global extinctions in the history of life: facts and hypotheses.

Paleontologists define global extinctions on Earth as a loss of about three-quarters of plant and animal species over a relatively short period of time. At least five global extinctions are documented in the Phanerozoic fossil record (~500-million-year period): ~65, 200, 260, 380, and 440 million years ago. In addition, there is evidence of global extinctions in earlier periods of life on Earth - during the Late Cambrian (~500 million years ago) and Ediacaran periods (more than 540 million years ago). There is still no common opinion on the causes of their occurrence. The current study is a systematized review of the data on recorded extinctions of complex life forms on Earth from the moment of their occurrence during the Ediacaran period to the modern period. The review discusses possible causes for mass extinctions in the light of the influence of abiogenic factors, planetary or astronomical, and the consequences of their actions. We evaluate the pros and cons of the hypothesis on the presence of periodicity in the extinction of Phanerozoic marine biota. Strong evidence that allows us to hypothesize that additional mechanisms associated with various internal biotic factors are responsible for the emergence of extinctions in the evolution of complex life forms is discussed. Developing the idea of the internal causes of periodicity and discontinuity in evolution, we propose our own original hypothesis, according to which the bistability phenomenon underlies the complex dynamics of the biota development, which is manifested in the form of global extinctions. The bistability phenomenon arises only in ecosystems with predominant sexual reproduction. Our hypothesis suggests that even in the absence of global abiotic catastrophes, extinctions of biota would occur anyway. However, our hypothesis does not exclude the possibility that in different periods of the Earth's history the biota was subjected to powerful external influences that had a significant impact on its further development, which is reflected in the Earth's fossil record.

[Molecular Mechanisms of Non-Inherited Antibiotic Tolerance in Bacteria and Archaea].

The phenomenon of bacterial persistence, also known as non-inherited antibiotic tolerance in a part of bacterial populations, was described more than 70 years ago. This type of tolerance contributes to the chronization of infectious diseases, including tuberculosis. Currently, the emergence of persistent cells in bacterial populations is associated with the functioning of some stress-induced molecular triggers, including toxin-antitoxin systems. In the presented review, genetic and metabolic peculiarities of persistent cells are considered and the mechanisms of their occurrence are discussed. The hypothesis of the origin of persister cells based on bistability, arising due to the non-linear properties of a coupled transcription-translation system, was proposed. Within this hypothesis, the phenomenon of the bacterial persistence of modern cells is considered as a result of the genetic fixation of the phenotypic multiplicity that emerged in primitive cells in the process of neutrally coupled co-evolution (genetic drift of multiple neutrally coupled mutations). Our hypothesis explains the properties of persister cells, as well as their origin and "ineradicable" nature.

Persister Cells - a Plausible Outcome of Neutral Coevolutionary Drift.

The phenomenon of bacterial persistence - a non-inherited antibiotic tolerance in a minute fraction of the bacterial population, was observed more than 70 years ago. Nowadays, it is suggested that "persister cells" undergo an alternative scenario of the cell cycle; however, pathways involved in its emergence are still not identified. We present a mathematically grounded scenario of such possibility. We have determined that population drift in the space of multiple neutrally coupled mutations, which we called "neutrally coupled co-evolution" (NCCE), leads to increased dynamic complexity of bacterial populations via appearance of cells capable of carrying out a single cell cycle in two or more alternative ways and that universal properties of the coupled transcription-translation system underlie this phenotypic multiplicity. According to our hypothesis, modern persister cells have derived from such cells and regulatory mechanisms that govern the consolidation of this phenomenon represented the trigger. We assume that the described type of neutrally coupled co-evolution could play an important role in the origin of extremophiles, both in bacteria and archaea.

Dynamic landscape of the local translation at activated synapses.

The mammalian target of rapamycin (mTOR) signaling pathway is the central regulator of cap-dependent translation at the synapse. Disturbances in mTOR pathway have been associated with several neurological diseases, such as autism and epilepsy. RNA-binding protein FMRP, a negative regulator of translation initiation, is one of the key components of the local translation system. Activation and inactivation of FMRP occurs via phosphorylation by S6 kinase and dephosphorylation by PP2A phosphatase, respectively. S6 kinase and PP2A phosphatase are activated in response to mGluR receptor stimulation through different signaling pathways and at different rates. The dynamic aspects of this system are poorly understood. We developed a mathematical model of FMRP-dependent regulation of postsynaptic density (PSD) protein synthesis in response to mGluR receptor stimulation and conducted in silico experiments to study the regulatory circuit functioning. The modeling results revealed the possibility of generating oscillatory (cyclic and quasi-cyclic), chaotic and even hyperchaotic dynamics of postsynaptic protein synthesis as well as the presence of multiple attractors in a wide range of parameters of the local translation system. The results suggest that autistic disorders associated with mTOR pathway hyperactivation may be due to impaired proteome stability associated with the formation of complex dynamic regimes of PSD protein synthesis in response to stimulation of mGluR receptors on the postsynaptic membrane of excitatory synapses on pyramidal hippocampal cells.

