Rational in silico design of aptamers for organophosphates based on the example of paraoxon

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its threedimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed

Reactive oxygen species in pathogenesis of atherosclerosis.

Co-author affiliations: - Sechenov Institute of Evolutionary Physiology and Biochemistry RAS, Saint Petersburg, Russia - Research Institute of Hygiene, Occupational Pathology and Human Ecology, Saint Petersburg, Russia - Koltzov Institute of Developmental Biology RAS, Moscow, Russia - Institute of Cell Biophysics RAS, Pushchino, Russia - Institute of General Pathology and Pathophysiology RAMS, Moscow, RussiaThe volume of publications on the role of reactive oxygen species (ROS) in biological processes has been increasing exponentially over the last decades. ROS in large amounts clearly have detrimental effects on cell physiology, whereas low concentrations of ROS are permanently produced in cells and play a role as signaling molecules. An imbalance in ROS production and defense mechanisms can lead to pathological vascular remodeling, atherosclerosis being among them. The aim of this review is to examine different sources of ROS from the point of view of their participation in pathogenesis of atherosclerosis and related cardiovascular risk. Among the possible sources of ROS discussed here are mitochondria, NADPH-oxidases, xanthine oxidase, peroxidases, NO-synthases, cytochrome P450, cyclooxygenases, lipoxygenases, and hemoglobin of red blood cells. A great challenge for future research is to establish interrelations, feedback and feed-forward regulation mechanisms of various sources of ROS in development of atherosclerosis and other vascular pathologies

Chemical pretreatment of cells for enhanced MALDI-TOF-MS discrimination of clinical staphylococci including MRSA

BACKGROUND: Limited success has been reported for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) differentiation of staphylococci, including methicillin resistant Staphylococcus aureus (MRSA) strains. Chemical pretreatment of cells prior to MALDI-TOF-MS analysis has not been systematically investigated for enhanced discrimination of S.aureus strains. OBJECTIVES: To evaluate various chemical pretreatment of cells for MALDI-TOF-MS discrimination of clinical staphylococcal isolates, with a focus on differentiation of MRSA from methicillin sensitive S. aureus (MSSA) strains and from other staphylococcal species. METHOD: MALDI-TOF-MS of a well-characterised S. aureus strain(s) was optimised with respect to matrix chemical(s), matrix solvent and target plating method. Various chemical pretreatments (solvents, reductants, detergents) and pretreatment application methods were then evaluated for enhancement of spectral richness. The three most promising pretreatments were applied to MALDI-TOF-MS discrimination of three set of clinical isolates comprising non-S.aureus staphylococci (77 isolates ), MSSA (36) and MRSA (43), with analysis by total or set specific resolved peaks. RESULTS: The optimized MALDI-TOF-MS protocol involved α-cyano-4-hydroxycinnamic acid (CHCA) as matrix chemical (in 1:2 acetonitrile:H2O and 2% trifluoroacetic acid), with application as an overlay onto smeared cells (on-probe). On-probe application of chemical pretreatment was most effective at enhancing MALDI-TOF-MS spectral richness. Use of reductants and detergents as pretreatments were ineffective. The three most effective solvents/acid pretreatments - ethanol:formate, ethanol:acetate and formate:isopropanol - each generated reproducible and distinct spectra over the 2,000 -10,000 m/z range. For the combined sets of clinical isolates (114), all three of these pretreatments increased the total number of resolved peaks in comparison with no pretreatment controls. The ethanol:formate pretreatment gave 100% clustering of non-S. aureus staphylococci, based on total resolved peaks. The formate:isopropanol pretreatment generated the largest increase in number of MRSA set specific peaks (from 18 to 32; 78% increase) and clustered the majority (77%) of the MRSA strains together, although compete discrimination of the MSSA and MRSA was not achieved. CONCLUSION: MALDI-TOF-MS discrimination of clinical isolates of staphylococci is enhanced through chemical pretreatment of cells. Three chemical pretreatments, not previously applied to staphylococci, are highlighted for enhancing spectral richness and offering new opportunities for improved discrimination of staphylococci, including MRSA and MSSA strains

Chemical pretreatment of cells for enhanced MALDI-TOF-MS discrimination of clinical staphylococci including MRSA

BACKGROUND: Limited success has been reported for matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) differentiation of staphylococci, including methicillin resistant Staphylococcus aureus (MRSA) strains. Chemical pretreatment of cells prior to MALDI-TOF-MS analysis has not been systematically investigated for enhanced discrimination of S.aureus strains. OBJECTIVES: To evaluate various chemical pretreatment of cells for MALDI-TOF-MS discrimination of clinical staphylococcal isolates, with a focus on differentiation of MRSA from methicillin sensitive S. aureus (MSSA) strains and from other staphylococcal species. METHOD: MALDI-TOF-MS of a well-characterised S. aureus strain(s) was optimised with respect to matrix chemical(s), matrix solvent and target plating method. Various chemical pretreatments (solvents, reductants, detergents) and pretreatment application methods were then evaluated for enhancement of spectral richness. The three most promising pretreatments were applied to MALDI-TOF-MS discrimination of three set of clinical isolates comprising non-S.aureus staphylococci (77 isolates ), MSSA (36) and MRSA (43), with analysis by total or set specific resolved peaks. RESULTS: The optimized MALDI-TOF-MS protocol involved α-cyano-4-hydroxycinnamic acid (CHCA) as matrix chemical (in 1:2 acetonitrile:H2O and 2% trifluoroacetic acid), with application as an overlay onto smeared cells (on-probe). On-probe application of chemical pretreatment was most effective at enhancing MALDI-TOF-MS spectral richness. Use of reductants and detergents as pretreatments were ineffective. The three most effective solvents/acid pretreatments - ethanol:formate, ethanol:acetate and formate:isopropanol - each generated reproducible and distinct spectra over the 2,000 -10,000 m/z range. For the combined sets of clinical isolates (114), all three of these pretreatments increased the total number of resolved peaks in comparison with no pretreatment controls. The ethanol:formate pretreatment gave 100% clustering of non-S. aureus staphylococci, based on total resolved peaks. The formate:isopropanol pretreatment generated the largest increase in number of MRSA set specific peaks (from 18 to 32; 78% increase) and clustered the majority (77%) of the MRSA strains together, although compete discrimination of the MSSA and MRSA was not achieved. CONCLUSION: MALDI-TOF-MS discrimination of clinical isolates of staphylococci is enhanced through chemical pretreatment of cells. Three chemical pretreatments, not previously applied to staphylococci, are highlighted for enhancing spectral richness and offering new opportunities for improved discrimination of staphylococci, including MRSA and MSSA strains

