The RoxyScan is a novel measurement of red blood cell deformability under oxidative and shear stress.

Exposure to both oxidative and shear stress, a condition that the red blood cell (RBC) continuously experiences in the circulation in vivo can be mimicked in a Couette type viscometer and monitored by ektacytometry. RBCs maintain their deformation and orientation under shear stress and oxidative stress until a threshold is reached at which these conditions appear to overwhelm the elaborate and complex pathways that maintain a proper redox environment in the cell. Oxidative stress under shear alters the ability of the cell to deform, changes cell morphology, its orientation in the shear stress field, and appears to alter intracellular and membrane characteristics. The application of the RoxyScan technology allows the comparison of oxidant effects and the role of antioxidant systems. This provides the opportunity to study the ability of RBC to deal with oxidative stress in various conditions, including RBC disorders such as sickle cell disease (SCD).

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration.

Pathologies such as malaria, hemorrhagic stroke, sickle cell disease, and thalassemia are characterized by the release of hemoglobin degradation products from damaged RBCs. Hematin (liganded with OH-) and hemin (liganded with Cl-)-are the oxidized forms of heme with toxic properties due to their hydrophobicity and the presence of redox-active Fe3. In the present study, using the original LaSca-TM laser particle analyzer, flow cytometry, and confocal microscopy, we showed that both hematin and hemin induce dose-dependent RBC spherization and hemolysis with ghost formation. Hematin and hemin at nanomolar concentrations increased [Ca2+]i in RBC; however, spherization and hemolysis occurred in the presence and absence of calcium, indicating that both processes are independent of [Ca2+]i. Both compounds triggered acute phosphatidylserine exposure on the membrane surface, reversible after 60 min of incubation. A comparison of hematin and hemin effects on RBCs revealed that hematin is a more reactive toxic metabolite than hemin towards human RBCs. The toxic effects of heme derivatives were reduced and even reversed in the presence of albumin, indicating the presence in RBCs of the own recovery system against the toxic effects of heme derivatives.

Oxidative stress's impact on red blood cells: Unveiling implications for health and disease.

Oxidative stress, a condition characterized by an imbalance between reactive oxygen species (ROS) production and the body's ability to detoxify them, has emerged as a pivotal factor in the pathophysiology of various diseases. Red blood cells (RBCs), essential components of the circulatory system, are particularly susceptible to oxidative damage due to their high oxygen-carrying capacity and the abundance of vulnerable biomolecules. This review comprehensively explores the intricate mechanisms underlying oxidative stress-induced damage to red blood cells and the subsequent implications for overall health and disease. We delve into the sources of ROS generation within RBCs, including metabolic processes and external factors, shedding light on the delicate redox balance that governs cellular homeostasis. The impact of oxidative stress on red blood cells extends beyond the confines of their primary physiological role, as these cells actively participate in immune responses, inflammation modulation, and nitric oxide metabolism. Consequently, understanding the implications of oxidative stress on RBCs provides valuable insights into the broader landscape of health and disease. In conclusion, this review underscores the critical role of oxidative stress in influencing red blood cell physiology and its far-reaching implications for human health. Elucidating the molecular intricacies of this relationship not only enhances our understanding of fundamental biological processes but also paves the way for the development of targeted therapeutic interventions to mitigate the adverse effects of oxidative stress on red blood cells and, by extension, on overall health.

New view on the compatibility of hemoglobin function in the erythrocytes.

OBJECTIVE: Aim: To study the process of hemoglobin oxidation and the enzymatic reactions associated with it. PATIENTS AND METHODS: Materials and Methods: Heparinized human blood (15 IU/ml) was obtained from the clinical department. The concentration of oxy- and methemoglobin, auto-oxidation of hemoglobin was determined spectrophotometrically spectrophotometrically. Autooxidation of hemoglobin was recorded spectrophotometrically, and protein concentration was determined by the Lowry method. Monooxygenase activity of hemoglobin was also measured by the method described by Lowry spectrophotometrically. The concentration of O2 and H2O2 in the reaction media was determined on a biomicroanalyzer OR 210/3 (Redelkis). RESULTS: Results: The obtained experimental data allow us to propose a mechanism of "spontaneous autooxidation" of oxyhemoglobin, which can be described by the following equations: Hb2+O2 → Hb3+ + O2 - (1) Hb2+O2 + 2e - + 2H+ →Hb3+ + H2O2 (2) Hb2+O2 + 2e - + 2H+ →Hb2+ + H2O2 (3) Hb2+ + O2 →Hb2+O2 (4) Spectral characteristics of the process of "spontaneous auto-oxidation" indicate the formation of a metform of hemoglobin, the depletion of oxygen by the system was established, at pH 5.6, an increase in the monooxygenase activity of hemoglobin is observed 3-4 times compared to the physiological level. CONCLUSION: Сonclusions: In addition to the main, previously known functions of hemoglobin (gas transport, peroxidase, monooxygenase), it catalyzes a two-electron oxidase reaction in which O2 is reduced to H2O2. This is confirmed by experimental data on the formation of one of the products of "spontaneous autoxidation" of oxyhemoglobin _ deoxyform at pH 5.6 _ 8.9.

