[Influence of standard treatment on changes in the structural and functional properties of circulation red blood cells in obstructive and non-obstructive acute pyelonephritis].

AIM: To determine the effect of standard treatment on changes in the structural and functional properties of erythrocytes in obstructive and non-obstructive acute pyelonephritis. MATERIALS AND METHODS: The structural and functional properties of erythrocytes and their intracellular metabolism in 78 patients with a diagnosis of primary non-obstructive and secondary obstructive acute pyelonephritis, randomized by age, gender, and the minimum number of concomitant diseases were investigated. RESULTS AND DISCUSSION: In acute non-obstructive pyelonephritis, changes of the content of proteins in circulating erythrocytes responsible for the structure formation and stabilization of the plasma membrane (-spectrin, anion transport protein, pallidin, protein 4.1), intracellular metabolism (anion transport protein, glutathione-S-transferase), membrane flexibility and shape (actin, tropomyosin) are insignificant, alike from acute obstructive pyelonephritis. In addition, processes of lipid peroxidation inside red blood cells are intensified, and oxidative stress develops with a decrease in the sorption capacity of erythrocytes, as well as the content and ratio of lipid fractions in the plasma membrane, which form the basis of the lipid components and play the main role in the sequencing of protein macromolecules and the normal metabolism of red blood cells. CONCLUSION: In acute obstructive pyelonephritis, changes in the content and ratio of proteins and lipids in the erythrocyte membrane lead to functional rearrangements that are not corrected by standard treatment.

The associations of erythrocyte membrane polyunsaturated fatty acids with skeletal muscle loss: A prospective cohort study.

BACKGROUND & AIMS: Polyunsaturated fatty acids (PUFAs) may play a vital role in maintaining skeletal muscle mass in the aged population. This study investigated the longitudinal relationship between the concentrations of erythrocyte membrane PUFAs and age-related changes in skeletal muscle mass over an average 6.5 years of follow-up in a Chinese middle-aged and older adult population. METHODS: A total of 1494 participants aged 57.4 ± 4.7 years were included in this study. Skeletal muscle mass was determined using dual-energy X-ray absorptiometry. Per year percent changes in the skeletal muscle index (Δ% SMI), appendicular skeletal muscle index (Δ% ASMI), and total body lean mass index (Δ% TBLMI) from baseline were calculated. Concentrations of total and individual cis-n-3 and cis-n-6 PUFAs of the erythrocyte membrane were determined using gas-liquid chromatography. RESULTS: Fully adjusted linear regression models showed that per unit increases in the concentrations of C18:2 n-6, C20:4 n-6, C22:4 n-6, and total n-6 PUFAs resulted in increases of 0.022%-0.155 % in the Δ% SMI (P for linearity: <0.001-0.006). Restricted cubic spline analysis revealed an inverted U-shaped relationship between the concentrations of C20:2 n-6, C22:5 n-3, C22:6 n-3, and total n-3 PUFAs and the Δ% SMI (P for non-linearity: <0.001-0.036). In addition, an inverted U-shaped curve was also detected for the relationships of the linoleic acid/α-linolenic acid ratio (P for non-linearity = 0.010) and n-6/n-3 PUFA ratio (P for non-linearity = 0.013) with the Δ% SMI, with the Δ% SMI peaking at respective ratios of 124.96 and 3.69. Similar associations were revealed by the Bayesian kernel machine regression model. No interaction effect was detected between the individual PUFAs for the Δ% SMI in the bivariate exposure-response analysis. Overall, similar results were observed for the Δ% ASMI and Δ% TBLMI. CONCLUSIONS: The associations between different individual PUFAs and age-related muscle loss in middle-aged and older adults may be different. Our results suggest that high concentrations of erythrocyte membrane n-6 PUFAs may be correlated with less skeletal muscle mass loss, whereas extremely high concentrations of n-3 PUFAs may be correlated with more muscle loss.

Alterations in plasma and erythrocyte membrane fatty acid composition following exposure to toxic copper level affect membrane deformability and fluidity in female wistar rats.