A Novel C1q Domain-Containing Protein Isolated from the Mollusk Modiolus kurilensis Recognizing Glycans Enriched with Acidic Galactans and Mannans

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman’s degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca(2+)-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein

Effects of exposure of rat erythrocytes to a hypogeomagnetic field

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Background:Hypomagnetic fields can disrupts the normal functioning of living organisms by a mechanism thought to involve oxidative stress. In erythrocytes, oxidative stress can inter alia lead to changes to hemoglobin content and to hemolysis. Objective:To study the effects of hypomagnetism on the state of rat erythrocytes in vitro. Methods:Rat erythrocytes were exposed to an attenuated magnetic field (AMF) or Earth’s magnetic field (EMF), in the presence of tert-butyl hydroperoxide (TBHP) as inducer of oxidative stress. Determinations: total hemoglobin (and its three forms – oxyhemoglobin, methemoglobin, and hemichrome) released from erythrocytes, spectral data (500–700 nm); oxygen radical concentrations, electron paramagnetic resonance. Results:AMF and EMF exposed erythrocytes were compared. After 4 h incubation at high TBHP concentrations (>700 μM), AMF exposed erythrocytes released significantly more (p<0.05) hemoglobin (Hb), mostly as methemoglobin (metHb). Conversely, after 24 h incubation at low TBHP concentrations (⩽350 μM), EMF exposed erythrocytes released significantly more (p<0.001) hemoglobin, with metHb as a significant proportion of the total Hb. Erythrocytes exposed to AMF generated more radicals than those exposed to the EMF. Conclusion:Under particular conditions of oxidative stress, hypomagnetic fields can disrupt the functional state of erythrocytes and promote cell death; an additive effect is implicated

Inhibition of protein tyrosine phosphatases unmasks vasoconstriction and potentiates calcium signalling in rat aorta smooth muscle cells in response to an agonist of 5-HT2B receptors BW723C86

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.In blood vessels, serotonin 5-HT2B receptors mainly mediate relaxation, although their activation by the selective agonist BW723C86 is known to exert contraction of aorta in deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NAME) hypertensive rats [Russel et al., 2002; Banes et al., 2003] and in mice with type 2 diabetes [Nelson et al., 2012]. The unmasking effect on vasoconstriction can be caused by a shift in the balance of tyrosine phosphorylation in smooth muscle cells (SMC) due to oxidative stress induced inhibition of protein tyrosine phosphatases (PTP). We have demonstrated that BW723C86 which does not cause contraction of rat aorta and mesenteric artery rings, evoked a vasoconstrictor effect in the presence of PTP inhibitors sodium orthovanadate (Na3VO4) or BVT948. BW723C86 induced a weak rise of [Ca2+]i in the SMC isolated from rat aorta; however, after pre-incubation with Na3VO4 the response to BW723C86 increased more than 5-fold. This effect was diminished by protein tyrosine kinase (PTK) inhibitor genistein, inhibitor of Src-family kinases PP2, inhibitor of NADPH-oxidase VAS2870 and completely suppressed by N-acetylcysteine and 5-HT2B receptor antagonist RS127445. Using fluorescent probe DCFH-DA we have shown that Na3VO4 induces oxidative stress in SMC. In the presence of Na3VO4 BW723C86 considerably increased formation of reactive oxygen species while alone had no appreciable effect on DCFH oxidation. We suggest that oxidative stress causes inhibition of PTP and unmasking of 5-HT2B receptors functional activity

Spectroscopy 19 (2005) 235-246 235 IOS Press A new method for studying platelets, based upon the low-angle light scattering technique. 1. Theoretical and experimental foundations of the method

Abstract. A new method is presented for simultaneous monitoring of changes in shape and aggregation of platelets. The signal of light scattering alterations at angles below six degrees was shown to be determined by platelet aggregation dynamics (aggregation, disaggregation, coagulation). Over a range of larger angles (6-15 degrees), cell shape changes also contributed to the signal: (i) spherization, and (ii) pseudopodia formation. The first stage was shown to be fast (t 1/2 of few seconds) and correlated with [Ca 2+ ] increase. It was characterised by a narrow signal fluctuation and by a rapid increase (30-40%) in signal intensity. During the second stage, which was much slower, the signal decreased describing the aggregation process. The EC 50 value for ADP-induced spherization was 40 nmol l −1 . Aggregation kinetics in saline solution under turbulent flow showed second order kinetics in relation to initial cell concentration. The rate constant depended on stirring conditions and on calcium concentration in the medium. Standardisation of the testing conditions made it possible to characterize the initial functional state of platelets by their sensitivity to agonists, with estimation of EC 50 and maximum velocity of aggregation (Umax) values. The method has potential applications in pharmacology and toxicology research and in clinical practice, as a simple and highly sensitive functionality test for platelets