IN SILICO MODELING OF THE REDOX METABOLISM IN HUMAN ERYTHROCYTES

There was elaborated the mathematical model of erythrocytes metabolism, including glycolysis (Embden-Meyerhof pathway), pentose phosphate pathway, metHb restoration pathway, Н2О2 metabolism reaction. The final model includes 50 reactions and 60 metabolites. Within the model was studied the change of activity of some enzymes and concentrations of metabolites in stationary state, that take part in the processes of utilization of oxygen active forms and restoration of metgemoglobin, depending on amount of exogenous and endogenous Н2О2. There was demonstrated the threshold character of changes of the many studied parameters, that testifies that the cells can be practically in physiological state at the change of external conditions for rather long time. There was carried out an assessment of redox-state of erythrocytes at oxidizing load: was demonstrated the change of EGSSG/2GSH, ENADP+/NADPH and ENAD+/NADH from the concentration of endogenous Н2О2. There was established that in the studied diapason of concentrations of endogenous Н2О2 was observed the high slope of the change of EGSSG/2GSH, that was not observed for ENADP+/NADPH and the other redox-pairs. The results of modeling coincide with existing views on the functioning of enzymes of antioxidant protection in human erythrocytes and testify to the possibility of practical use of the mode

Reaction-diffusion kinetics on lattice at the microscopic scale

Lattice-based stochastic simulators are commonly used to study biological reaction-diffusion processes. Some of these schemes that are based on the reaction-diffusion master equation (RDME), can simulate for extended spatial and temporal scales but cannot directly account for the microscopic effects in the cell such as volume exclusion and diffusion-influenced reactions. Nonetheless, schemes based on the high-resolution microscopic lattice method (MLM) can directly simulate these effects by representing each finite-sized molecule explicitly as a random walker on fine lattice voxels. The theory and consistency of MLM in simulating diffusion-influenced reactions have not been clarified in detail. Here, we examine MLM in solving diffusion-influenced reactions in 3D space by employing the Spatiocyte simulation scheme. Applying the random walk theory, we construct the general theoretical framework underlying the method and obtain analytical expressions for the total rebinding probability and the effective reaction rate. By matching Collins-Kimball and lattice-based rate constants, we obtained the exact expressions to determine the reaction acceptance probability and voxel size. We found that the size of voxel should be about 2% larger than the molecule. MLM is validated by numerical simulations, showing good agreement with the off-lattice particle-based method, eGFRD. MLM run time is more than an order of magnitude faster than eGFRD when diffusing macromolecules with typical concentrations in the cell. MLM also showed good agreements with eGFRD and mean-field models in case studies of two basic motifs of intracellular signaling, the protein production-degradation process and the dual phosphorylation cycle. Moreover, when a reaction compartment is populated with volume-excluding obstacles, MLM captures the non-classical reaction kinetics caused by anomalous diffusion of reacting molecules

Correcting Glucose-6-Phosphate Dehydrogenase Deficiency with a Small-Molecule Activator

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency

Μελέτη διαταραχών της ομοιόστασης των ερυθροκυττάρων σε κλινικά σημαντικές συνθήκες