BACKGROUND: Deformability and fluidity function of the red blood cell membrane are properties defined by the lipid composition. Toxic copper level induces membrane lipid peroxidation which could cause membrane instability. This study therefore investigated the effect of exposure to toxic copper level for 30 days on red blood cell membrane deformability and fluidity in female Wistar rats. METHODS: Twelve (12) female Wistar rats (160 ± 10 g) were randomly grouped (n = 6) into control (given 0.1 ml distilled water p.o.) and copper-toxic (100 mg/kg Copper Sulphate, p.o.), and treated for 30 days. Plasma obtained and RBC membrane prepared from blood collected over EDTA post-treatment were assayed for total cholesterol (TC), phospholipids and fatty acid profile using spectrophotometry and Gas chromatography while heparinized blood was subjected to fragility test. Data were analyzed using student T-test for statistical significance at p < 0.05. RESULTS AND CONCLUSION: Plasma TC increased by 4.33% while RBC membrane TC decreased by 20.32% in copper-toxic group compared to control. Compared to control, excess copper significantly increased membrane phospholipids level (0.72 ± 0.01 vs 0.59 ± 0.04 mg/dL) but reduced membrane cholesterol/phospholipid ratio (46.61 ± 4.72 vs 72.66 ± 6.47) and stability (by 23.53%). Number of cis- and saturated fatty acids increased in copper-treated plasma and RBC membrane compared to control. Exposure to toxic copper level alters erythrocyte membrane fluidity and deformability by disrupting membrane lipid composition, saturation, bond configuration in phospholipids and permeability.

Acarbose Mitigates Age-Dependent Alterations in Erythrocyte Membrane Transporters During Aging in Rats.

Acarbose (ACA), a well-studied and effective inhibitor of α-amylase and α-glucosidase, is a postprandial-acting antidiabetic medicine. The membrane of the erythrocyte is an excellent tool for analyzing different physiological and biochemical activities since it experiences a range of metabolic alterations throughout aging. It is uncertain if ACA modulates erythrocyte membrane activities in an age-dependent manner. As a result, the current study was conducted to explore the influence of ACA on age-dependent deteriorated functions of transporters/exchangers, disrupted levels of various biomarkers such as lipid hydroperoxides (LHs), protein carbonyl (PCO), sialic acid (SA), total thiol (-SH), and erythrocyte membrane osmotic fragility. In addition to a concurrent increase in Na+/H+ exchanger activity and concentration of LH, PCO, and osmotic fragility, we also detected a considerable decrease in membrane-linked activities of Ca2+-ATPase (PMCA) and Na+/K+-ATPase (NKA), as well as concentrations of SA and -SH in old-aged rats. The aging-induced impairment of the activities of membrane-bound ATPases and the changed levels of redox biomarkers were shown to be effectively restored by ACA treatment.

Erythrocyte-cancer hybrid membrane-coated reduction-sensitive nanoparticles for enhancing chemotherapy efficacy in breast cancer.

Cell-membrane-coated biomimetic nanoparticles (NPs) have attracted great attention due to their prolonged circulation time, immune escape mechanisms and homotypic targeting properties. Biomimetic nanosystems from different types of cell -membranes (CMs) can perform increasingly complex tasks in dynamic biological environments thanks to specific proteins and other properties inherited from the source cells. Herein, we coated doxorubicin (DOX)-loaded reduction-sensitive chitosan (CS) NPs with 4T1 cancer cell -membranes (CCMs), red blood cell -membranes (RBCMs) and hybrid erythrocyte-cancer membranes (RBC-4T1CMs) to enhance the delivery of DOX to breast cancer cells. The physicochemical properties (size, zeta potential and morphology) of the resulting RBC@DOX/CS-NPs, 4T1@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs, as well as their cytotoxic effect and cellular NP uptake in vitro were thoroughly characterized. The anti-cancer therapeutic efficacy of the NPs was evaluated using the orthotopic 4T1 breast cancer model in vivo. The experimental results showed that DOX/CS-NPs had a DOX-loading capacity of 71.76 ± 0.87 %, and that coating of DOX/CS-NPs with 4T1CM significantly increased the NP uptake and cytotoxic effect in breast cancer cells. Interestingly, by optimizing the ratio of RBCMs:4T1CMs, it was possible to increase the homotypic targeting properties towards breast cancer cells. Moreover, in vivo tumor studies showed that compared to control DOX/CS-NPs and free DOX, both 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs significantly inhibited tumor growth and metastasis. However, the effect of 4T1@DOX/CS-NPs was more prominent. Moreover, CM-coating reduced the uptake of NPs by macrophages and led to rapid clearance from the liver and lungs in vivo, compared to control NPs. Our results suggest that specific self-recognition to source cells resulting in homotypic targeting increased the uptake and the cytotoxic capacity of 4T1@DOX/CS-NPs by breast cancer cells in vitro and in vivo. In conclusion, tumor-disguised CM-coated DOX/CS-NPs exhibited tumor homotypic targeting and anti-cancer properties, and were superior over targeting with RBC-CM or RBC-4T1 hybrid membranes, suggesting that the presence of 4T1-CM is critical for treatment outcome.

