Alexander A Vassilevski

Vavilov Institute of General Genetics, Moskva, Moscow, Russia

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Publications (41)134.29 Total impact

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    ABSTRACT: Yellow sac spiders (Cheiracanthium punctorium, family Miturgidae) are unique in terms of venom composition, because, as we show here, two-domain toxins have replaced the usual one-domain peptides as the major constituents. We report the structure of the two-domain Che. punctorium toxins (CpTx), along with the corresponding cDNA and genomic DNA sequences. At least three groups of insecticidal CpTx were identified, each consisting of several members. Unlike many cone snail and snake toxins, accelerated evolution is not typical of cptx genes, which instead appear to be under the pressure of purifying selection. Both CpTx modules present the inhibitor cystine knot (ICK), or knottin signature; however, the sequence similarity between the domains is low. Conversely, notable similarity was found between separate domains of CpTx and one-domain toxins from spiders of the Lycosidae family. The observed chimerism is a landmark of exon shuffling events, but in contrast to many families of multidomain protein genes no introns were found in the cptx genes. Considering the possible scenarios, we suggest that an early transcription-mediated fusion event between two related one-domain toxin genes led to the emergence of a primordial cptx-like sequence. We conclude that evolution of toxin variability in spiders appears to be quite different from other venomous animals.
    Insect Molecular Biology 05/2014; · 3.04 Impact Factor
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    ABSTRACT: In this paper we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide is found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule Tk-hefu and was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that while the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ≈35 μM) to κ-hefutoxin 1 (IC50 ≈40 μM). We conclude that α-hairpinins are attractive in their simplicity structural templates, which may be used for functional engineering and drug design.
    Journal of Biological Chemistry 03/2014; · 4.65 Impact Factor
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    ABSTRACT: Spiderines are comparatively long polypeptide toxins (∼110 residues) from lynx spiders (genus Oxyopes). They are built of an N-terminal linear cationic domain (∼40 residues) and a C-terminal knottin domain (∼60 residues). The linear domain empowers spiderines with strong cytolytic activity. In the present work we report 16 novel spiderine sequences from O. takobius and O. lineatus classified into two subfamilies. Strikingly, negative selection acts on both linear and knottin domains. Genes encoding Oxyopes two-domain toxins were sequenced and found to be intronless. We further discuss a possible scenario of lynx spider modular toxin evolution.
    FEBS letters 01/2014; · 3.54 Impact Factor
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    ABSTRACT: Apart from the conventional neurotoxins and cytotoxins, venom of the lynx spider Oxyopes takobius was found to contain two-domain modular toxins named spiderines OtTx 1a, 1b, 2a and 2b. These toxins show both insecticidal activity (median lethal dose against flesh fly larvae of 75 µg/g) and potent antimicrobial effects (minimal inhibitory concentrations in the range of 0.1-10 µM). Full sequences of the purified spiderines were established by a combination of Edman degradation, mass spectrometry and cDNA cloning. They are relatively large molecules (~110 residues; 12.0-12.5 kDa) and consist of two distinct modules separated by a short linker. The N-terminal part (~40 residues) contains no cysteine residues, is highly cationic, forms amphipathic α-helical structures in membrane-mimicking environment, and shows potent cytolytic effects on cells of different origin. The C-terminal part is disulfide-rich (~60 residues; 5 S-S bonds) and contains the inhibitor cystine knot (ICK/knottin) signature. The N-terminal part of spiderines is much alike linear cytotoxic peptides found in different organisms, whereas the C-terminal part corresponds to the usual spider neurotoxins. We synthesized the modules of OtTx 1a and compared their activity to that of the full-length mature toxin produced recombinantly, highlighting the importance of the N-terminal part that in both insecticidal and antimicrobial assays retained full-length toxin activity. The unique structure of spiderines fills up the missing part of the two-domain spider toxin ensemble. This article is protected by copyright. All rights reserved.
    FEBS Journal 10/2013; · 4.25 Impact Factor
