Bardoxolone methyl displays detrimental effects on endothelial bioenergetics, suppresses endothelial ET-1 release, and increases endothelial permeability in human microvascular endothelium

Nrf2 is a master regulator of antioxidant cellular defence, and agents activating the Nrf2 pathway have been tested in various diseases. However, unexpected side effects of cardiovascular nature reported for bardoxolone methyl in patients with type 2 diabetes mellitus and stage 4 chronic kidney disease (the BEACON trial) still have not been fully explained. Here, we aimed to characterize the effects of bardoxolone methyl compared with other Nrf2 activators- dimethyl fumarate and Lsulforaphane - on human microvascular endothelium. Endothelial toxicity, bioenergetics, mitochondrial membrane potential, endothelin-1 (ET-1) release, endothelial permeability, Nrf2 expression, and ROS production were assessed in human microvascular endothelial cells (HMEC-1) incubated for 3 and 24 hours with 100nM-5 μM of either bardoxolone methyl, dimethyl fumarate, or L-sulforaphane. Three-hour incubation with bardoxolone methyl (100nM-5 μM), although not toxic to endothelial cells, significantly affected endothelial bioenergetics by decreasing mitochondrial membrane potential (concentrations ≥ 3 μM), decreasing spare respiratory capacity (concentrations ≥ 1 μM), and increasing proton leak (concentrations ≥ 500nM), while dimethyl fumarate and L-sulforaphane did not exert such actions. Bardoxolone methyl at concentrations ≥ 3 μM also decreased cellular viability and induced necrosis and apoptosis in the endothelium upon 24-hour incubation. In turn, endothelin-1 decreased permeability in endothelial cells in picomolar range, while bardoxolone methyl decreased ET-1 release and increased endothelial permeability even after short-term (3 hours) incubation. In conclusion, despite that all three Nrf2 activators exerted some beneficial effects on the endothelium, as evidenced by a decrease in ROS production, bardoxolone methyl, the most potent Nrf2 activator among the tested compounds, displayed a distinct endothelial profile of activity comprising detrimental effects on mitochondria and cellular viability and suppression of endothelial ET-1 release possibly interfering with ET-1-dependent local regulation of endothelial permeability

Nondestructive detection method for the calcium and nitrogen content of living plants based on Convolutional Neural Networks (CNN) using multispectral images

Herein, we present the novel method targeted for determination of plant nutritional state with the use of computer vision and Neural Networks. The method is based on multispectral imaging performed by an exclusively designed Agroscanner and a dedicated analytical system for further data analysis with Neural Networks. An Agroscanner is a low-cost mobile construction intended for multispectral measurements at macro-scale, operating at four wavelengths: 470, 550, 640 and 850 nm. Together with developed software and implementation of a Neural Network it was possible to design a unique approach to process acquired plant images and assess information about plant physiological state. The novelty of the developed technology is focused on the multispectral, macro-scale analysis of individual plant leaves, rather than entire fields. Such an approach makes the method highly sensitive and precise. The method presented herein determines the basic physiological deficiencies of crops with around 80% efficiency

Temporal sequence of the human RBCs' vesiculation observed in nano-scale with application of AFM and complementary techniques

Based on the multimodal characterization of human red blood cells (RBCs), the link between the storage-related sequence of the nanoscale changes in RBC membranes in the relation to their biochemical profile as well as mechanical and functional properties was presented. On the background of the accumulation of RBCs waste products, programmed cell death and impaired rheological properties, progressive alterations in the RBC membranes including changes in their height and diameter as well as the in situ characterization of RBC-derived microparticles (RMPs) on the RBCs surface were presented. The advantage of atomic force microscopy (AFM) in RMPs visualization, even at the very early stage of vesiculation, was shown based on the results revealed by other reference techniques. The nanoscale characterization of RMPs was correlated with a decrease in cholesterol and triglycerides levels in the RBC membranes, proving the link between the lipids leakage from RBCs and the process of vesiculation

An insight into the stages of ion leakage during red blood cell storage

Packed red blood cells (pRBCs), the most commonly transfused blood product, are exposed to environmental disruptions during storage in blood banks. In this study, temporal sequence of changes in the ion exchange in pRBCs was analyzed. Standard techniques commonly used in electrolyte measurements were implemented. The relationship between ion exchange and red blood cells (RBCs) morphology was assessed with use of atomic force microscopy with reference to morphological parameters. Variations observed in the Na+, K+, Cl−, H+, HCO3−, and lactate ions concentration show a complete picture of singly-charged ion changes in pRBCs during storage. Correlation between the rate of ion changes and blood group type, regarding the limitations of our research, suggested, that group 0 is the most sensitive to the time-dependent ionic changes. Additionally, the impact of irreversible changes in ion exchange on the RBCs membrane was observed in nanoscale. Results demonstrate that the level of ion leakage that leads to destructive alterations in biochemical and morphological properties of pRBCs depend on the storage timepoint