Activity of Na+/K+- and Ca2+-ATPases in human erythrocyte membranes: Protocol improvement, relation to cholesterol content, and effects of polyphenols.

It is known that the activities of Na+/K+- and Ca2+-ATPases in the plasma membrane with an excess of cholesterol are compromised. Our main goal was to find out whether quercetin, resveratrol, or caffeic acid, in the nano- and low micromolar concentration ranges, can improve the ATPase activity in human erythrocyte membranes with excess cholesterol. These molecules belong to different chemical classes of polyphenols and are widely present in plant foods. Also, due to some variations in the protocol for determining the ATPase activity, we first analyzed several key parameters of the protocol to improve the accuracy of the results. The activities of Na+/K+- and Ca2+-ATPases were reduced in membranes with moderate and high cholesterol levels compared to membranes from normocholesterolemic subjects (p < 0.01). All three polyphenols affected the ATPase activity in a similar biphasic manner. Namely, the ATPase activity gradually increased with increasing polyphenol concentration up to 80-200 nM, and then gradually decreased with further increase in polyphenol concentration. Moreover, the stimulating effect of the polyphenols was highest in membranes with high cholesterol content, making ATPase activity values close/equal to those in normal cholesterol membranes. In other words, quercetin, resveratrol, and caffeic acid at nanomolar concentrations were able to improve/restore the functioning of Na+/K+- and Ca2+-ATPases in erythrocyte membranes with high cholesterol levels. This suggests a common membrane-mediated mechanism of action for these polyphenols, related to the content of membrane cholesterol.

Effect of Cell Membrane-cloaked Nanoparticle Elasticity on Nano-Bio Interaction.

Cell membrane-cloaked nanoparticles are exploited as a promising drug carrier to enhance circulation, accumulation, penetration into tumor sites and cellular internalization. However, the effect of physicochemical properties (e.g., size, surface charge, shape, and elasticity) of cell membrane-cloaked nanoparticles on nano-bio interaction is rarely studied. In the present study, keeping the other parameters constant, erythrocyte membrane (EM)-cloaked nanoparticles (nanoEMs) with different Young's moduli are fabricated by altering different kinds of nano-core (i.e., aqueous phase core, gelatin nanoparticles, and platinum nanoparticles). The designed nanoEMs are used to investigate the effect of nanoparticle elasticity on nano-bio interaction including cellular internalization, tumor penetration, biodistribution, blood circulation, and so on. The results demonstrate that the nanoEMs with intermediate elasticity (≈95 MPa) have a relatively higher increase in cellular internalization and inhibition of tumor cells migration than the soft (≈11 MPa) and stiff (≈173 MPa) ones. Furthermore, in vivo studies show that nanoEMs with intermediate elasticity preferentially accumulate and penetrate into tumor sites than the soft and stiff ones, while in circulation, softer nanoEMs show a longer blood circulation time. This work provides an insight for optimizing the design of biomimetic carriers and may further contribute to the selection of nanomaterials on biomedical application.

Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells.