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    ABSTRACT: Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C(1)X3C(2)XnC(3)X3C(4)), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C(1)-C(4) and C(2)-C(3). Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal "tail" regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S-S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.
    Plant Molecular Biology 10/2013; · 3.52 Impact Factor
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    ABSTRACT: A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk-AMP-X1 and Tk-AMP-X2, were completely sequenced and shown to belong to the α-hairpinin structural family of plant peptides with a characteristic C1xxxC2-X(n)-C3xxxC4 motif. The peptides inhibit spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk-AMP-X peptides. They code for precursor proteins of unusual multimodular structure consisting of a signal peptide, several α-hairpinin (4-Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C-terminal prodomain. Three types of precursor proteins, with five, six or seven 4-Cys peptide modules were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk-AMP-X precursors have no introns in the protein-coding regions and are upregulated by fungal pathogens and abiotic stress providing conclusive evidence for their role in stress response. A combined PCR-based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically related Triticum and Aegilops species including polyploid wheat genome donors. Association of the Tk-AMP-X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk-AMP-X-related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize. This article is protected by copyright. All rights reserved.
    FEBS Journal 05/2013; · 4.25 Impact Factor
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    ABSTRACT: To gain success in the evolutionary "arms race", venomous animals such as scorpions produce diverse neurotoxins selected to hit targets in the nervous system of prey. Scorpion α-toxins affect insect and/or mammalian voltage-gated sodium channels (Nav's) and thereby modify the excitability of muscle and nerve cells. Although more than a hundred α-toxins are known and a number of them have been studied into detail, the molecular mechanism of their interaction with Nav's is still poorly understood. Here, we employ extensive molecular dynamics simulations and spatial mapping of hydrophobic/hydrophilic properties distributed over the molecular surface of α-toxins. It is revealed that in spite of the small size and relatively rigid structure, these toxins possess modular organization from structural, functional and evolutionary perspectives. The more conserved and rigid "core module" is supplemented with the "specificity module" (SM) that is comparatively flexible and variable, and determines the taxon (mammal vs. insect) specificity of α-toxin activity. We further show that SMs in mammal toxins are more flexible and hydrophilic than in insect toxins. Concomitant sequence-based analysis of Nav's extracellular loops suggests that α-toxins recognize the channels using both modules. We propose that the core module binds to the voltage-sensing domain IV, whereas the more versatile SM interacts with the pore domain in repeat I of Nav's. These findings corroborate and expand the hypothesis on different functional epitopes of toxins that has been reported previously. In effect, we propose that the modular structure in toxins evolved to match the domain architecture of Nav's.
    Journal of Biological Chemistry 05/2013; · 4.65 Impact Factor
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    ABSTRACT: Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.
    Analytical and Bioanalytical Chemistry 01/2013; · 3.66 Impact Factor
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    ABSTRACT: Antichlamydial activity of cyto-insectotoxin 1a (CIT 1a), representative of a unique class of antimicrobial peptides from the venom of the Central Asian spider Lachesana tarabaevi, was studied. A plasmid vector expressing the cit 1a gene controlled by a human cytomegalovirus tetracycline-dependent promoter was constructed. Impressive inhibition of Chlamydia trachomatis infection in HEK 293 cells transfected by the cit 1a-harboring vector was achieved. With the use of various schemes of cell infection and gene expression induction, it was shown for the first time that an antimicrobial peptide exerts its potent antichlamydial action at an early stage of the pathogen life cycle.
    Archives of Microbiology 01/2013; · 1.91 Impact Factor
