Is It Possible to Reverse the Storage-Induced Lesion of Red Blood Cells?

Cold-storage of packed red blood cells (PRBCs) in the blood bank is reportedly associated with alteration in a wide range of RBC features, which change cell storage each on its own timescale. Thus, some of the changes take place at an early stage of storage (during the first 7 days), while others occur later. We still do not have a clear understanding what happens to the damaged PRBC following their transfusion. We know that some portion (from a few to 10%) of transfused cells with a high degree of damage are removed from the bloodstream immediately or in the first hour(s) after the transfusion. The remaining cells partially restore their functionality and remain in the recipient’s blood for a longer time. Thus, the ability of transfused cells to recover is a significant factor in PRBC transfusion effectiveness. In the present review, we discuss publications that examined RBC lesions induced by the cold storage, aiming to offer a better understanding of the time frame in which these lesions occur, with particular emphasis on the question of their reversibility. We argue that transfused RBCs are capable (in a matter of a few hours) of restoring their pre-storage levels of ATP and 2,3-DPG, with subsequent restoration of cell functionality, especially of those properties having a more pronounced ATP-dependence. The extent of reversal is inversely proportional to the extent of damage, and some of the changes cannot be reversed

Hemolytic Activity of Nanoparticles as a Marker of Their Hemocompatibility

The potential use of nanomaterials in medicine offers opportunities for novel therapeutic approaches to treating complex disorders. For that reason, a new branch of science, named nanotoxicology, which aims to study the dangerous effects of nanomaterials on human health and on the environment, has recently emerged. However, the toxicity and risk associated with nanomaterials are unclear or not completely understood. The development of an adequate experimental strategy for assessing the toxicity of nanomaterials may include a rapid/express method that will reliably, quickly, and cheaply make an initial assessment. One possibility is the characterization of the hemocompatibility of nanomaterials, which includes their hemolytic activity as a marker. In this review, we consider various factors affecting the hemolytic activity of nanomaterials and draw the reader’s attention to the fact that the formation of a protein corona around a nanoparticle can significantly change its interaction with the red cell. This leads us to suggest that the nanomaterial hemolytic activity in the buffer does not reflect the situation in the blood plasma. As a recommendation, we propose studying the hemocompatibility of nanomaterials under more physiologically relevant conditions, in the presence of plasma proteins in the medium and under mechanical stress

System-Forming Dominant of Professional Qualities Formation of a Teacher on the Basis of Contextual-Network Technology

Due to the rapid globalization of the modern educational space, pedagogical education in Russia characterized by the process of large-scale modernization, which has led to growing problem of educator training as a multicultural personality capable of network collaboration with educators of both domestic and foreign countries. Orientation on the educator training capable of expressing responsibility and self-independence in the decision making, creativity and initiative is becoming the primary trend in the State policy in the context of educational sphere, that is viewed as the factor of development not only of the personality of a student, but also of an educator. The goal of the paper is to argue a theoretical foundation of the professional qualities of an educator in the context of contextual-network technology, the basis of which are the ideas of contextual education, androgogy, ideas of network learning. These trends are more focused on the problems of the professional qualities formation of an educator in the context of a health-saving educational environment in which the personalities of education interact to obtain a holistic picture of the world. However, the practical experience of professional qualities formation showed insufficient development of this problem in accordance with the requirements of the FSES HE and the introduction of digital technologies in the learning process, which is confirmed by regulatory documents, in particular the Russian Government's Resolution on the Approval of the State Program of the Russian Federation "Education Development (from 26.2017 No.1642)."All abovementioned trends have updated the need to develop the problem of professional qualities formation of an educator.Research methods of research are: theoretical (analysis of philosophical, psychological and educational literature, theoretical modeling of structural and meaningful basis of the study process; empirical (questionnaire, business games, projects); diagnostic (analysis of activity products, summative phase, pedagogical experiment, methods of mathematical statistics).The concept of "professional qualities of an educator is revealed" and the system of professional qualities of an educator is argued, and its effectiveness is proved.Contextual and network technology is developed and the author’s program «Professional Qualities Formation of an Educator in the Context of Health-Saving Educational Environment" contributed to the health saving and the natural potential of an educator and motivated an educator to achieve the results and pedagogical comfort

System-Forming Dominant of Professional Qualities Formation of a Teacher on the Basis of Contextual-Network Technology