Red blood cell (RBC)-derived systems offer a potential platform for delivery of biomedical cargos. Although the importance of specific proteins associated with the biodistribution and pharmacokinetics of these particles has been recognized, it remains to be explored whether some of the key transmembrane and cytoskeletal proteins responsible for immune-modulatory effects and mechanical integrity of the particles are retained. Herein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative tandem mass tag mass spectrometry in conjunction with bioinformatics analysis, we have examined the proteomes of micro- and nanosized erythrocyte ghosts doped with indocyanine green and compared them with those of RBCs. We identified a total of 884 proteins in each set of RBCs, micro-, and nanosized particles, of which 8 and 45 proteins were expressed at significantly different relative abundances when comparing micro-sized particles vs RBCs and nanosized particles vs RBCs, respectively. We found greater differences in relative abundances of some mechano-modulatory proteins, such as band 3 and protein 4.2, and immunomodulatory proteins like CD44, CD47, and CD55 in nanosized particles as compared to RBCs. Our findings highlight that the methods utilized in fabricating RBC-based systems can induce substantial effects on their proteomes. Mass spectrometry data are available at ProteomeXchange with the identifier PXD038780.

Probing the Interaction Between Supercarrier RBC Membrane and Nanoparticles for Optimal Drug Delivery.

Red blood cell (RBC) membrane-hitchhiking nanoparticles (NPs) have been an increasingly popular supercarrier for targeted drug delivery. However, the kinetic details of the shear-induced NP detachment process from RBC in blood flow remain unclear. Here, we perform detailed computational simulations of the traversal dynamics of an RBC-NP composite supercarrier with tunable properties. We show that the detachment of NPs from RBC occurs in a shear-dependent manner which is consistent with previous experiment results. We quantify the NP detachment rate in the microcapillary flow, and our simulation results suggest that there may be an optimal adhesion strength span of 25-40 µJ/m2 for rigid spherical NPs to improve the supercarrier performance and targeting efficiency. In addition, we find that the stiffness and the shape of NPs alter the detachment efficiency by changing the RBC-NP contact areas. Together, these findings provide unique insights into the shear-dependent NP release from the RBC surface, facilitating the clinical utility of RBC-NP composite supercarriers in targeted and localized drug delivery with high precision and efficiency.

Mediation analysis of erythrocyte lipophilic index on the association between BMI and risk of oral cancer.

AIMS: To explore the relationship between the fatty acid lipophilic index (LI) of the erythrocyte membrane and oral cancer risk, as well as to evaluate the possibility of LI acting as a mediator of the association between body mass index (BMI) and oral cancer. METHOD: Twenty-three fatty acids (FAs) of the erythrocyte membrane were measured using gas chromatography in 380 patients with oral cancer and 387 control subjects. The LI was calculated based on the FA proportion and FA melting points. The association of BMI and erythrocyte LI with oral cancer risk was analysed using logistic regression. The mediation effect of LI on the association between BMI and oral cancer risk was evaluated using mediation analysis. RESULTS: Among the control group, 46.0% were overweight or obese, which was significantly higher than that of oral cancer patients (29.5%). Significant differences in erythrocyte membrane saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) were observed between the patient and control groups. The proportion of C18:1 n-9 from the MUFA family increased in oral cancer patients (12.67%) compared with controls (12.21%). While the total proportion of n-3 PUFAs decreased in oral cancer patients compared with controls, with C20:5 n-3 decreasing from 0.66 to 0.47%, and C22:6 n-3 decreasing from 5.82 to 4.86%. The LI was lower in the control participants (M = 27.6, IQR: 27.3-27.9) than in the oral cancer patients (M = 28.2, IQR: 27.9-28.5). BMI was inversely associated with oral cancer risk with a fully adjusted OR of 0.59 (95% CI: 0.43-0.83), while LI was positively associated with oral cancer risk with a fully adjusted OR of 1.99 (95% CI:1.36-2.94). LI explained 7% of the variance in the relationship between BMI and oral cancer risk. CONCLUSIONS: The distribution of the FA profile in erythrocyte membranes differed between the oral cancer patients and the control group. The LI derived from the profile of FAs was positively associated with the risk of oral cancer, and the associations between BMI and oral cancer risk can be explained, at least in part, by LI.

Unravelling the genetic and phenotypic heterogeneity of SPTA1 gene variants in Hereditary Elliptocytosis and Hereditary Pyropoikilocytosis patients using next-generation sequencing.

Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP) are clinically and genetically heterogeneous red cell membranopathies that result from the defects in the horizontal linkage between RBC (red blood cell) membrane and cytoskeletal proteins affecting its mechanical stability and deformability thereby reducing its lifespan. The principal defect in HE and HPP is due to dysfunction or deficiency of RBC cytoskeletal proteins namely, α-spectrin (SPTA1), ß-spectrin (SPTB) and protein 4.1R (EPB41R). This study reports the genetic and phenotypic heterogeneity of 10 Indian patients (5 with HE and 5 with HPP)harboringSPTA1 gene variants. We used targeted next-generation sequencing (t-NGS) to characterize the causative genetic variants in 10 HE/HPP suspected patients and studied the correlation between the identified variants with their corresponding phenotypic features.t-NGS detected 12 SPTA1 variants, out of which 8 are novel. Nearly all of the detected variants have a damaging effect on the protein stability and function, as shown by the insilico analysis. The possible effect of the detected variants on the protein structure was studied using the HOPE software and DynaMut tools wherever possible. To the best of our knowledge, this is the first report on HE/HPP cases confirmed by a genetic study from India. To conclude, HE is caused by monoallelic mutations while HPP, the more severe form, is typically caused by biallelic (homozygous or compound heterozygous) mutations justifying the phenotypic heterogeneity associated with patients. Moreover, analysis at the molecular level by NGS permits diagnosis in these disorders with highly variable heterogeneity requiring regular transfusions and may facilitate prognostic contemplations.

Architecture of the human erythrocyte ankyrin-1 complex.

The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2, glycophorin A and glycophorin B. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and, unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.

Structure, dynamics and assembly of the ankyrin complex on human red blood cell membrane.

The cytoskeleton of a red blood cell (RBC) is anchored to the cell membrane by the ankyrin complex. This complex is assembled during RBC genesis and comprises primarily band 3, protein 4.2 and ankyrin, whose mutations contribute to numerous human inherited diseases. High-resolution structures of the ankyrin complex have been long sought-after to understand its assembly and disease-causing mutations. Here, we analyzed native complexes on the human RBC membrane by stepwise fractionation. Cryo-electron microscopy structures of nine band-3-associated complexes reveal that protein 4.2 stabilizes the cytoplasmic domain of band 3 dimer. In turn, the superhelix-shaped ankyrin binds to this protein 4.2 via ankyrin repeats (ARs) 6-13 and to another band 3 dimer via ARs 17-20, bridging two band 3 dimers in the ankyrin complex. Integration of these structures with both prior data and our biochemical data supports a model of ankyrin complex assembly during erythropoiesis and identifies interactions essential for the mechanical stability of RBC.

Nonalcoholic Fatty Liver Disease Patients Exhibit Reduced CD47 and Increased Sphingosine, Cholesterol, and Monocyte Chemoattractant Protein-1 Levels in the Erythrocyte Membranes.

Background: Nonalcoholic fatty liver disease (NAFLD) constitutes a significant cause of deaths, liver transplantations, and economic costs worldwide. Despite extended research, investigations on the role of erythrocytes are scarce. Red blood cells from experimental animals and human patients with NAFLD present phosphatidylserine exposure, which is then recognized by Kupffer cells. This event leads to erythrophagocytosis and amplification of inflammation through iron disposition. In addition, it has been shown that erythrocytes from NAFLD patients release the chemokine monocyte chemoattractant protein-1 (MCP1), leading to increased tumor necrosis factor alpha release from macrophages RAW 264.7. However, erythrophagocytosis can also be caused by reduced CD47 levels. Moreover, increased MCP1 release could be either signal-induced or caused by higher MCP1 levels on the erythrocyte membrane. Finally, erythrocyte efferocytosis could provide additional inflammatory metabolites. Methods: In this study, we measured the erythrocyte membrane levels of CD47 and MCP1 by enzyme-linked immunosorbent assay, and cholesterol and sphingosine with thin-layer chromatography. Eighteen patients (8 men and 10 women, aged 56.7 ± 11.5 years) and 14 healthy controls (7 men and 7 women, aged 39.3 ± 15.6 years) participated in our study. Results: The erythrocyte CD47 levels were decreased in the erythrocyte membranes of NAFLD patients (844 ± 409 pg/mL) compared with healthy controls (2969 ± 1936 pg/mL) with P = 0.012. Levels of MCP1 increased in NAFLD patients (389 ± 255 pg/mL) compared with healthy controls (230 ± 117 pg/mL) with P = 0.0274, but low statistical power. Moreover, in erythrocyte membranes, there was a statistically significant accumulation of sphingosine and cholesterol in NAFLD patients compared with healthy controls. Conclusions: Our results imply that erythrocytes release chemotactic "find me" signals (MCP1) while containing reduced "do not eat me" signals (CD47). These molecules can lead to erythrophagocytosis. Next, increased "goodbye" signals (sphingosine and cholesterol) could augment inflammation by metabolic reprogramming.

