Inhibition of oxidative hemolysis in erythrocytes by mitochondria-targeted antioxidants of SkQ series

In the present work we studied the effect of antioxidants of the SkQ1 family (10-(6′-plastoquinonyl)decyltriphenylphosphonium) on the oxidative hemolysis of erythrocytes induced by a lipophilic free radical initiator 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN) and a water-soluble free radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH). SkQ1 was found to protect erythrocytes from hemolysis, 2 μM being the optimal concentration. Both the oxidized and reduced SkQ1 forms exhibited protective properties. Both forms of SkQ1 also inhibited lipid peroxidation in erythrocytes induced by the lipophilic free radical initiator AMVN as detected by accumulation of malondialdehyde. However, in the case of induction of erythrocyte oxidation by AAPH, the accumulation of malondialdehyde was not inhibited by SkQ1. In the case of AAPH-induced hemolysis, the rhodamine-containing analog SkQR1 exerted a comparable protective effect at the concentration of 0.2 μM. At higher SkQ1 and SkQR1 concentrations, the protective effect was smaller, which was attributed to the ability of these compounds to facilitate hemolysis in the absence of oxidative stress. We found that plastoquinone in the oxidized form of SkQ1 could be reduced by erythrocytes, which apparently accounted for its protective action. Thus, the protective effect of SkQ in erythrocytes, which lack mitochondria, proceeded at concentrations that are two to three orders of magnitude higher than those that were active in isolated mitochondria.     

Impact of Conditional miRNA126 Overexpression on Apoptosis-Resistant Endothelial Cell Production

The activation of endothelial cells is essential to repair damage caused by atherosclerosis via endothelial cell proliferation and migration. Overexpression of VEGF (vascular endothelial growth factor) and the downstream gene, B-cell lymphoma-2 (BCL-2) could result in apoptosis-resistant endothelial cells, which are responsible for aggravated hyperplasia and instable plaques generation. Previous studies have shown that miRNA126 could regulate the expression of VEGF. Here, we verified the existence of a miRNA126 binding site in VEGF’s 3’UTR. Additionally, VEGF regulated BCL-2 expression via AP1 (Activator Protein 1) binding site in BCL-2’s promoter. Next, we established an apoptosis-resistant endothelial cell line and constructed a lentiviral vector to express miRNA126 under the control of the BCL-2 promoter to investigate whether conditional expression of miRNA126 could modulate VEGF and BCL-2 expression in apoptosis-resistant endothelial cells. This lentiviral system specifically expressed miRNA126 in cells with high BCL-2 levels, downregulated VEGF expression, inhibited MAPK pathway activation and downregulated BCL-2 expression via suppression of AP1, and as a whole, reduced apoptosis-resistant endothelial cells, while the effects of miRNA126 on normal endothelial cells were relatively small. Our results demonstrate that conditional miRNA126 overexpression under the control of the downstream BCL-2 promoter provides a flexible regulatory strategy for reducing the apoptosis-resistant endothelial cells without having a significant impact on normal endothelial cells.     

Subgroups of Paediatric Acute Lymphoblastic Leukaemia Might Differ Significantly in Genetic Predisposition to Asparaginase Hypersensitivity

L-asparaginase (ASP) is a key element in the treatment of paediatric acute lymphoblastic leukaemia (ALL). However, hypersensitivity reactions (HSRs) to ASP are major challenges in paediatric patients. Our aim was to investigate genetic variants that may influence the risk to Escherichia coli-derived ASP hypersensitivity. Sample and clinical data collection was carried out from 576 paediatric ALL patients who were treated according to protocols from the Berlin—Frankfurt—Münster Study Group. A total of 20 single nucleotide polymorphisms (SNPs) in GRIA1 and GALNT10 genes were genotyped. Patients with GRIA1 rs4958351 AA/AG genotype showed significantly reduced risk to ASP hypersensitivity compared to patients with GG genotype in the T-cell ALL subgroup (OR = 0.05 (0.01–0.26); p = 4.70E-04), while no such association was found in pre-B-cell ALL. In the medium risk group two SNPs of GRIA1 (rs2055083 and rs707176) were associated significantly with the occurrence of ASP hypersensitivity (OR = 0.21 (0.09–0.53); p = 8.48E-04 and OR = 3.02 (1.36–6.73); p = 6.76E-03, respectively). Evaluating the genders separately, however, the association of rs707176 with ASP HSRs was confined only to females. Our results suggest that genetic variants of GRIA1 might influence the risk to ASP hypersensitivity, but subgroups of patients can differ significantly in this respect.     

Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP^Sc. Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP^Sc were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrP^C to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties.     

Advanced lymphoblastic clones detection in T-cell leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant neoplasm of the lymphocyte precursors that suffered malignant transformation arresting the lymphoid cell differentiation. Clinical studies revealed monoor, more rarely, oligoclonal nature of the disease. A precise identification of malignant clone markers is both the crucial stage of early diagnostics and the essential prognostic factor for therapeutic treatment. Here we present an improved system for unbiased detection of lymphoblastic clones in bone marrow aspirates of T-ALL patients. The system based on multiplex PCR of rearranged T-cell receptor locus (TRB) and straightforward sequencing of the resulted PCR fragments. Testing of the system on genomic DNA from Jurkat cell line and four clinical bone marrow aspirates revealed a set of unique TRB rearrangements that precisely characterize each of tested samples. Therefore, the outcome of the system produces highly informative molecular genetic markers for further monitoring of minimal residual disease in T-ALL patients.     

A real-time computational model for estimating kinematics of ankle ligaments

Background: An accurate assessment of ankle ligament kinematics is crucial in understanding the injury mechanisms and can help to improve the treatment of an injured ankle, especially when used in conjunction with robot-assisted therapy. A number of computational models have been developed and validated for assessing the kinematics of ankle ligaments. However, few of them can do real-time assessment to allow for an input into robotic rehabilitation programs. Method: An ankle computational model was proposed and validated to quantify the kinematics of ankle ligaments as the foot moves in real-time. This model consists of three bone segments with three rotational degrees of freedom (DOFs) and 12 ankle ligaments. This model uses inputs for three position variables that can be measured from sensors in many ankle robotic devices that detect postures within the foot–ankle environment and outputs the kinematics of ankle ligaments. Validation of this model in terms of ligament length and strain was conducted by comparing it with published data on cadaver anatomy and magnetic resonance imaging. Results: The model based on ligament lengths and strains is in concurrence with those from the published studies but is sensitive to ligament attachment positions. Conclusions: This ankle computational model has the potential to be used in robot-assisted therapy for real-time assessment of ligament kinematics. The results provide information regarding the quantification of kinematics associated with ankle ligaments related to the disability level and can be used for optimizing the robotic training trajectory.