[New evidence of an old problem: the coupling of genome replication to cell growth in bacteria].

The problem of coordinating genome replication with cell growth in bacteria was posed over four decades ago. Unlike for eukaryotes, this problem has not been completely solved even for Escherichia coli, which has been comprehensively studied by molecular biologists, to say nothing of other bacteria. Current models of the bacterial life cycle solve the coupling problem by introducing a phenomenological hypothesis that considers the dynamic coordination of growth and replication but does not unveil the underlying molecular mechanisms. Here we review the mechanisms regulating genome replication initiation with regards to their coupling to growth processes in the three best investigated bacterial species: E. coli, Bacillus subtilis, and Caulobacter crescentus. A putative correlation between the type of cell growth laws and the actual mechanisms regulating the replication of DNA formed during the process of evolution in various classes of bacteria, is discussed, including those intracellular parasites in which degenerative evolution his discarded most of their genomes. We contemplate the concept of a universal growth law for bacterial cells and some features in the formation of a primitive negative replication regulating mechanism in the context of the coupling problem.

[A mathematical model of the intracellular reproduction of the influenza virus].

A mathematical model of the intracellular reproduction of the influenza virus has been constructed, which describes the regulation of transcription, translation, replication, and assembly of the virus in a sensitive noninterferonogenic cell. A structural and parametric verification of the model has been performed, which involves an analysis of alternative mechanisms of regulation of the key events of the reproduction of the influenza virus at the stages of the primary and secondary transcription. It was shown that the dynamics of the concentrations of the system components is in qualitative agreement with the experimental data. An analysis of the model parameters showed a high sensitivity of the model to changes in the parameters that control the efficiency of the binding of the viral polymerase to virus-specific RNAs at the stages of initiation and elongation of transcription and replication of the viral genome. The results obtained suggest that the stages of the virus reproduction sensitive to changes in the parameters are potential targets for drugs. Using the model, a minimal multiplicity of infection at which the formation of viral particles is observed was predicted.

[Statistical analysis of nucleosome formation sites].

The prediction of the DNA capacity to form nucleosome structure based on sequence statistics is of importance in the analysis of gene expression regulation in eukaryotes. A context analysis of nucleotide sequences of experimentally defined sites of nucleosome formation made it possible to determine the sequence preference for nucleosome formation on the basis of statistical information. An improved version of the Markov model was developed to predict the preference of DNA sequences to be within a nucleosome structure. The developed VMM (Variable Memory Markov model) program computes the nucleosome formation potential for genomic DNA sequences of arbitrary lengths, including the short transcription factor binding sites. Differences in nucleosome potential for exons, introns, and promoters were revealed. A correlation of the nucleosome potential estimate with text complexity was established. The VMM is available at http://wwwmgs. bionet.nsc.ru/programs/VMM/.

SITECON: a tool for detecting conservative conformational and physicochemical properties in transcription factor binding site alignments and for site recognition.

The local DNA conformation in the region of transcription factor binding sites, determined by context, is one of the factors underlying the specificity of DNA-protein interactions. Analysis of the local conformation of a set of functional DNA sequences may allow for determination of the conservative conformational and physicochemical parameters reflecting molecular mechanisms of interaction. The web resource SITECON is designed to detect conservative conformational and physicochemical properties in transcription factor binding sites, contains a knowledge base of conservative properties for >100 high-quality sample sites and allows for recognition of potential transcription factor binding sites based on conservative properties from both the knowledge base and the results of analysis of a sample proposed by a user. The resource SITECON is available at http://wwwmgs.bionet.nsc.ru/mgs/programs/sitecon/.

[Age-related specifics of aldosterone reception in distal segments of the rat nephrons and of Na(+), K(+)-ATPase expression induction]. / Vozrastnye osobennosti retseptsii al'dosterona v distal'nykh segmentakh nefronov krys i induktsii ékspressii Na(+), K(+)-ATFazy.

