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The Faculty of Computer Systems and Technology at Technical University – Sofia undertook analyses and experiments on the use of Artificial Intelligence methods in the field of Information Security. In the course of the study a Cyber Defence related to the classification consisting of three phases has been proposed: Operative Cyber Intelligence, Tactical Cyber Intelligence and Incident Handling. It has been found that there is no universal Artificial Intelligence method effective for all phases mentioned above and for all applications. In each case, a set of criteria should be developed to select (and then experiment) an appropriate method (or combination of methods). A selection of methods for the first two stages of Cyber Defence was described in previous work. In the present paper are considered some of the considerations related to effective use of appropriate methods for the Incident Handling phase.

The Drosophila abnormal spindle ( asp ) gene was discovered about 40 years ago and shown to be required for both mitotic and meiotic cell division. Subsequent studies showed that asp is highly conserved and that mutations in its human ortholog ASPM ( Abnormal Spindle-like Microcephaly-associated ; or MCPH5 ) are the most common cause of autosomal recessive primary microcephaly. This finding greatly stimulated research on ASPM and its fly and mouse ( Aspm ) orthologs. The three Asp orthologous proteins bind the microtubules (MTs) minus ends during cell division and also function in interphase nuclei. Investigations on different cell types showed that Asp/Aspm/ASPM depletion disrupts one or more of the following mitotic processes: aster formation, spindle pole focusing, centrosome-spindle coupling, spindle orientation, metaphase-to-anaphase progression, chromosome segregation, and cytokinesis. In addition, ASPM physically interacts with components of the DNA repair and replication machineries and is required for the maintenance of chromosomal DNA stability. We propose the working hypothesis that the asp / Aspm / ASPM genes play the same conserved functions in Drosophila , mouse, and human cells. Human microcephaly is a genetically heterogeneous disorder caused by mutations in 30 different genes that play a variety of functions required for cell division and chromosomal DNA integrity. Our hypothesis postulates that ASPM recapitulates the functions of most human microcephaly genes and provides a justification for why ASPM is the most frequently mutated gene in autosomal recessive primary microcephaly., Competing Interests: The authors declare no conflict of interest.

Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and Drosophila cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal Drosophila S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: mast / orbit / chb ( CLASP1 ), mei-38 ( TPX2 ), mars ( HURP ), dgt6 ( HAUS6 ), Eb1 ( MAPRE1/EB1 ), Patronin ( CAMSAP2 ), asp ( ASPM ), and Klp10A ( KIF2A ). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.

Moonlighting proteins can perform one or more additional functions besides their primary role. It has been posited that a protein can acquire a moonlighting function through a gradual evolutionary process, which is favored when the primary and secondary functions are exerted in different cellular compartments. Transcription factors (TFs) and splicing factors (SFs) control processes that occur in interphase nuclei and are strongly reduced during cell division, and are therefore in a favorable situation to evolve moonlighting mitotic functions. However, recently published moonlighting protein databases, which comprise almost 400 proteins, do not include TFs and SFs with secondary mitotic functions. We searched the literature and found several TFs and SFs with bona fide moonlighting mitotic functions, namely they localize to specific mitotic structure(s), interact with proteins enriched in the same structure(s), and are required for proper morphology and functioning of the structure(s). In addition, we describe TFs and SFs that localize to mitotic structures but cannot be classified as moonlighting proteins due to insufficient data on their biochemical interactions and mitotic roles. Nevertheless, we hypothesize that most TFs and SFs with specific mitotic localizations have either minor or redundant moonlighting functions, or are evolving towards the acquisition of these functions.