Due to the rapid globalization of the modern educational space, pedagogical education in Russia characterized by the process of large-scale modernization, which has led to growing problem of educator training as a multicultural personality capable of network collaboration with educators of both domestic and foreign countries. Orientation on the educator training capable of expressing responsibility and self-independence in the decision making, creativity and initiative is becoming the primary trend in the State policy in the context of educational sphere, that is viewed as the factor of development not only of the personality of a student, but also of an educator. The goal of the paper is to argue a theoretical foundation of the professional qualities of an educator in the context of contextual-network technology, the basis of which are the ideas of contextual education, androgogy, ideas of network learning. These trends are more focused on the problems of the professional qualities formation of an educator in the context of a health-saving educational environment in which the personalities of education interact to obtain a holistic picture of the world. However, the practical experience of professional qualities formation showed insufficient development of this problem in accordance with the requirements of the FSES HE and the introduction of digital technologies in the learning process, which is confirmed by regulatory documents, in particular the Russian Government's Resolution on the Approval of the State Program of the Russian Federation "Education Development (from 26.2017 No.1642)."All abovementioned trends have updated the need to develop the problem of professional qualities formation of an educator.Research methods of research are: theoretical (analysis of philosophical, psychological and educational literature, theoretical modeling of structural and meaningful basis of the study process; empirical (questionnaire, business games, projects); diagnostic (analysis of activity products, summative phase, pedagogical experiment, methods of mathematical statistics).The concept of "professional qualities of an educator is revealed" and the system of professional qualities of an educator is argued, and its effectiveness is proved.Contextual and network technology is developed and the author’s program «Professional Qualities Formation of an Educator in the Context of Health-Saving Educational Environment" contributed to the health saving and the natural potential of an educator and motivated an educator to achieve the results and pedagogical comfort

The Deletion of NHR1 Region of the AML1-ETO Protein Significantly Decreases Its Ability To Promote Proliferation and Self-Renewal of Early Hematopoietic Cells in Culture

Abstract Although the transcriptional product of the (8;21) chromosomal translocation, the AML1-ETO protein, has numerous well established effects on the behavior of human hematopoietic cells, the specific cellular pathways perturbed by it are only partly known. Recently AML1-ETO has been shown to bind E-proteins (members of the class I basic helix-loop-helix family) via its NHR1 (nervy homology region one) domain, thus causing a replacement of the co-activator p300/CBP complex by the co-repressor hystone deacytelase complexes and abrogating the E-protein induced transcriptional activation (Zhang et al, Science 2004). The removal of amino acids 93 to 189 from the NHR1 domain (the ΔNHR1 mutant form) has completely reversed this inhibitory effect, and thus we have studied the biological effects of this deletion. To assess the difference in self-renewal capacity between cells transduced with the full length and the ΔNHR1 forms of AML1-ETO, CD34+ cells were plated into methylcellulose culture medium. On days 14 and 28 myeloid and erythroid colonies were scored, and the cells harvested and re-plated. The scoring on day 14 showed no significant difference in either the overall number or the type of colonies, with both the full-length AML1-ETO and the ΔNHR1 mutant showing a decreased erythroid colony formation, thus indicating a preserved negative effect on hematopoietic cell differentiation. However, on days 28 and 42 cells expressing the full-length AML1-ETO scored a significantly higher number of colonies than cells expressing the ΔNHR1 mutant, whereas cells transduced with the empty MIGR1 vector were unable to form any colonies after second re-plating. To assess the difference in growth potential, we have plated cells containing each of the constructs in IMD medium supplemented with 20% BIT and cytokines (SCF, FLT-3L, IL-6, TPO). Following one week in culture the cells were harvested, counted and then serially re-plated for five weeks. We observed a significantly higher number of cells expressing the full-length AML1-ETO as compared to the ΔNHR1 mutant at each weekly time point. Cells growing in the liquid culture were also weekly re-plated into methylcellulose culture medium. In this assay the loss of NHR1 domain significantly diminished the ability of AML1-ETO to maintain colony-forming progenitors in culture. To assess the difference in transcriptional activity of the different constructs, we have measured the expression of p21, and several other potential AML1-ETO target genes, by Real Time PCR. The expression of p21 was increased 14-fold by the full-length AML1-ETO, but only 2-fold by the ΔNHR1 mutant. We conclude that partial deletion of the NHR1 lowers the self-renewal capacity of transduced hematopoietic cells, decreases the proliferative advantage conferred by AML1-ETO, undermines its ability to maintain colony-forming units, and affects the transcriptional activation of AML1-ETO target genes. As the loss of NHR1 abolishes the E-protein induced transcriptional activation without affecting the binding of AML1-ETO to DNA, our findings identify an additional mechanism of action for AML1-ETO, independent of its dominant-negative effect on AML1 target genes

Do We Store Packed Red Blood Cells under “Quasi-Diabetic” Conditions?

