Search Results (16)

Water molecules in the cytosol of red blood cells (RBCs) may exist in a free or bound state. The ratio between the free and bound water depends on the composition of the cytoplasm, particularly on the hemoglobin concentration. Microwave dielectric spectroscopy (MDS) provides information on the state of intracellular water in red blood cell suspension and the erythrocyte cytosol state. In the presented study, we used MDS to assess the differences in the free-to-bound water ratio in subpopulations of freshly donated human erythrocytes of different ages (young, mature, and senescent cells) obtained by fractionation in a Percoll density gradient. The obtained MDS parameters (dielectric strength ∆ε, the relaxation time τ, and the broadening parameter α) were compared with the red blood cell indices and single cell deformability measurements obtained for each subpopulation. We demonstrated that the unique hematological indices and deformability of red blood cells of different ages are well-correlated with the specific values of dielectric fitting parameters. The obtained results indicate that the dielectric properties of cytosolic water can serve as a sensitive marker of changes occurring in the cytosol of red blood cells during cell aging. Full article

N6–methyladenosine (m⁶A) is a widespread post–transcriptional modification in eukaryotic mRNAs. Proteins with the YTH structural domain act as m⁶A–binding proteins by recognizing the m⁶A modification and regulating mRNA through this recognition. In this study, SlYTHDF2, a prototypical m⁶A –binding protein gene in the YTH family was expressed in various tissues, and subcellular localization analyses indicated that the SlYTHDF2 protein was localized in the nucleus and cytoplasm. SlYTHDF2 knockout lines were obtained using CRISPR/Cas9 technology and showed the senesced leaves prematurely increased endogenous ABA accumulation compared with the wild type. Moreover, we found that dark promoted leaf senescence in SlYTHDF2 knockout lines and exogenous ABA further accelerated leaf senescence under dark conditions. The qRT–PCR analysis revealed significant alterations in the expression of genes associated with the ABA pathway. Relative to the wild type, the CR–slythdf2 plants exhibited reduced levels of photosynthetic pigments, higher accumulation of reactive oxygen species, and increased damage to cell membranes. Additionally, we discovered that SlYTHDF2 interacts with the chloroplast–binding protein SlRBCS3 through yeast two–hybrid and BiFC experiments. Overall, our data suggest the important role of SlYTHDF2 in regulating tomato leaf senescence. Full article

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16− CD62L+) and aged (senescent) neutrophils (CD16+ CD62L−) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions. Full article

Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na⁺ and Ca²⁺. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca²⁺ ATPase and an increase in Ca²⁺ and Na⁺ leaks through ion channels in senescent-like cells. Our data revealed that Ca²⁺ overload in these cells is a result of reduced PMCA activity and facilitated Ca²⁺ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca²⁺-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca²⁺ reduced the initially high selectivity of Piezo1 channel for Ca²⁺ and allowed higher Na⁺ uptake, Na⁺ accumulation, and swelling. Full article

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity. Full article

Preeclampsia (PE) presents with maternal de novo hypertension and significant proteinuria and is one of the leading causes of maternal and perinatal morbidity and mortality with unknown etiology. The disease is associated with inflammatory vascular response and severe red blood cell (RBC) morphology changes. This study examined the nanoscopic morphological changes of RBCs from PE women versus normotensive healthy pregnant controls (PCs) and non-pregnant controls (NPCs) applying atomic force microscopy (AFM) imaging. The results revealed that the membrane of fresh PE RBCs differed significantly from healthy ones by the presence of invaginations and protrusions and an increased roughness value (R_rms) (4.7 ± 0.8 nm for PE vs. 3.8 ± 0.5 nm and 2.9 ± 0.4 nm for PCs and NPCs, respectively). PE-cells aging resulted in more pronounced protrusions and concavities, with exponentially increasing R_rms values, in contrast to the controls, where the R_rms parameter decreased linearly with time. The R_rms, evaluated on a 2 × 2 µm² scanned area, for senescent PE cells (13 ± 2.0 nm) was significantly higher (p < 0.01) than that of PCs (1.5 ± 0.2 nm) and NPCs (1.9 ± 0.2 nm). Furthermore, the RBCs from PE patients appeared fragile, and often only ghosts were observed instead of intact cells at 20–30 days of aging. Oxidative-stress simulation on healthy cells led to RBC membrane features similar to those observed for PE cells. The results demonstrate that the most pronounced effects on RBCs in PE patients are related to impaired membrane homogeneity and strongly altered roughness values, as well as to vesiculation and ghost formation in the course of cell aging. Full article