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    ABSTRACT: Membrane-active peptides (MAPs) represent a broad variety of molecules, and biological functions of most are directly associated with their ability to interact with membranes. Taking into account the effect of MAPs on living cells they can be nominally divided into three major groups - fusion (FPs), antimicrobial/cytolytic (AMPs/CPs) and cellpenetrating (CPPs) peptides. Although spatial structure of different MAPs varies to a great extent, linear α-helical peptides represent the most studied class. These peptides possess relatively simple structural organization and share a set of similar molecular features, which make them very attractive to both experimental and computational studies. Here, we review different molecular modeling methods in prospective of their applications to study of α-helical MAPs. The most sophisticated of them, such as molecular dynamics simulations, give atomistic information about molecular interactions driving peptide binding to the water-lipid interface, cooperative mechanisms of membrane destabilization and thermodynamics of these processes. Significant progress has been achieved in this field during the last few years, resulting in a possibility to observe computationally MAPs action in realistic peptide-to-lipid ratios and over the microsecond timescale. Other relatively simple but powerful approaches allow assessment of important characteristics of MAPs such as α-helical propensity, amphiphilicity, total hydrophobicity, and spatial distribution of charge and hydrophobic/hydrophilic properties, etc. Altogether, computational methods provide efficient basis for rational design of MAPs with predefined properties and a spectrum of biological activities.
    Current Protein and Peptide Science 11/2012; 13(7):644-57. · 2.33 Impact Factor
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    ABSTRACT: Venom of Lachesana tarabaevi (Zodariidae, "ant spiders") exhibits high insect toxicity and serves a rich source of potential insecticides. Five new peptide toxins active against insects were isolated from the venom by means of liquid chromatography and named latartoxins (LtTx). Complete amino acid sequences of LtTx (60-71 residues) were established by a combination of Edman degradation, mass spectrometry and selective proteolysis. Three toxins have eight cysteine residues that form four intramolecular disulfide bridges, and two other molecules contain an additional cystine; three LtTx are C-terminally amidated. Latartoxins can be allocated to two groups with members similar to CSTX and LSTX toxins from Cupiennius salei (Ctenidae) and Lycosa singoriensis (Lycosidae). The interesting feature of the new toxins is their modular organization: they contain an N-terminal cysteine-rich (knottin or ICK) region as in many neurotoxins from spider venoms and a C-terminal linear part alike some cytolytic peptides. The C-terminal fragment of one of the most abundant toxins LtTx-1a was synthesized and shown to possess membrane-binding activity. It was found to assume amphipathic α-helical conformation in membrane-mimicking environment and exert antimicrobial activity at micromolar concentrations. The tails endow latartoxins with the ability to bind and damage membranes; LtTx show cytolytic activity in fly larvae neuromuscular preparations. We suggest a membrane-dependent mode of action for latartoxins with their C-terminal linear modules acting as anchoring devices.
    Biochimica et Biophysica Acta 10/2012; · 4.66 Impact Factor
  • Biochemical Journal 09/2012; 446(2):331. · 4.65 Impact Factor
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    ABSTRACT: Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.
    Biochimica et Biophysica Acta 07/2012; 1818(11):2868-75. · 4.66 Impact Factor
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    ABSTRACT: A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.
    Biochemical Journal 05/2012; 446(1):69-77. · 4.65 Impact Factor
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    ABSTRACT: Hevein-like plant defense peptides WAMP-1a/b with a unique 10-Cys motif are found in the wheat Triticum kiharae seeds. Three different wamp genomic and cDNA sequences were derived from T. kiharae; no introns were spotted in the protein-coding regions of the genes. The deduced Wamp precursor proteins consist of a signal peptide, mature peptide (WAMP) and C-terminal prosequence. Origin of WAMPs from class I/IV chitinases via deletion of the catalytic domain is proposed based on homology between their genes. Genome screening of several cereals and goatgrasses from the genera Triticum and Aegilops was performed. No wamp homologues were identified in Triticum monococcum (A(b)A(b)) or Triticum urartu (A(u)A(u)), diploid species with an A genome. To the contrary, highly homologous wamp genes were discovered in hexaploid Triticum aestivum (A(u)A(u)BBDD) and T. kiharae (A(b)A(b)GGDD), and the putative genome donors Triticum timopheevii (A(b)A(b)GG), Aegilops speltoides (BB), and Aegilops tauschii (DD), providing strong evidence for the ancient origin of these genes and their association with the B, D and G genomes. The role of T. kiharae WAMPs in biotic stress is suggested by their antifungal activity and increased accumulation of wamp transcripts in response to phytopathogen challenge. Differential reaction to fungi was demonstrated: Fusarium oxysporum enhanced expression of wamp genes, whereas Aspergillus niger induced transcription reprogramming and alternative polyadenylation. WAMPs participate in plant response also to abiotic stress. Although no changes were noted at elevated or decreased temperatures, high salt concentrations enhanced wamp expression, the first indication of hevein-type peptide participation in salinity stress.