Building Synthetic Biosensors Using Red Blood Cell Proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.

Human erythrocytes under stress. Spectroscopic fingerprints of known oxidative mechanisms and beyond.

In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (H2O2) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than H2O2 and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of ß-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and H2O2 caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into ß-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.

High linoleic acid levels in red blood cells predict a poor response to neoadjuvant chemotherapy in human epidermal growth factor receptor type 2-positive breast cancer patients.

OBJECTIVE: Polyunsaturated fatty acids are categorized as ω-3 or ⍵-6. Previous studies demonstrate that breast cancers display a high expression of fatty acid synthase and high fatty acid levels. Our study sought to determine if changes in plasma or red blood cell membrane fatty acid levels were associated with the response to preoperative (neoadjuvant) chemotherapy in non-metastatic breast cancer patients. METHODS: Our prospective study assessed fatty acid levels in plasma and red blood cell membrane. Response to neoadjuvant chemotherapy was evaluated by the presence or absence of pathologic complete response and/or residual cancer burden. RESULTS: A total of 28 patients were included. First, patients who achieved pathologic complete response had significantly higher neutrophil-to-lymphocyte ratio versus no pathologic complete response (P = 0.003). Second, total red blood cell membrane polyunsaturated fatty acids were higher in the absence of pathologic complete response (P = 0.0028). Third, total red blood cell membrane ⍵-6 polyunsaturated fatty acids were also higher in no pathologic complete response (P < 0.01). Among ⍵-6 polyunsaturated fatty acids, red blood cell membrane linoleic acid was higher in the absence of pathologic complete response (P < 0.01). Notably, plasma polyunsaturated fatty acid, ⍵-6, and linoleic acid levels did not have significant differences. A multivariate analysis confirmed red blood cell membrane linoleic acid was associated with no pathologic complete response; this was further confirmed by receiver operating characteristic analysis (specificity = 92.3%, sensitivity = 76.9%, and area under the curve = 0.855). CONCLUSIONS: Pending further validation, red blood cell membrane linoleic acid might serve as a predictor biomarker of poorer response to neoadjuvant chemotherapy in non-metastatic human epidermal growth factor receptor type 2-positive breast cancer. Measuring fatty acids in red blood cell membrane could offer a convenient, minimally invasive strategy to identifying patients more likely to respond or those with chemoresistance.

The Development and Characterisation of ssDNA Aptamers via a Modified Cell-SELEX Methodology for the Detection of Human Red Blood Cells.

Blood is one of the most commonly found biological fluids at crime scenes, with the detection and identification of blood holding a high degree of evidential value. It can provide not only information about the nature of the crime but can also lead to identification via DNA profiling. Presumptive tests for blood are usually sensitive but not specific, so small amounts of the substrate can be detected, but false-positive results are often encountered, which can be misleading. Novel methods for the detection of red blood cells based on aptamer-target interactions may be able to overcome these issues. Aptamers are single-stranded DNA or RNA sequences capable of undergoing selective antigen association due to three-dimensional structure formation. The use of aptamers as a target-specific moiety poses several advantages and has the potential to replace antibodies within immunoassays. Aptamers are cheaper to produce, display no batch-to-batch variation and can allow for a wide range of chemical modifications. They can help limit cross-reactivity, which is a hindrance to current forensic testing methods. Within this study, a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process was used to generate aptamers against whole red blood cells. Obtained aptamer pools were analysed via massively parallel sequencing to identify viable sequences that demonstrate a high affinity for the target. Using bioinformatics platforms, aptamer candidates were identified via their enrichment profiles. Binding characterisation was also conducted on two selected aptamer candidates via fluorescent microscopy and qPCR to visualise and quantify aptamer binding. The potential for these aptamers is broad as they can be utilised within a range of bioassays for not only forensic applications but also other analytical science and medical applications. Potential future work includes the incorporation of developed aptamers into a biosensing platform that can be used at crime scenes for the real-time detection of human blood.

Smoking is associated with increased eryptosis, suicidal erythrocyte death, in a large population-based cohort.