[Clinical effect, changes in the lipid structure of the erythrocyte membrane and microcirculation in patients with arterial hypertension in combination with coronary heart disease under the influence of laser therapy]. / Klinicheskii effekt, izmenenie lipidnoi struktury eritrotsitarnoi membrany i mikrotsirkulyatsii u bol'nykh arterial'noi gipertoniei v sochetanii s ishemicheskoi bolezn'yu serdtsa pod vliyaniem lazeroterapii.

The high prevalence of the combination of arterial hypertension (AH) with coronary heart disease (CHD) suggests the improvement of their treatment methods. In this regard, it is of interest to assess the dynamics of the clinical picture of patients against the background of pathogenetically determined subcellular and systemic changes under the influence of laser therapy (LT). OBJECTIVE: To evaluate the clinical effect of LT in patients with hypertension in combination with coronary artery disease and trace its relationship with the dynamics of the structure of the lipid bilayer of the erythrocyte membrane and changes in the microvascular bed. MATERIAL AND METHODS: We examined 65 male patients (mean age 50.9±6.3 years) with II-III degree AH in combination with coronary artery disease with angina pectoris. Among them, 40 patients received a 10-day course of LT, and 25 patients underwent simulated laser irradiation. At the initial stage and after 1 month, all patients underwent a bicycle exercise test, a study of the lipid composition of the erythrocyte membrane, including the main fractions of phospholipids and free cholesterol, as well as the level of intracellular Ca2+ and lipid peroxidation products - malondialdehyde and diene conjugates. Microcirculation was assessed using the method of conjunctival biomicroscopy. RESULTS: One month after the course of LT, patients showed a significant increase in exercise tolerance by 37.8%, a decrease in systolic blood pressure with a standard load by 9.9%. The improvement of the clinical picture occurred against the background of a decrease in the activity of lipid peroxidation and structural changes in the cell membrane: an increase in polyunsaturated fractions of phospholipids and a decrease in the cholesterol content, as well as a decrease in the Ca2+ level in the cell from 0.23 [0.19; 0.32] to 0.20 [0.16; 0.26] mmol/l. The results of the analysis of conjunctival biomicroscopy demonstrated a statistically significant decrease in the ratio of arteriolovenular calibers, a limitation of the severity of sludge syndrome by 59%, and an almost twofold (from 3.9±0.52 to 7.2±1.23 cap/mm2) increase in capillary density. CONCLUSION: The data obtained showed that in patients with hypertension in combination with coronary artery disease, LT causes positive changes in the lipid structure of the cell membrane and microcirculation parameters, which is accompanied by a hypotensive effect and an improvement in the clinical and functional state of patients.

Erythro-VLPs: Anchoring SARS-CoV-2 spike proteins in erythrocyte liposomes.

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/µm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future.

Effect of Fe3+ on Na,K-ATPase: Unexpected activation of ATP hydrolysis.