Η ανεπάρκεια του μεταβολικού ενζύμου αφυδρογονάση της 6-φωσφορικής γλυκόζης (G6PD) επηρεάζει πάνω από 400 εκατομμύρια ανθρώπους παγκοσμίως, γεγονός που καθιστά τη μελέτη της καταλληλότητας των αιμοδοτών με ανεπάρκεια G6PD σημαντική για τις μεταγγίσεις αίματος. Η παρούσα εργασία είχε ως στόχο την μελέτη των αλλαγών που συντελούνται στα αποθηκευμένα ερυθροκύτταρα δοτών με ανεπάρκεια G6PD (G6PD-), συγκριτικά με εκείνα των αιμοδοτών που διαθέτουν φυσιολογική δραστικότητα του ενζύμου (G6PD+) και την εκτίμηση των αποτελεσμάτων της μετάγγισης αίματος από G6PD- δότες σε δέκτες, μέσω ενός μοντέλου in vitro προσομοίωσης της μετάγγισης, με πειράματα ανασύστασης. Αρχικά, οι δώδεκα αιμοδότες (Ν=6 δότες G6PD+ και N=6 δότες G6PD-) μελετήθηκαν ως προς το αιματολογικό και βιοχημικό προφίλ τους, με τη χρήση αυτόματων αναλυτών. Για τον προσδιορισμό των βιοχημικών παραμέτρων και στις δυο ομάδες απομονώθηκε ο ορός από το συνολικό αίμα. Η μέτρηση ενδοκυττάριων δραστικών ριζών οξυγόνου (ROS) με φθορισμομετρία, ο προσδιορισμός δεικτών οσμωτικής και μηχανικής ευθραυστότητας, καθώς και η μέτρηση του ποσοστού της αιμόλυσης συντελέστηκαν σε δείγματα πακεταρισμένων ερυθροκυττάρων που αντιστοιχούσαν σε διαφορετικά χρονικά σημεία της αποθηκευτικής πορείας. Ακόμη, στα πειράματα ανασύστασης χρησιμοποιήθηκαν αποθηκευμένα ερυθροκύτταρα συγκεκριμένων ημερών αποθήκευσης (ημέρες 21, 30 και 42) τριών δοτών G6PD- και τριών δοτών G6PD+ και πλάσμα υγιούς δότη και ακολούθησε 24h επώαση στους 370C. Στα προκύπτοντα δείγματα της ανασύστασης εφαρμόστηκαν οι ίδιες μεθοδολογικές προσεγγίσεις για τη διεξαγωγή αποτελεσμάτων. Συμπερασματικά, η αποθηκευτική ικανότητα των ερυθροκυττάρων G6PD- φαίνεται να είναι παρόμοια ή και καλύτερη από εκείνη των G6PD+ δοτών για συγκεκριμένες παραμέτρους. Το μοντέλο in vitro προσομοίωσης της μετάγγισης έθεσε σημαντικά ερωτήματα ως προς τα μετα-μεταγγισιακά αποτελέσματα που μπορεί να έχει η μετάγγιση με χρήση αιμοδοτών G6PD- σε ορισμένες ομάδες δεκτών, οι οποίοι χαρακτηρίζονται από αυξημένο οξειδωτικό φορτίο.Glucose-6-phosphate dehydrogenase deficiency affects more than 400 million people worldwide, a fact that justifies the importance of evaluating the suitability of G6PD deficient donors in blood transfusions. The current work, has aimed to describe the progressive physiological changes that occur in erythrocytes from G6PD- donors during their storage at blood bank conditions, compared to those donated by G6PD+ donors and to estimate the effects of transfusing G6PD- erythrocytes in recipients, via an in vitro model of transfusion. For these purposes, 12 blood volunteers were recruited (6 G6PD- and 6 G6PD+) and tested, at first place, for their hematological and biochemical profiles, by using automatic analyzers. The measurements of reactive oxygen species (ROS) (by fluorometry), of osmotic and mechanical fragility and of hemolysis were perfomed in stored red blood cells in fresh samples and following different storage periods. Also, in reconstitution experiments red blood cells, from three G6PD- donors and three G6PD+ donors, stored 21, 30 or 42 days at blood bank conditions were incubated with fresh plasma donated by healthy volunteers (“recipients”), overnight at body temperature (370C). The same methodological approaches were followed for the assessment of the reconstituted blood. According to the ex vivo results, the storage capacity of G6PD- donors seems to be very similar or even better in comparison to that of G6PD+ donors regarding those specific parameters, however, the measurements performed in the reconstituted blood raised critical questions regarding the post-transfusional efficiency and effects of G6PD- RBCs in recipients experiencing high levels of oxidative stress

Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

<div><p>Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.</p></div