BioUML-towards a universal research platform.

BioUML (https://www.biouml.org)-is a web-based integrated platform for systems biology and data analysis. It supports visual modelling and construction of hierarchical biological models that allow us to construct the most complex modular models of blood pressure regulation, skeletal muscle metabolism, COVID-19 epidemiology. BioUML has been integrated with git repositories where users can store their models and other data. We have also expanded the capabilities of BioUML for data analysis and visualization of biomedical data: (i) any programs and Jupyter kernels can be plugged into the BioUML platform using Docker technology; (ii) BioUML is integrated with the Galaxy and Galaxy Tool Shed; (iii) BioUML provides two-way integration with R and Python (Jupyter notebooks): scripts can be executed on the BioUML web pages, and BioUML functions can be called from scripts; (iv) using plug-in architecture, specialized viewers and editors can be added. For example, powerful genome browsers as well as viewers for molecular 3D structure are integrated in this way; (v) BioUML supports data analyses using workflows (own format, Galaxy, CWL, BPMN, nextFlow). Using these capabilities, we have initiated a new branch of the BioUML development-u-science-a universal scientific platform that can be configured for specific research requirements.

A Modular Mathematical Model of Exercise-Induced Changes in Metabolism, Signaling, and Gene Expression in Human Skeletal Muscle.

Skeletal muscle is the principal contributor to exercise-induced changes in human metabolism. Strikingly, although it has been demonstrated that a lot of metabolites accumulating in blood and human skeletal muscle during an exercise activate different signaling pathways and induce the expression of many genes in working muscle fibres, the systematic understanding of signaling-metabolic pathway interrelations with downstream genetic regulation in the skeletal muscle is still elusive. Herein, a physiologically based computational model of skeletal muscle comprising energy metabolism, Ca2+, and AMPK (AMP-dependent protein kinase) signaling pathways and the expression regulation of genes with early and delayed responses was developed based on a modular modeling approach and included 171 differential equations and more than 640 parameters. The integrated modular model validated on diverse including original experimental data and different exercise modes provides a comprehensive in silico platform in order to decipher and track cause-effect relationships between metabolic, signaling, and gene expression levels in skeletal muscle.

Genome-Wide Atlas of Promoter Expression Reveals Contribution of Transcribed Regulatory Elements to Genetic Control of Disuse-Mediated Atrophy of Skeletal Muscle.

The prevention of muscle atrophy carries with it clinical significance for the control of increased morbidity and mortality following physical inactivity. While major transcriptional events associated with muscle atrophy-recovery processes are the subject of active research on the gene level, the contribution of non-coding regulatory elements and alternative promoter usage is a major source for both the production of alternative protein products and new insights into the activity of transcription factors. We used the cap-analysis of gene expression (CAGE) to create a genome-wide atlas of promoter-level transcription in fast (m. EDL) and slow (m. soleus) muscles in rats that were subjected to hindlimb unloading and subsequent recovery. We found that the genetic regulation of the atrophy-recovery cycle in two types of muscle is mediated by different pathways, including a unique set of non-coding transcribed regulatory elements. We showed that the activation of "shadow" enhancers is tightly linked to specific stages of atrophy and recovery dynamics, with the largest number of specific regulatory elements being transcriptionally active in the muscles on the first day of recovery after a week of disuse. The developed comprehensive database of transcription of regulatory elements will further stimulate research on the gene regulation of muscle homeostasis in mammals.

Signaling Pathways Potentially Responsible for Foam Cell Formation: Cholesterol Accumulation or Inflammatory Response-What is First?