Despite lacking the central apoptotic machinery, senescent or damaged RBCs can undergo an unusual apoptosis-like cell death, termed eryptosis. This premature death can be caused by, or a symptom of, a wide range of diseases. However, various adverse conditions, xenobiotics, and endogenous mediators have also been recognized as triggers and inhibitors of eryptosis. Eukaryotic RBCs are unique among their cell membrane distribution of phospholipids. The change in the RBC membrane composition of the outer leaflet occurs in a variety of diseases, including sickle cell disease, renal diseases, leukemia, Parkinson’s disease, and diabetes. Eryptotic erythrocytes exhibit various morphological alterations such as shrinkage, swelling, and increased granulation. Biochemical changes include cytosolic Ca²⁺ increase, oxidative stress, stimulation of caspases, metabolic exhaustion, and ceramide accumulation. Eryptosis is an effective mechanism for the elimination of dysfunctional erythrocytes due to senescence, infection, or injury to prevent hemolysis. Nevertheless, excessive eryptosis is associated with multiple pathologies, most notably anemia, abnormal microcirculation, and prothrombotic risk; all of which contribute to the pathogenesis of several diseases. In this review, we provide an overview of the molecular mechanisms, physiological and pathophysiological relevance of eryptosis, as well as the potential role of natural and synthetic compounds in modulating RBC survival and death. Full article

Acute normoxic exercise impacts the rheological properties of red blood cells (RBC) and their senescence state; however, there is a lack of data on the effects of exercise performed in hypoxia on RBC properties. This crossover study compared the effects of acute hypoxia vs. normoxia on blood rheology, RBC senescence, and coagulation during exercise. Nine trained male cyclists completed both a session in normoxia (FiO₂ = 21%) and hypoxia (FiO₂ = 15.3% ≈ 2500 m). The two sessions were randomly performed, separated by one week, and consisted of an incremental and maximal exercise followed by a 20 min exercise at the first ventilatory threshold (VT1) on a home-trainer. Blood samples were taken before and after exercise to analyze hematological parameters, blood rheology (hematocrit, blood viscosity, RBC deformability and aggregation), RBC senescence markers (phosphatidylserine (PS) and CD47 exposure, intraerythrocyte reactive oxygen species (ROS), and calcium content), and blood clot viscoelastic properties. Hemoglobin oxygen saturation (SpO₂) and blood lactate were also measured. In both conditions, exercise induced an increase in blood viscosity, hematocrit, intraerythrocyte calcium and ROS content, and blood lactate concentration. We also observed an increase in blood clot amplitude, and a significant drop in SpO₂ during exercise in the two conditions. RBC aggregation and CD47 exposure were not modified. Exercise in hypoxia induced a slight decrease in RBC deformability which could be related to the slight increase in mean corpuscular hemoglobin concentration (MCHC). However, the values of RBC deformability and MCHC after the exercise performed in hypoxia remained in the normal range of values. In conclusion, acute hypoxia does not amplify the RBC and coagulation changes induced by an exercise bout. Full article

Eryptosis is a physiological mechanism for the clearance of senescent or damaged erythrocytes by phagocytes. Excessive eryptosis is stimulated under several pathologies and associated with endothelial injury and thrombosis. Cigarette smoke (CS) is an established risk factor for vascular diseases and cigarette smokers have high-levels of eryptotic erythrocytes. This study, for the first time, investigates the mechanism by which CS damages red blood cells (RBCs). CS extract (CSE) from commercial cigarettes was prepared and standardized for nicotine content. Cytofluorimetric analysis demonstrated that treatment of human RBCs with CSE caused dose-dependent, phosphatidylserine externalization and cell shrinkage, hallmarks of apoptotic death. CSE did not affect cellular levels of Ca²⁺, reactive oxygen species (ROS) or glutathione (GSH). Immununoprecipitation and immunoblotting revealed the assembly of the death-inducing signaling complex (DISC) and oligomerization of Fas receptor as well as cleaved caspase-8 and caspase-3 within 6 h from the treatment. At the same time-interval, CSE elicited neutral sphyngomielinase (nSMase) activity-dependent ceramide formation and phosphorylation of p38 MAPK. Through specific inhibitors’ nSMase, caspase-8 or p38 MAPK activities, we demonstrated that p38 MAPK activation is required for caspase-8-mediated eryptosis and that ceramide generation is initiator caspase-dependent. Finally, ex vivo analysis detected phosphorylated p38 MAPK (p-p38) and Fas-associated signaling complex in erythrocytes from cigarette smokers. In conclusion, our study demonstrates that CSE exposure induces in erythrocytes an extrinsic apoptotic pathway involving p38 MAPK-initiated DISC formation followed by activation of caspase-8/caspase-3 via ceramide formation. Full article