Mammalian oviducts contain smooth muscles and inward-facing ciliated epithelium. Muscular contractions, not ciliary beating, propel oocytes through the oviduct towards the uterus. In crawling gastropods (unique models for studying the functioning of phasic smooth muscles), muscular contractions, propagating along the foot sole, play a principal role in determining the crawling rate. We have described the muscular mechanisms controlling the crawling rate and hypothesize here that the same mechanisms provide embryo transportation through the human fallopian tube. The data collected for gastropods were transferred to the human fallopian tube, using embryo speed and tube length (tonus) instead of crawling speed and sole length. Smooth muscle active states: tonic contraction/relaxation and rhythmic contractions (peristalsis) are involved in ovum transport. The ovum/embryo speed is linearly correlated with the tonus of the longitudinal and/or the circular muscles of the tube. Some known bioactive substances control muscular tonus and we suppose embryo speed through the contraction force of muscle cells involved in peristaltic waves. Other known substances facilitate peristalsis and we suppose that they have no effect on muscular tonus and increase dose-dependently embryo speed through the number of muscle cells recruited in peristaltic waves (at a constant wave frequency). This number depends on the physiological state of the woman. The combination of all possible effects and the ability of muscles to contract rhythmically determine the embryo speed and its implantation site. This hypothesis is the first description of the tubal muscular mechanisms that determine normal and any type of ectopic pregnancy at chronic disorders of tubal contractility. How to predict ectopic pregnancy? We have reason to assume that the intensity of rhythmic uterine contractions reflects that in the fallopian tube. It is known that uterine contractility significantly increases during the preovulatory phase and decreases during the mid-luteal phase. The hypothesis supposes that in women with increased risk of ectopic pregnancy, decreased uterine contractility during the preovulatory phase in comparison with the norm indicates a future tubal/abdominal gestation; increased uterine contractility during this phase and/or the mid-luteal phase as compared to the norm points to a future cervical pregnancy. The optimal methods for recording uterine activity are up to clinicians., (Copyright © 2019. Published by Elsevier Ltd.)

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins., Competing Interests: The authors have declared that no competing interests exist.

Background: The calmodulin-regulated spectrin-associated proteins (CAMSAPs) belong to a conserved protein family, which includes members that bind the polymerizing mcrotubule (MT) minus ends and remain associated with the MT lattice formed by minus end polymerization. Only one of the three mammalian CAMSAPs, CAMSAP1, localizes to the mitotic spindle but its function is unclear. In Drosophila, there is only one CAMSAP, named Patronin. Previous work has shown that Patronin stabilizes the minus ends of non-mitotic MTs and is required for proper spindle elongation. However, the precise role of Patronin in mitotic spindle assembly is poorly understood., Results: Here we have explored the role of Patronin in Drosophila mitosis using S2 tissue culture cells as a model system. We show that Patronin associates with different types of MT bundles within the Drosophila mitotic spindle, and that it is required for their stability. Imaging of living cells expressing Patronin-GFP showed that Patronin displays a dynamic behavior. In prometaphase cells, Patronin accumulates on short segments of MT bundles located near the chromosomes. These Patronin "seeds" extend towards the cell poles and stop growing just before reaching the poles. Our data also suggest that Patronin localization is largely independent of proteins acting at the MT minus ends such as Asp and Klp10A., Conclusion: Our results suggest a working hypothesis about the mitotic role of Patronin. We propose that Patronin binds the minus ends within MT bundles, including those generated from the walls of preexisting MTs via the augmin-mediated pathway. This would help maintaining MT association within the mitotic bundles, thereby stabilizing the spindle structure. Our data also raise the intriguing possibility that the minus ends of bundled MTs can undergo a limited polymerization.

Background: Expression of the CNDP2 gene is frequently up- or down-regulated in different types of human cancers. However, how the product of this gene is involved in cell growth and proliferation is poorly understood. Moreover, our knowledge of the functions of the CNDP2 orthologs in well-established model organisms is scarce. In particular, the function of the D. melanogaster ortholog of CNDP2, encoded by the CG17337 gene (hereafter referred to as dCNDP2), is still unknown., Results: This study was aimed at developing a set of genetic and molecular tools to study the roles of dCNDP2. We generated a dCNDP2 null mutation (hereafter ∆dCNDP2) using CRISPR/Cas9-mediated homologous recombination (HR) and found that the ∆dCNDP2 mutants are homozygous viable, morphologically normal and fertile. We also generated transgenic fly lines expressing eGFP-tagged and non-tagged dCNDP2 protein, all under the control of the UAS promoter, as well as polyclonal antibodies specific to dCNDP2. Using these tools, we demonstrate that only one of the two predicted dCNDP2 isoforms is expressed throughout the different tissues tested. dCNDP2 was detected in both the cytoplasm and the nucleus, and was found to be associated with multiple sites in the salivary gland polytene chromosomes., Conclusions: The dCNDP2 gene is not essential for fly viability under standard laboratory conditions. The subcellular localization pattern of dCNDP2 suggests that this protein might have roles in both the cytoplasm and the nucleus. The genetic and molecular tools developed in this study will allow further functional characterization of the conserved CNDP2 protein using D. melanogaster as a model system.