Iron is a key element in cell function; however, its excess in iron overload conditions can be harmful through the generation of reactive oxygen species (ROS) and cell oxidative stress. Activity of Na,K-ATPase has been shown to be implicated in cellular iron uptake and iron modulates the Na,K-ATPase function from different tissues. In this study, we determined the effect of iron overload on Na,K-ATPase activity and established the role that isoforms and conformational states of this enzyme has on this effect. Total blood and membrane preparations from erythrocytes (ghost cells), as well as pig kidney and rat brain cortex, and enterocytes cells (Caco-2) were used. In E1-related subconformations, an enzyme activation effect by iron was observed, and in the E2-related subconformations enzyme inhibition was observed. The enzyme's kinetic parameters were significantly changed only in the Na+ curve in ghost cells. In contrast to Na,K-ATPase α2 and α3 isoforms, activation was not observed for the α1 isoform. In Caco-2 cells, which only contain Na,K-ATPase α1 isoform, the FeCl3 increased the intracellular storage of iron, catalase activity, the production of H2O2 and the expression levels of the α1 isoform. In contrast, iron did not affect lipid peroxidation, GSH content, superoxide dismutase and Na,K-ATPase activities. These results suggest that iron itself modulates Na,K-ATPase and that one or more E1-related subconformations seems to be determinant for the sensitivity of iron modulation through a mechanism in which the involvement of the Na, K-ATPase α3 isoform needs to be further investigated.

Omega-3 index is directly associated with a healthy red blood cell distribution width.

Low red blood cell (RBC) membrane content of EPA and DHA, i.e., the omega-3 index (O3I), and elevated RBC distribution width (RDW) are risk factors for all-cause mortality. O3I and RDW are related with membrane fluidity and deformability. Our objective was to determine if there is a relationship between O3I and RDW in healthy adults. Subjects without inflammation or anemia, and with values for O3I, RDW, high-sensitivity C-reactive protein (CRP), body mass index (BMI), age and sex were identified (n = 25,485) from a clinical laboratory dataset of  > 45,000 individuals. RDW was inversely associated with O3I in both sexes before and after (both p < 0.00001) adjusting models for sex, age, BMI and CRP. Stratification by sex revealed a sex-O3I interaction with the RDW-O3I slope (p < 0.00066) being especially steep in females with O3I ≤ 5.6%. In healthy adults of both sexes, the data suggested that an O3I of > 5.6% may help maintain normal RBC structural and functional integrity.

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy.

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from -40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.

Erythrocyte fatty acid membrane composition in children on long-term parenteral nutrition enriched with ω-3 fatty acids.

BACKGROUND: Composite lipid emulsions containing soybean oil (30%), medium-chain triglycerides (30%), olive oil (25%), and fish oil (15%) (SMOF) are now widely used. OBJECTIVES: We aimed to evaluate the tolerance, the efficiency, and the erythrocyte fatty acid (FA) profile for children on long-term home parenteral nutrition (HPN) receiving a composite fish oil-based emulsion (FOLE). METHODS: At baseline, children (n = 46) with severe intestinal failure highly dependent on parenteral nutrition (PN) for ≥1 y were included in the study when they had received the composite FOLE for >6 mo. Out of this baseline group, only 25 children remained highly PN-dependent (SMOF1, n = 25) and could be assessed a second time, 2.4 y later (SMOF2, n = 25). An independent control group ("weaned off PN" group; n = 24) included children who had been weaned off PN for >2 y (median: 4 y). RBC-FA composition was established by GC-MS. Growth parameters, plasma citrulline, conjugated bilirubin, FA profiles, and the Holman ratio (20:3ω-9/20:4ω-6) were compared between groups. RESULTS: No difference for growth parameters, citrulline, and bilirubin was observed between the SMOF groups after 2.4 y (0.2 < P < 0.8). The weaned-off group did not differ from the SMOF groups for growth parameters (0.2 < P < 0.4) but citrulline was higher (P < 0.0001) and conjugated bilirubin lower (P < 0.01). The composite FOLE induced higher proportions of EPA (20:5n-3) (8.4% ± 2.9%) and DHA (22:6n-3) (11.7% ± 2.2%) than what was observed in weaned-off children (0.8% ± 0.4% and 6.6% ± 2.3%, respectively) but lower proportions of arachidonic acid (20:4n-6). However, the Holman ratio did not vary between groups (P = 0.9), whereas the PUFA concentrations varied widely. CONCLUSIONS: Long-term use of the composite FOLE was well tolerated in HPN-dependent children. The RBC-FA profile alterations were consistent with the ω-3 PUFA-enriched composition of this emulsion without evidence of essential FA deficiency.