Total body irradiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated radiation-induced iron in the spleens of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed increased splenic Fe³⁺ and altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not affect iron deposition in the spleen or modulate iron-binding proteins. Caspase-3 was activated after ~7–14 days, indicating apoptosis had occurred. We also identified markers of iron-dependent apoptosis known as ferroptosis. The p21/Waf1 accelerated senescence marker was not upregulated. Macrophage inflammation is an effect of TBI. We investigated the effects of radiation and Fe³⁺ on the J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, after ~24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron-binding proteins and triggers apoptosis and possible ferroptosis. Full article

Human red blood cells (RBCs), senescent or damaged due to particular stress, can be removed by programmed suicidal death, a process called eryptosis. There are various molecular mechanisms underlying eryptosis. The most frequent is the increase in the cytoplasmic concentration of Ca²⁺ ions, later exposure of erythrocytes to oxidative stress, hyperosmotic shock, ceramide formation, stimulation of caspases, and energy depletion. Phosphatidylserine (PS) exposed by eryptotic RBCs due to interaction with endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor, causes the RBCs to adhere to vascular wall with consequent damage to the microcirculation. Eryptosis can be triggered by various xenobiotics and endogenous molecules, such as high cholesterol levels. The possible diseases associated with eryptosis are various, including anemia, chronic kidney disease, liver failure, diabetes, hypertension, heart failure, thrombosis, obesity, metabolic syndrome, arthritis, and lupus. This review addresses and collates the existing ex vivo and animal studies on the inhibition of eryptosis by food-derived phytochemicals and natural compounds including phenolic compounds (PC), alkaloids, and other substances that could be a therapeutic and/or co-adjuvant option in eryptotic-driven disorders, especially if they are introduced through the diet. Full article

The chloroplast comprises the outer and inner membranes that are composed of the translocon protein complexes Toc and Tic (translocon at the outer/inner envelope membrane of chloroplasts), respectively. Tic55, a chloroplast Tic protein member, was shown to be not vital for functional protein import in Arabidopsis from previous studies. Instead, Tic55 was revealed to be a dark-induced senescence-related protein in our earlier study. To explore whether Tic55 elicits other biological functions, a tic55-II knockout mutant (SALK_086048) was characterized under different stress treatments. Abiotic stress conditions, such as cold, heat, and high osmotic pressure, did not cause visible effects on tic55-II mutant plant, when compared to the wild type (WT). In contrast, senescence was induced in the individually darkened leaves (IDLs), resulting in the differential expression of the senescence-related genes PEROXISOME DEFECTIVE 1 (PED1), BLUE COPPER-BINDING PROTEIN (BCB), SENESCENCE 1 (SEN1), and RUBISCO SMALL SUBUNIT GENE 2B (RBCS2B). The absence of Tic55 in tic55-II knockout mutant inhibited expression of the senescence-related genes PED1, BCB, and SEN1 at different stages of dark adaptation, while causing stimulation of RBCS2B gene expression at an early stage of dark response. Finally, yeast one-hybrid assays located the ANAC003 promoter region with cis-acting elements are responsible for binding to the different AtbHLH proteins, thereby causing the transactivation of an HIS3 reporter gene. ANAC003 was shown previously as a senescence-related protein and its activation would lead to expression of senescence-associated genes (SAGs), resulting in plant senescence. Thus, we propose a hypothetical model in which three signaling pathways may be involved in controlling the expression of ANAC003, followed by expression of SAGs that in turn leads to leaf senescence in Arabidopsis by this study and previous data. Full article