Accumulation of lipid-laden (foam) cells in the arterial wall is known to be the earliest step in the pathogenesis of atherosclerosis. There is almost no doubt that atherogenic modified low-density lipoproteins (LDL) are the main sources of accumulating lipids in foam cells. Atherogenic modified LDL are taken up by arterial cells, such as macrophages, pericytes, and smooth muscle cells in an unregulated manner bypassing the LDL receptor. The present study was conducted to reveal possible common mechanisms in the interaction of macrophages with associates of modified LDL and non-lipid latex particles of a similar size. To determine regulatory pathways that are potentially responsible for cholesterol accumulation in human macrophages after the exposure to naturally occurring atherogenic or artificially modified LDL, we used transcriptome analysis. Previous studies of our group demonstrated that any type of LDL modification facilitates the self-association of lipoprotein particles. The size of such self-associates hinders their interaction with a specific LDL receptor. As a result, self-associates are taken up by nonspecific phagocytosis bypassing the LDL receptor. That is why we used latex beads as a stimulator of macrophage phagocytotic activity. We revealed at least 12 signaling pathways that were regulated by the interaction of macrophages with the multiple-modified atherogenic naturally occurring LDL and with latex beads in a similar manner. Therefore, modified LDL was shown to stimulate phagocytosis through the upregulation of certain genes. We have identified at least three genes (F2RL1, EIF2AK3, and IL15) encoding inflammatory molecules and associated with signaling pathways that were upregulated in response to the interaction of modified LDL with macrophages. Knockdown of two of these genes, EIF2AK3 and IL15, completely suppressed cholesterol accumulation in macrophages. Correspondingly, the upregulation of EIF2AK3 and IL15 promoted cholesterol accumulation. These data confirmed our hypothesis of the following chain of events in atherosclerosis: LDL particles undergo atherogenic modification; this is accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. This chain of events may explain the relationship between cholesterol accumulation and inflammation. The primary sequence of events in this chain is related to inflammatory response rather than cholesterol accumulation.

Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis.

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.

BioUML: an integrated environment for systems biology and collaborative analysis of biomedical data.

BioUML (homepage: http://www.biouml.org, main public server: https://ict.biouml.org) is a web-based integrated environment (platform) for systems biology and the analysis of biomedical data generated by omics technologies. The BioUML vision is to provide a computational platform to build virtual cell, virtual physiological human and virtual patient. BioUML spans a comprehensive range of capabilities, including access to biological databases, powerful tools for systems biology (visual modelling, simulation, parameters fitting and analyses), a genome browser, scripting (R, JavaScript) and a workflow engine. Due to integration with the Galaxy platform and R/Bioconductor, BioUML provides powerful possibilities for the analyses of omics data. The plug-in-based architecture allows the user to add new functionalities using plug-ins. To facilitate a user focus on a particular task or database, we have developed several predefined perspectives that display only those web interface elements that are needed for a specific task. To support collaborative work on scientific projects, there is a central authentication and authorization system (https://bio-store.org). The diagram editor enables several remote users to simultaneously edit diagrams.

Computational master-regulator search reveals mTOR and PI3K pathways responsible for low sensitivity of NCI-H292 and A427 lung cancer cell lines to cytotoxic action of p53 activator Nutlin-3.

BACKGROUND: Small molecule Nutlin-3 reactivates p53 in cancer cells by interacting with the complex between p53 and its repressor Mdm-2 and causing an increase in cancer cell apoptosis. Therefore, Nutlin-3 has potent anticancer properties. Clinical and experimental studies of Nutlin-3 showed that some cancer cells may lose sensitivity to this compound. Here we analyze possible mechanisms for insensitivity of cancer cells to Nutlin-3. METHODS: We applied upstream analysis approach implemented in geneXplain platform ( genexplain.com ) using TRANSFAC® database of transcription factors and their binding sites in genome and using TRANSPATH® database of signal transduction network with associated software such as Match™ and Composite Module Analyst (CMA). RESULTS: Using genome-wide gene expression profiling we compared several lung cancer cell lines and showed that expression programs executed in Nutlin-3 insensitive cell lines significantly differ from that of Nutlin-3 sensitive cell lines. Using artificial intelligence approach embed in CMA software, we identified a set of transcription factors cooperatively binding to the promoters of genes up-regulated in the Nutlin-3 insensitive cell lines. Graph analysis of signal transduction network upstream of these transcription factors allowed us to identify potential master-regulators responsible for maintaining such low sensitivity to Nutlin-3 with the most promising candidate mTOR, which acts in the context of activated PI3K pathway. These finding were validated experimentally using an array of chemical inhibitors. CONCLUSIONS: We showed that the Nutlin-3 insensitive cell lines are actually highly sensitive to the dual PI3K/mTOR inhibitor NVP-BEZ235, while no responding to either PI3K -specific LY294002 nor Bcl-XL specific 2,3-DCPE compounds.