Smoking has multiple detrimental effects on health, and is a major preventable cause of premature death and chronic disease. Despite the well-described effect of inhaled substances from tobacco smoke on cell toxicity, the association between smoking and suicidal erythrocyte death, termed eryptosis, is virtually unknown. Therefore, the blood samples of 2023 participants of the German National Cohort Study (NAKO) were analyzed using flow cytometry analysis to determine eryptosis from fluorescent annexin V-FITC-binding to phosphatidylserine-exposing erythrocytes. Blood analyses were complemented by the measurement of hematologic parameters including red blood cell count, hematocrit, hemoglobin, mean corpuscular cell volume (MCV) and mean corpuscular hemoglobin (MCH). Eryptosis was higher in smokers than in non- and ex-smokers, and positively associated with the number of cigarettes smoked daily (r = 0.08, 95% CI [0.03, 0.12]). Interestingly, despite increased eryptosis, smokers had higher red blood cell indices than non-smokers. To conclude, smokers were characterized by higher eryptosis than non-smokers, without showing any obvious detrimental effect on classic hematological parameters.

Microvesicles from stored red blood cells induce P-selectin and von Willebrand factor release from endothelial cells via a protein kinase C-dependent mechanism.

INTRODUCTION: The use of packed red blood cells (pRBCs) for resuscitation is limited by the red blood cell storage lesion, a series of biochemical and physiological changes that occur during the storage and aging of blood. Microvesicles (MVs) shed from pRBCs during this process are one component of the red blood cell storage lesion and lead to acute lung injury and pulmonary vascular microthrombi. We hypothesized that MVs from stored pRBCs lead to the release of P-selectin and von Willebrand factor (vWF) from endothelial cells and that this mechanism is mediated via activation of protein kinase C (PKC) or protein kinase A (PKA). METHODS: Leukoreduced, platelet-poor murine pRBCs were isolated from C57BL/6 8-12 week-old male mice via cardiac puncture, prepared via centrifugation using a Ficoll gradient, and stored for up to 14 days, the equivalent of 42 days of storage in humans. MVs were isolated from the stored pRBC units via sequential high-speed centrifugation. Murine lung endothelial cells (MLECs) were cultured and grown to confluence, then treated with MVs and either calphostin C, a PKC inhibitor (10 µg/mL), or PKI 14-22 amide, a PKA inhibitor (10 µM). The supernatant was collected after 1 h. P-selectin and vWF A2 concentrations were quantified via ELISA. Immunofluorescent staining for vWF was performed on MLECs. Statistical analysis was performed via unpaired t-test or ANOVA as indicated and reported as mean ± SD. Concentration is reported as pg/mL. RESULTS: MLECs treated with MVs isolated from stored pRBCs demonstrated increased release of P-selectin and vWF A2 in a dose-dependent fashion. MLECs treated with MVs prepared from stored as compared to fresh pRBCs demonstrated increased release of P-selectin (3751 ± 726 vs 359 ± 64 pg/mL, p < 0.0001) and vWF A2 (3141 ± 355 vs 977 ± 75 pg/mL, p < 0.0001) with increasing duration of storage. The treatment of MVs with calphostin C decreased the amount of P-selectin (1471 ± 444 vs 3751 ± 726 pg/mL, p < 0.0001) and VWF A2 (2401 ± 289 vs 3141 ± 355 pg/mL, p = 0.0017) released into the supernatant by MLECs compared to MVs alone. The treatment of MVs with PKI 14-22 increased the amount of P-selectin released compared to MVs alone (1999 ± 67 vs 1601 ± 135 pg/mL, p = 0.0018). CONCLUSIONS: MVs from stored pRBCs stimulate the release of P-selectin and VWF A2 from endothelial cells. The effect of MVs increases with both dose of MVs and age of stored pRBCs from which they are formed. This mechanism is dependent on activation of PKC and inhibition of this enzyme represents a potentially significant strategy to modulate the inflammatory response to resuscitation with stored pRBCs.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

BACKGROUND: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied. STUDY DESIGN AND METHODS: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery. RESULTS: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity. DISCUSSION: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed.

Transcriptomics of early responses to purified Piscine orthoreovirus-1 in Atlantic salmon (Salmo salar L.) red blood cells compared to non-susceptible cell lines.