Sex-specific differences of adenosine triphosphate levels in red blood cells isolated from ApoE/LDLR double-deficient mice

In this study for the first time, we investigated the correlation between sex-specific differences in adenosine triphosphate (ATP) levels in red blood cells (RBCs) and their mechanical, biochemical, and morphological alterations during the progression of atherosclerosis in ApoE/LDLR double-deficient ($ApoE/LDLR^{-/-}$) mice. Our results indicate that both sex and age affect alterations in RBCs of both $ApoE/LDLR^{-/-}$ and C57BL/6J mice. When compared with male RBCs, female RBCs were characterized by lower basal ATP and mean corpuscular hemoglobin concentration (MCHC), higher hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), deformability, and phosphatidylserine (PS) exposure levels, regardless of age in both, $ApoE/LDLR^{-/-}$ and C57BL/6J mice. $ApoE/LDLR^{-/-}$ mice compared with age-matched controls showed lower basal ATP levels regardless of age and sex. Intracellular ATP level of RBCs was decreased solely in senescent female C57BL/6J mice, while it was elevated in males. Basal extracellular ATP levels were 400 times lower than corresponding intracellular level. In conclusion, basal ATP levels, RBC morphology, deformability, PS exposure levels alterations are sex-dependent in mice. Changes in basal ATP levels were correlated with PS exposure and trends of changes in MCV. Trends of changes of the most RBC parameters were similar in both sexes of $ApoE/LDLR^{-/-}$ mice compared with age-matched controls, however, their kinetics and levels vary greatly between different stages of disease progression

Label-free testing strategy to evaluate packed red blood cell quality before transfusion to leukemia patients

Abstract Patients worldwide require therapeutic transfusions of packed red blood cells (pRBCs), which is applied to the high-risk patients who need periodic transfusions due to leukemia, lymphoma, myeloma and other blood diseases or disorders. Contrary to the general hospital population where the transfusions are carried out mainly for healthy trauma patients, in case of high-risk patients the proper quality of pRBCs is crucial. This leads to an increased demand for efficient technology providing information on the pRBCs alterations deteriorating their quality. Here we present the design of an innovative, label-free, noninvasive, rapid Raman spectroscopy-based method for pRBCs quality evaluation, starting with the description of sample measurement and data analysis, through correlation of spectroscopic results with reference techniques' outcomes, and finishing with methodology verification and its application in clinical conditions. We have shown that Raman spectra collected from the pRBCs supernatant mixture with a proper chemometric analysis conducted for a minimum one ratio of integral intensities of the chosen Raman marker bands within the spectrum allow evaluation of the pRBC quality in a rapid, noninvasive, and free-label manner, without unsealing the pRBCs bag. Subsequently, spectroscopic data were compared with predefined reference values, either from pRBCs expiration or those defining the pRBCs quality, allowing to assess their utility for transfusion to patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL)

Spectroscopic signature of red blood cells in a D-galactose-induced accelerated aging model

This work presents a semi-quantitative spectroscopic approach, including FTIR-ATR and Raman spectroscopies, for the biochemical analysis of red blood cells (RBCs) supported by the biochemical, morphological and rheological reference techniques. This multi-modal approach provided the description of the RBC alterations at the molecular level in a model of accelerated aging induced by administration of D-galactose (D-gal), in comparison to natural aging. Such an approach allowed to conclude that most age-related biochemical RBC membrane changes (a decrease in lipid unsaturation and the level of phospholipids, or an increase in acyl chain shortening) as well as alterations in the morphological parameters and RBC deformability are well reflected in the D-gal model of accelerated aging. Similarly, as in natural aging, a decrease in LDL level in blood plasma and no changes in the fraction of glucose, creatinine, total cholesterol, HDL, iron, or triglycerides were observed during the course of accelerated aging. Contrary to natural aging, the D-gal model led to an increase in cholesterol esters and the fraction of total esterified lipids in RBC membranes, and evoked significant changes in the secondary structure of the membrane proteins. Moreover, a significant decrease in the phosphorous level of blood plasma was specific for the D-gal model. On the other hand, natural aging induced stronger changes in the secondary structures of the proteins of the RBCs' interior. This work proves that research on the aging mechanism, especially in circulation-related diseases, should employ the D-gal model with caution. Nonetheless, the D-gal model enables to imitate age-related rheological alterations in RBCs, although they are partially derived from different changes observed in the RBC membrane at the molecular level