Red blood cell (RBC) transfusion is one of the most common therapeutic procedures in modern medicine. Although frequently lifesaving, it often has deleterious side effects. RBC quality is one of the critical factors for transfusion efficacy and safety. The role of various factors in the cells’ ability to maintain their functionality during storage is widely discussed in professional literature. Thus, the extra- and intracellular factors inducing an accelerated RBC aging need to be identified and therapeutically modified. Despite the extensively studied in vivo effect of chronic hyperglycemia on RBC hemodynamic and metabolic properties, as well as on their lifespan, only limited attention has been directed at the high sugar concentration in RBCs storage media, a possible cause of damage to red blood cells. This mini-review aims to compare the biophysical and biochemical changes observed in the red blood cells during cold storage and in patients with non-insulin-dependent diabetes mellitus (NIDDM). Given the well-described corresponding RBC alterations in NIDDM and during cold storage, we may regard the stored (especially long-stored) RBCs as “quasi-diabetic”. Keeping in mind that these RBC modifications may be crucial for the initial steps of microvascular pathogenesis, suitable preventive care for the transfused patients should be considered. We hope that our hypothesis will stimulate targeted experimental research to establish a relationship between a high sugar concentration in a storage medium and a deterioration in cells’ functional properties during storage

Double-Facet Effect of Artificial Mechanical Stress on Red Blood Cell Deformability: Implications for Blood Salvage

The use of intra-operative blood salvage, dialysis, and artificial organs are associated with the application of non-physiological mechanical stress on red blood cells (RBCs). To explore the effect of these procedures on red cell deformability, we determined it before and after the mechanical stress application both in an in vitro system and following a blood-saving procedure. RBC from eight healthy donors and fifteen packed RBC units were subjected to mechanical stress. RBCs from five patients undergoing orthopedic surgery were also collected. We measured the percent of undeformable cells (%UDFC) in the red cell samples using our cell flow properties image analyzer, which provides the distribution of RBC deformability in a large cell population. Mechanical stress systematically reduced the cell deformability and increased the %UDFC, while simultaneously causing hemolysis of rigid, undeformable RBCs. Ultimately, the overall result depended on the initial level of the undeformable cells; the stress-induced change in the proportion of rigid cells (Δ%UDFC) increased (Δ%UDFC > 0) when its initial value was low, and decreased (Δ%UDFC < 0) when its initial value was high. This suggests that the final impact of mechanical stress on the percent of rigid cells in the RBC population is primarily determined by their initial concentration in the sample

Red Blood Cell Deformability Is Expressed by a Set of Interrelated Membrane Proteins

Red blood cell (RBC) deformability, expressing their ability to change their shape, allows them to minimize their resistance to flow and optimize oxygen delivery to the tissues. RBC with reduced deformability may lead to increased vascular resistance, capillary occlusion, and impaired perfusion and oxygen delivery. A reduction in deformability, as occurs during RBC physiological aging and under blood storage, is implicated in the pathophysiology of diverse conditions with circulatory disorders and anemias. The change in RBC deformability is associated with metabolic and structural alterations, mostly uncharacterized. To bridge this gap, we analyzed the membrane protein levels, using mass spectroscopy, of RBC with varying deformability determined by image analysis. In total, 752 membrane proteins were identified. However, deformability was positively correlated with the level of only fourteen proteins, with a highly significant inter-correlation between them. These proteins are involved in membrane rafting and/or the membrane–cytoskeleton linkage. These findings suggest that the reduction of deformability is a programmed (not arbitrary) process of remodeling and shedding of membrane fragments, possibly mirroring the formation of extracellular vesicles. The highly significant inter-correlation between the deformability-expressing proteins infers that the cell deformability can be assessed by determining the level of a few, possibly one, of them

The Acetylation of AML1-ETO Is Required for Leukemogenesis

Abstract Abstract 1588 Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures: No relevant conflicts of interest to declare