Piscine red blood cells (RBC) are nucleated and have been characterized as mediators of immune responses in addition to their role in gas exchange. Salmonid RBC are major target cells of Piscine orthoreovirus-1 (PRV-1), the etiological agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). PRV-1 replicates in RBC ex vivo, but no viral amplification has been possible in available A. salmon cell lines. To compare RBC basal transcripts and transcriptional responses to PRV-1 in the early phase of infection with non-susceptible cells, we exposed A. salmon RBC, Atlantic salmon kidney cells (ASK) and Salmon head kidney cells (SHK-1) to PRV-1 for 24 h. The RNA-seq analysis of RBC supported their previous characterization as pluripotent cells, as they expressed a wide repertoire of genes encoding pattern recognition receptors (PRRs), cytokine receptors, and genes implicated in antiviral activities. The comparison of RBC to ASK and SHK-1 revealed immune cell features exclusively expressed in RBC, such as genes involved in chemotactic activity in response to inflammation. Differential expression analysis of RBC exposed to PRV-1 showed 46 significantly induced genes (≥ 2-fold upregulation) linked to the antiviral response pathway, including RNA-specific PRRs and interferon (IFN) response factors. In SHK-1, PRV induced a more potent or faster antiviral response (213 genes induced). ASK cells showed a differential response pattern (12 genes induced, 18 suppressed) less characterized by the dsRNA-induced antiviral pathway. Despite these differences, the RIG-I-like receptor 3 (RLR3) in the family of cytosolic dsRNA receptors was significantly induced in all PRV-1 exposed cells. IFN regulatory factor 1 (IRF1) was significantly induced in RBC only, in contrast to IRF3/IRF7 induced in SHK-1. Differences in IRF expression and activity may potentially affect viral propagation.

Effect of leukoreduction on the metabolism of equine packed red blood cells during refrigerated storage.

BACKGROUND: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited. OBJECTIVE: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC). ANIMALS: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks. METHODS: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses. RESULTS: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC. CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC.

Glycophorin B-PfEMP1 interaction mediates robust rosetting in Plasmodium falciparum.

P. falciparumerythrocyte membrane protein 1 (PfEMP1) is the major parasite protein responsible for rosetting by binding to host receptors such as heparan sulfate, CR1 on RBC surface. Usually monomeric protein-carbohydrate interactions are weak [1], therefore PfEMP1 binds to plasma proteins like IgM or α2-macroglobulin that facilitate its clustering on parasitized RBC surface and augment rosetting [2,3]. We show that 3D7A expresses PfEMP1, PF3D7_0412900, and employs its CIDRγ2 domain to interact with glycophorin B on uninfected RBC to form large rosettes but more importantly even in the absence of plasma proteins. Overall, we established the role of PF3D7_0412900 in rosetting as antibodies against CIDRγ2 domain reduced rosetting and also identified its receptor, glycophorin B which could provide clue why glycophorin B null phenotype, S-s-U- RBCs prevalent in malaria endemic areas is protective against severe malaria.

Investigation of the cytotoxicity, genotoxicity and antioxidant prospects of JM-20 on human blood cells: A multi-target compound with potential therapeutic applications.

JM-20 is a 1,5-benzodiazepine compound fused to a dihydropyridine fraction with different pharmacological properties. However, its potential toxic effects on blood cells have not yet been reported. Thus, the present study aimed to investigate, for the first time, the possible cytotoxicity of JM-20 through cell viability, cell cycle, morphology changes, reactive species (RS) to DCFH-DA, and lipid peroxidation in human leukocytes, its hemolytic effect on human erythrocytes, and its potential DNA genotoxicity using plasmid DNA in vitro. Furthermore, the compound's ability to reduce the DPPH radical was also measured. Human blood was obtained from healthy volunteers (30 ± 10 years old), and the leukocytes or erythrocytes were immediately isolated and treated with different concentrations of JM-20. A cytoprotective effect was exhibited by 10 µM JM-20 against 1 mM tert-butyl hydroperoxide (t-but-OOH) in the leukocytes. However, the highest tested concentrations of the compound (20 and 50 µM) changed the morphology and caused a significant decrease in the cell viability of leukocytes (p < 0.05, in comparison with Control). All tested concentrations of JM-20 also resulted in a significant increase in intracellular RS as measured by DCFH-DA in these cells (p < 0.05, in comparison with Control). On the other hand, the results point out a potent antioxidant effect of JM-20, which was similar to the classical antioxidant α-tocopherol. The IC50 value of JM-20 against the lipid peroxidation induced by (FeII) was 1.051 µM ± 0.21, while the IC50 value of α-tocopherol in this parameter was 1.065 µM ± 0.34. Additionally, 50 and 100 µM JM-20 reduced the DPPH radical in a statistically similar way to the 100 µM α-tocopherol (p < 0.05, in comparison with the control). No significant hemolysis in erythrocytes, no cell cycle changes in leukocytes, and no genotoxic effects in plasmid DNA were induced by JM-20 at any tested concentration. The in silico pharmacokinetic and toxicological properties of JM-20, derivatives, and nifedipine were also studied. Here, our findings demonstrate that JM-20 and its putative metabolites exhibit similar characteristics to nifedipine, and the in vitro and in silico data support the low toxicity of JM-20 to mammals.