7-hydroxymethyl chlorophyll (Chl) a reductase (HCAR) plays critical roles in the Chl cycle and degradation during leaf senescence, however, its function in horticultural crops remains unknown. Here, we identified an HCAR gene (CsHCAR) from cucumber (Cucumis sativus L.) and investigated its roles in response to dark-induced Chl degradation. CsHCAR encoded 459 amino acids, which were orthologous to Arabidopsis HCAR, had the conserved domains, and localized in the chloroplast. Gene expression analysis showed that CsHCAR expression was the highest in senescent leaves and was responsive to different stresses and phytohormone treatments. Overexpression of CsHCAR in tobacco accelerated dark-induced Chl degradation through enhancing the expression of Chl catabolic genes. After 10 d of darkness treatment, the biomass of CsHCAR overexpression plants was reduced. Furthermore, the value of net photosynthetic rate, maximum quantum yield of photosystem II, and effective quantum yield of photosystem II in CsHCAR overexpression plants was significantly reduced in comparison to that in wild-type (WT) plants. The photosynthetic protein content, including Lhcb1, Lhcb2, Lhcb4, RbcS, and RbcL in CsHCAR overexpression plants exhibited a lower level as compared to that observed in WT plants. In addition, the expression of genes encoding these proteins in CsHCAR overexpression plants was significantly lower than that in WT plants. Moreover, CsHCAR overexpression plants inhibited the dark-induced accumulation of reactive oxygen species (ROS). These results indicate that CsHCAR affects the stability of photosynthetic proteins in chloroplasts, positively regulates Chl degradation, and plays an important role in maintaining ROS homeostasis in leaves. Full article

BACKGROUND: Blood transfusion remains a key treatment for managing occlusive episodes and painful crises in sickle-cell disease (SCD). In that clinical context, red blood cells (RBCs) from donors and transfused to patients, may be affected by plasma components in the recipients’ blood. Senescence lesion markers appear on the red cells after transfusion, shortening the RBC lifespan in circulation. In the specific context of SCD, senescence signals can also trigger the occlusive painful events, typical of the disease. This work follows through our previous data that described a RBC senescence process, rapidly detected after challenge with SCD pathological plasmas. In this clinical context, we wanted here to further explore the characteristics and physiologic consequences of AA RBC lesions associated with senescence, as lesions caused by RBCs after transfusion may have adverse consequences for SCD patients. METHODS: Plasma samples from SCD patients, with acute symptoms (n = 20) or steady-state disease (n = 34) were co-incubated with donor AA RBCs from blood units for 24 to 48 h. Specific markers signing RBC senescence were quantified after the incubation with SCD plasma samples. The physiologic in-flow adhesion was investigated on senescent RBCs, an in vitro technic into biochips that mimic adherence of RBCs during the occlusive events of SCD. RESULTS: Senescence markers on AA RBCs, together with their in-flow adhesion to the plasma-bridging protein thrombospondin, were associated with the clinical status of the SCD patients from whom plasma was obtained. In these experiments, the highest values were obtained for SCD acute plasma samples. Adhesion of senescent RBCs into biochips, which is not reversed by a pre-treatment with recombinant Annexin V, can be reproduced with the use of chemical agents acting on RBC membrane channels that regulate either Ca²⁺ entry or modulating RBC hydration. CONCLUSION: We found that markers on red cells are correlated, and that the senescence induced by SCD plasma provokes the adhesion of RBCs to the vessel wall protein thrombospondin. In-flow adhesion of senescent red cells after plasma co-incubations can be reproduced with the use of modulators of RBC membrane channels; activating the Piezo1 Ca²⁺ mechanosensitive channel provokes RBC adhesion of normal (non-senescent) RBCs, while blocking the Ca²⁺-dependent K⁺ Gardos channel, can reverse it. Clinically modulating the RBC adhesion to vascular wall proteins might be a promising avenue for the treatment of painful occlusive events in SCD. Full article

In mammals, 2 × 10¹² red blood cells (RBCs) are produced every day in the bone marrow to ensure a constant supply of iron to maintain effective erythropoiesis. Impaired iron absorption in the duodenum and inefficient iron reutilization from senescent RBCs by macrophages contribute to the development of anemia. Ferroportin (Fpn), the only known cellular iron exporter, as well as hephaestin (Heph) and ceruloplasmin, two copper-dependent ferroxidases involved in the above-mentioned processes, are key elements of the interaction between copper and iron metabolisms. Crosslinks between these metals have been known for many years, but metabolic effects of one on the other have not been elucidated to date. Neonatal iron deficiency anemia in piglets provides an interesting model for studying this interplay. In duodenal enterocytes of young anemic piglets, we identified iron deposits and demonstrated increased expression of ferritin with a concomitant decline in both Fpn and Heph expression. We postulated that the underlying mechanism involves changes in copper distribution within enterocytes as a result of decreased expression of the copper transporter—Atp7b. Obtained results strongly suggest that regulation of iron absorption within enterocytes is based on the interaction between proteins of copper and iron metabolisms and outcompetes systemic regulation. Full article