Expression and molecular characterization of the Mycobacterium tuberculosis PII protein.

The signal transduction protein PII plays an important role in cellular nitrogen assimilation and regulation. The molecular characteristics of the Mycobacterium tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The binding characteristics of the purified protein with ATP and ADP were investigated using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44 microM. This binding strength was not significantly affected by the presence of 2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII exists in homotrimeric state which is consistent with other PII proteins. Phylogenetic analysis showed that Mtb PII consistently grouped with other actinobacterial PII proteins.

Phylogenetic analysis of the p53 and p63/p73 gene families.

Proteins of the relative families p53 and p63/p73 are transcriptional factors that are involved in the signaling pathway in cells. The wide spectrum of their functions includes cell cycle arrest and apoptosis in response to DNA damage. The p53 protein also participates in development of particular tissues during embryogenesis. Thus, it is of high importance to establish the relation between structure, function and evolution of these proteins. In the current computational study, the evolutionary mode of the p63/p73 protein family is investigated. Search for the adaptive branches of the phylogenetic tree and the adaptive codons in the nucleotide sequences was performed using the codem1 program from the PAML package, version 3.14. The results obtained were compared with those of our previous phylogenetic analysis of the p53 protein. Evidence was obtained that the evolutionary history of the p63/p73 proteins has been under positive selection. An attempt is made to associate the current evidence with the previous for positive selection in the p53 family. Recently the G245C substitution has been assumed to result in formation of a novel Zn(2+)-binding site in the p53 protein. The molecular mechanics simulations were performed to estimate energy of zinc binding to its site in two dominant-negative p53 mutants--G245C and R175H--in comparison with the wild-type p53. The results of the estimation provided evidence of the novel Zn(2+)-binding site functionality in G245C mutant form.

PDBSite: a database of the 3D structure of protein functional sites.

The PDBSite database provides comprehensive structural and functional information on various protein sites (post-translational modification, catalytic active, organic and inorganic ligand binding, protein-protein, protein-DNA and protein-RNA interactions) in the Protein Data Bank (PDB). The PDBSite is available online at http://wwwmgs.bionet.nsc.ru/mgs/gnw/pdbsite/. It consists of functional sites extracted from PDB using the SITE records and of an additional set containing the protein interaction sites inferred from the contact residues in heterocomplexes. The PDBSite was set up by automated processing of the PDB. The PDBSite database can be queried through the functional description and the structural characteristics of the site and its environment. The PDBSite is integrated with the PDBSiteScan tool allowing structural comparisons of a protein against the functional sites. The PDBSite enables the recognition of functional sites in protein tertiary structures, providing annotation of function through structure. The PDBSite is updated after each new PDB release.

PDBSiteScan: a program for searching for active, binding and posttranslational modification sites in the 3D structures of proteins.

PDBSiteScan is a web-accessible program designed for searching three-dimensional (3D) protein fragments similar in structure to known active, binding and posttranslational modification sites. A collection of known sites we designated as PDBSite was set up by automated processing of the PDB database using the data on site localization in the SITE field. Additionally, protein-protein interaction sites were generated by analysis of atom coordinates in heterocomplexes. The total number of collected sites was more than 8100; they were assigned to more than 80 functional groups. PDBSiteScan provides automated search of the 3D protein fragments whose maximum distance mismatch (MDM) between N, Calpha and C atoms in a fragment and a functional site is not larger than the MDM threshold defined by the user. PDBSiteScan requires perfect matching of amino acids. PDBSiteScan enables recognition of functional sites in tertiary structures of proteins and allows proteins with functional information to be annotated. The program PDBSiteScan is available at http://wwwmgs.bionet.nsc.ru/mgs/systems/fastprot/pdbsitescan.html.