Crawling gastropods are unique models for studying the functioning of smooth muscles and ciliated epithelia, since they cover the foot sole and are involved in locomotion, allowing for direct investigation. Two types of crawling are known: creeping by muscular waves in terrestrial gastropods such as Helix and сiliary gliding in aquatic gastropods such as Lymnaea. It was found that the smooth muscles that underlie the ciliated epithelium in Lymnaea are involved in gliding and contribute significantly to fast crawling. Thus, the locomotor apparatus is fundamentally the same in both snails and the difference between crawling reflects an adaptation to a habitat. The control of crawling speed is also the same. Tonic contraction, relaxation, and rhythmic contractions are involved in this control. During a locomotor episode, the sole length and crawling speed spontaneously change and directly correlate with each other via the contraction force of the muscle cells in the locomotory waves. Dopamine, unlike ergometrine, decreases the sole length and crawling speed. Serotonin stimulates, increases crawling and determines the number of muscle cells involved in the locomotory waves for each locomotor episode. This control (taking into account heterogeneity) apparently might exist in any other phasic smooth muscle, including vertebrates.

Background: S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM)., Results: We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase., Conclusions: We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.

The Drosophila S2 tissue culture cells are a widely used system for studies on mitosis. S2 cells are particularly sensitive to gene silencing by RNA interference (RNAi), allowing targeted inactivation of mitotic genes. S2 cells are also well suited for high-resolution light microscopy analysis of mitosis in fixed cells, and can be easily immunostained to detect mitotic components. In addition, S2 cells are amenable to transformation with plasmid encoding fluorescently tagged mitotic proteins, allowing in vivo analysis of their behavior throughout cell division. However, S2 cells have not been widely used for transmission electron microscopy (TEM) analysis, which provides ultrastructural details on the morphology of the mitotic apparatus that cannot be obtained with high-resolution confocal microscopy. Here, we describe a simple method for the ultrastructural analysis of mitosis in Drosophila S2 cells. •Our method, which involves fixation and sectioning of a cell pellet, provides excellent preservation of mitotic structures and allows analysis of a higher number of mitotic divisions per sample, compared to correlative light-electron microscopy.•Dividing cells are randomly oriented within the pellet and are sectioned along different planes, providing all-around information on the structure of the mitotic apparatus.

We analyzed the pattern of spindle microtubule (MT) regrowth after cold- or colcemid-induced MT depolymerization in Drosophila S2 cells. Cold-induced MT disassembly at low temperature (–2 °C) destroyed kinetochore-driven MT regrowth without affecting astral MT formation. Conversely, colcemid-induced MT depolymerization strongly impaired centrosome-dependent MT nucleation, allowing kinetochore-driven MT regrowth. Collectively, these results indicate that the kinetochore- and the centrosome-mediated MT assembly pathways exploit molecular mechanisms that are at least in part different.

A comprehensive examination and treatment of 49 students aged 20-25 years diagnosed chronic generalized catarrhal gingivitis (CGCG) were held. Depending on the methods of treatment were created into two groups of observation. The main group comprised 24 patients who along with dental sanitation and treatment of gingivitis took the course of salt treatment for 17 days, as opposed to control group. Hygienic researches and mineralthcrapy were held in a special room (23.6 m2). equipped with sylvite blocks with a total reaction surface 5 m2, salt filters with air ducts filled with wooden plates with mineral fragments. The study of the effectiveness of a comprehensive treatment of young patients with CGCG allowsto gel an information of the positive impact of salt therapyon the clinical condition of marginal periodontal tissues and indeces of oral cavity local immunity. The main curative factorsforming the internal environment of silvinite structures are multicomponent highly dispersed salt aerosol with a defined particle size and aeroionization. Natural salts complex consisting of chlorides of potassium, sodium and magnesium has an anti-inflammatory and immuno-modulating effects.

In this study, we evaluated the force of the cilium on the foot sole of a medium-sized pond snail Lymnaea stagnalis, crawling up in the anaerobic water. It was found that there are 1.3 x 10(9) cilia on the foot sole, their force is 1.47 x 10(-6) W, the force of one cilium is 1.1 x 10(-15) W and the force of single ciliary beat is 1.1 x 10(-16) W.