The reason of unresponsiveness of young 10-day rats kidney to aldosterone was explored. The aldosterone binding in distal segments of renal nephrons and the influence of hormonal induction on the mRNA of the alpha and beta subunits of the Na+, K(+)-ATPase in 10-day and 2-month old rats were investigated. There was no age related difference in the aldosterone specific binding in presence of RU-38486 (10(-7) M; Russel Uclaf) in the cortical collecting tubules: 0.26 +/- 0.04 (n = 9) and 0.22 +/- 0.03 (n = 8) mMol/mm of tubule lengths in 10 day and adult rats, respectively. By Nozern blot analysis and RT-PCR more then three and two fold increase of the mRNA abundance of both subunits was found in young and adult renal cortex compare to the adrenalectomized control after aldosterone induction (5 micrograms/100 g. v. b. w. 4 times i/p injections in 3 hour interval between injections) (p < 0.01). By RT-PCR no expression of the alpha 2, alpha 3 and beta 2 isoforms has been observed in all experimental conditions. The age difference was discovered when aldosterone was injected together with spironolactone (5 micrograms and 12 mg per 100 g. b. w. respectively). It was shown, that spironolactone inhibits the effect of aldosterone in adults whereas the latter was unaffected in young rats. The scheme of the age-related differences in the aldosterone regulation of the sodium pump induction in the target cell of the distal part of the rat nephrons is presented.

[How cells protect themselves against stress?]. / Kak kletki zashchishchaiutsia ot stressa?

The current evidence on the mechanisms underlying cell response to heat shock is reviewed. The response dynamics, induction, and attenuation as well as heat shock proteins and the mechanisms through which they protect cells from stress are considered. The role of these proteins in regulating the signaling cascades, including apoptosis suppression, is shown.

[The dopamine system stress reaction in adults of Drosophila virilis is controlled by genes of Chromosome 6]. / Stress-reaktivnost' sistemy dofamina u imago Drosophila virilis kontroliruetsia genom khromosomy 6.

A linkage group of the gene responsible for changes of DA titer under stress in adults of D. virilis was determined. Line 160 of D. virilis, all autosomes of which bear visible recessive mutations, was used as an analyzer. Flies of lines 160 and 147 were shown to differ in DA content under normal conditions and in the way DA metabolic system reacts to stress. Among the offspring of the analyzing cross, groups of flies were found with one testable autosome from line 147 and all other chromosomes from line 160. Results of the DA titer measurements in flies of these groups under stress conditions have proved that the gene in question is linked to chromosome 6.

[Some regularities in the genetic control of the stress reaction in Drosophila]. / O nekotorykh zakonomernostiakh geneticheskogo kontrolia stress-reaktsii drozofily.

The heat-shock response was studied in Drosophila virilis strains with normal and impaired neurohormonal stress reaction. Flies from the latter strain were shown to have the impaired heat-shock response. In this strain, transcription of the heat shock gene hsp83 is reduced and synthesis of all heat shock proteins is suppressed. The neurohormonal stress reaction (status of dopamine, octopamine, and juvenile hormone metabolic systems) was examined in D. melanogaster strains having normal and impaired heat-shock response. The impairment of this response did not prevent the development of the stress reaction: in flies of both strains, the stress exposure resulted in an increase in the dopamine content and in a decrease in the activity of tyrosine decarboxylase (the first enzyme of dopamine synthesis) and in the level of juvenile hormone degradation. However, stress reactivity in mutant individuals differed from that in flies that did not carry stress-related mutations.

[The heat-shock reaction is disturbed in a Drosophila virilis strain incapable of a neurohormonal stress reaction]. / Reaktsiia teplovogo shoka narushena u ne sposobnoi k neirogormonal'noi stress-reaktsii linii Drosophila virilis.

Cellular stress response was investigated in two lines of D. virilis: wild type and line with disturbed neurohormonal stress-reaction. Analysis of proteins, synthesized in salivary glands of larvae of both lines under heat stress, revealed malfunction in heat shock reaction of mutant specimen. This malfunction expresses in decreased level of heat shock protein synthesis. Analysis of electrophoretic spectra of proteins from homogenates of imagoes of both lines maintained under normal conditions and those exposed to heat (38 degrees C, 60 min.) revealed correlation between protein spectrum and physiological state of the organism. Interlinear differences by proteins spectra in normal condition, controlled by a single gene (or by block of closely linked genes), were found. The question if there is a common genetic control for the neurohormonal stress-reaction and cellular stress response is discussed.