    Biochimie 04/2012; 94(4):1009-16. · 3.14 Impact Factor
  • Yaroslav A Andreev, Alexander A Vassilevski, Sergey A Kozlov
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    ABSTRACT: Receptors that are involved in generation and transduction of pain signals attract much interest from the scientific and corporate communities. Good commercial prospects for successful development of effective analgesic drugs stimulate significantly the research. This article provides a brief overview of the key molecular targets, i.e. cell receptors, inhibition of which can lead to analgesia. Today transient receptor potential (TRP), purinergic (P2X) receptors and acidsensing ion channels (ASIC) are considered to be the most important proteins for perception of pain stimuli. These ionotropic receptors also participate in the development of inflammation; their hyperactivity leads to many pathological conditions and is closely associated with acute and inflammatory pain. Development of molecules capable to selectively modulate these receptors, their in vitro and in vivo effects, as well as perspectives for practical application described in patents and research articles are reviewed in this paper.
    Recent Patents on Inflammation & Allergy Drug Discovery 01/2012; 6(1):35-45.
  • Alexander A Vassilevski, Eugene V Grishin
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    ABSTRACT: Spiders are one of the most intriguing groups of venomous animals. Substances found in their venom vary from simple inorganic compounds to large multi-domain proteins. In this article, we review some of the latest work presenting active principles that add to the known spider toxin universe. Two aspects of novelty are addressed in particular, structural (novel types of molecules in terms of structure) and functional (novel types of biological targets hit by substances from spider venom and novel mechanisms of action).
    Acta Chimica Slovenica 12/2011; 58(4):717-723. · 1.14 Impact Factor
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    ABSTRACT: A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs.
    FEBS Journal 09/2011; 278(22):4382-93. · 4.25 Impact Factor
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    ABSTRACT: Spider venoms are vast natural pharmacopoeias selected by evolution. The venom of the ant spider Lachesana tarabaevi contains a wide variety of antimicrobial peptides. We tested six of them (latarcins 1, 2a, 3a, 4b, 5, and cytoinsectotoxin 1a) for their ability to suppress Chlamydia trachomatis infection. HEK293 cells were transfected with plasmid vectors harboring the genes of the selected peptides. Controlled expression of the transgenes led to a significant decrease of C. trachomatis viability inside the infected cells.
    Antimicrobial Agents and Chemotherapy 08/2011; 55(11):5367-9. · 4.57 Impact Factor
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    ABSTRACT: This study presents purification, activity characterization, and 1H NMR study of the novel antifungal peptide EcAMP1 from kernels of barnyard grass Echinochloa crus-galli. The peptide adopts a disulfide-stabilized α-helical hairpin structure in aqueous solution and thus represents a novel fold among naturally occurring antimicrobial peptides. Micromolar concentrations of EcAMP1 were shown to inhibit growth of several fungal phytopathogens. Confocal microscopy revealed intensive EcAMP1 binding to the surface of fungal conidia followed by internalization and accumulation in the cytoplasm without disturbance of membrane integrity. Close spatial structure similarity between EcAMP1, the trypsin inhibitor VhTI from seeds of Veronica hederifolia, and some scorpion and cone snail toxins suggests natural elaboration of different functions on a common fold.
    Journal of Biological Chemistry 07/2011; 286(28):25145-25153. · 4.65 Impact Factor

Publication Stats

266 Citations
134.29 Total Impact Points

Institutions

  • 2013
    • Vavilov Institute of General Genetics
      Moskva, Moscow, Russia
  • 2011
    • Kurchatov Institute
      Moskva, Moscow, Russia
  • 2006–2011
    • Russian Academy of Sciences
      • Institute of Inorganic Chemistry
      Moskva, Moscow, Russia
  • 2008–2010
    • KU Leuven
      • • Department of Pharmaceutical and Pharmacological Sciences
      • • Laboratory for Toxicology and Food Chemistry
      Leuven, VLG, Belgium