Macrocytic anemias.

PURPOSE OF REVIEW: Over the last century, the diseases associated with macrocytic anemia have been changing with more patients currently having hematological diseases including malignancies and myelodysplastic syndrome. The intracellular mechanisms underlying the development of anemia with macrocytosis can help in understanding normal erythropoiesis. Adaptations to these diseases involving erythroid progenitor and precursor cells lead to production of fewer but larger red blood cells, and understanding these mechanisms can provide information for possible treatments. RECENT FINDINGS: Both inherited and acquired bone marrow diseases involving primarily impaired or delayed erythroid cell division or secondary adaptions to basic erythroid cellular deficits that results in prolonged cell division frequently present with macrocytic anemia. SUMMARY OF FINDINGS: In marrow failure diseases, large accumulations of iron and heme in early stages of erythroid differentiation make cells in those stages especially susceptible to death, but the erythroid cells that can survive the early stages of terminal differentiation yield fewer but larger erythrocytes that are recognized clinically as macrocytic anemia. Other disorders that limit deoxynucleosides required for DNA synthesis affect a broader range of erythropoietic cells, but they also lead to macrocytic anemia. The source of macrocytosis in other diseases remains uncertain.

Possible Markers for Distinguishing benign and Malignant Thyroid Tumors and Predicting Malignancy in Patients with Genetic Predisposition to Cancer.

BACKGROUND: We hypothesized that mutations in several genes disrupt oxidative metabolism, increasing the risk of developing tumors and their malignancy in patients with a family predisposition to cancer. The purpose of our study was to assess the characteristics of oxidative metabolism in patients with malignant and benign tumor with and without a family history of cancer and identify the marker predicting the likelihood of malignancy. METHODS: We conducted a study on patients with thyroid pathology (thyrotoxicosis, benign tumor pathology of the thyroid gland, and thyroid cancer) who underwent treatment at LLC "Oncology Scientific Research Center" in Tbilisi, Georgia between 2020-2021.  In patients' blood the thyroid hormones content, the oxidative metabolism parameters (activity of nonenzymatic antioxidant system (TAA), malondialdehyde (MDA) content),  geometrical and rheological (deformability index (EDI), membrane proteins content) characteristics of erythrocytes were determined. RESULTS: in the patient's blood serum with benign tumor (47 patients) MDA exceeded (p<0.005) and TAA decreased (p<0.005) in comparison to the control level; in patients with thyroid cancer (35 patients), MDA also exceeded (p<0.005), while TAA increased (p<0.005) up to the control level. In patients with benign and malignant tumors, the size of erythrocytes increased compared to the control indicators (p<0.005); in patients with thyroid cancer and benign tumors with a family history of cancer (29 patients) EDI increased (p<0.005), content of GLUT1 in erythrocyte membranes decreased (p<0.005) compared to the control level. CONCLUSIONS: Alterations in redox metabolism play a crucial role in tumor formation; an imbalance between anti-/pro-oxidant systems may contribute to tumor formation and support its progression into a more malignant state. Thyroid cancer is characterized by a reduction in erythrocyte deformability, related to TSH levels. These alterations are less detectable in patients with benign thyroid tumors with a family history of cancer.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Basigin mediation of Plasmodium falciparum red blood cell invasion does not require its transmembrane domain or interaction with monocarboxylate transporter 1.

Plasmodium falciparum invasion of the red blood cell is reliant upon the essential interaction of PfRh5 with the host receptor protein basigin. Basigin exists as part of one or more multiprotein complexes, most notably through interaction with the monocarboxylate transporter MCT1. However, the potential requirement for basigin association with MCT1 and the wider role of basigin host membrane context and lateral protein associations during merozoite invasion has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate the ability to uncouple basigin ectodomain presentation from its transmembrane domain-mediated interactions, including with MCT1. Merozoite invasion of reticulocytes is unaffected by disruption of basigin-MCT1 interaction and by removal or replacement of the basigin transmembrane helix. Therefore, presentation of the basigin ectodomain at the red blood cell surface, independent of its native association with MCT1 or other interactions mediated by the transmembrane domain, is sufficient to facilitate merozoite invasion.