Structural basis of membrane skeleton organization in red blood cells.

The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/ß-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.

Membrane skeleton hyperstability due to a novel alternatively spliced 4.1R can account for ellipsoidal camelid red cells with decreased deformability.

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to ß-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phase I/II/III clinical trials. Erythrocytes do not have a nucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin­V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Glassfrogs conceal blood in their liver to maintain transparency.

Transparency in animals is a complex form of camouflage involving mechanisms that reduce light scattering and absorption throughout the organism. In vertebrates, attaining transparency is difficult because their circulatory system is full of red blood cells (RBCs) that strongly attenuate light. Here, we document how glassfrogs overcome this challenge by concealing these cells from view. Using photoacoustic imaging to track RBCs in vivo, we show that resting glassfrogs increase transparency two- to threefold by removing ~89% of their RBCs from circulation and packing them within their liver. Vertebrate transparency thus requires both see-through tissues and active mechanisms that "clear" respiratory pigments from these tissues. Furthermore, glassfrogs' ability to regulate the location, density, and packing of RBCs without clotting offers insight in metabolic, hemodynamic, and blood-clot research.

Hemolytic Response of Iron Oxide Magnetic Nanoparticles at the Interface and in Bulk: Extraction of Blood Cells by Magnetic Nanoparticles.

Surface-active and water-soluble magnetic nanoparticles (NPs) were synthesized in the presence of a series of amphiphilic molecules of different functional groups to determine the hemolytic response and their ability to extract blood cells across the interface and aqueous bulk while maintaining minimum hemolysis. Amphiphilic molecules such as Gemini surfactants of strong hydrophobicity and low hydrophilic-lipophilic balance produced surface-active magnetic NPs, which were highly cytotoxic even when placed at the blood suspension (aqueous)-air interface. A similar behavior was shown by water-soluble magnetic NPs produced using monomeric ionic and nonionic surfactants and different amino acids. The NPs produced using mild biological surfactants and mono- and oligosaccharides of the same functional group proved to be excellent blood cell extractors with minimum hemolysis. α/ß-cyclodextrin and dextrose-stabilized magnetic NPs induced negligible hemolysis and extracted more than 50% of blood cells. The results showed that nontoxic magnetic NPs are excellent blood cell extractors from the blood suspension when tagged with amphiphilic molecules possessing good biocompatibility with cell membranes without inducing hemolysis. The work highlights the biological applicability of nontoxic magnetic NPs at biointerfaces and in blood suspensions.

Interactive Dynamics of Cell Volume and Cell Death in Human Erythrocytes Exposed to α-Hemolysin from Escherichia coli.

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (Pf) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced Pf in control- and aquaporin 1-null red blood cells, although the relative Pf decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles.

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C-H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm-1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

The regulation roles of Ca2+ in erythropoiesis: What have we learned?

Calcium (Ca2+) is an important second messenger molecule in the body, regulating cell cycle and fate. There is growing evidence that intracellular Ca2+ levels play functional roles in the total physiological process of erythroid differentiation, including the proliferation and differentiation of erythroid progenitor cells, terminal enucleation, and mature red blood cell aging and clearance. Moreover, recent research on the pathology of erythroid disorders has made great progress in the past decades, indicating that calcium ion hemostasis is closely related to ineffective erythropoiesis and increased sensitivity to stress factors. In this review, we summarized what is known about the functional roles of intracellular Ca2+ in erythropoiesis and erythrocyte-related diseases, with an emphasis on the regulation of the intracellular Ca2+ homeostasis during erythroid differentiation. An understanding of the regulation roles of Ca2+ homeostasis in erythroid differentiation will facilitate further studies and eventually molecular identification of the pathways involved in the pathological process of erythroid disorders, providing new therapeutic opportunities in erythrocyte-related disease.

Red blood cell distribution width as a prognostic biomarker for viral infections: prospects and challenges.

Viral diseases remain a significant global health threat, and therefore prioritization of limited healthcare resources is required to effectively manage dangerous viral disease outbreaks. In a pandemic of a newly emerged virus that is yet to be well understood, a noninvasive host-derived prognostic biomarker is invaluable for risk prediction. Red blood cell distribution width (RDW), an index of red blood cell size disorder (anisocytosis), is a potential predictive biomarker for severity of many diseases. In view of the need to prioritize resources during response to outbreaks, this review highlights the prospects and challenges of RDW as a prognostic biomarker for viral infections, with a focus on hepatitis and COVID-19, and provides an outlook to improve the prognostic performance of RDW for risk prediction in viral diseases.

Erythropoietic properties of human induced pluripotent stem cells-derived red blood cells in immunodeficient mice.

Transfusion of red blood cells (RBCs) is a life-saving intervention for anemic patients. Human induced pluripotent stem cells (iPSC) have the capability to expand and differentiate into RBCs (iPSC-RBCs). Here we developed a murine model to investigate the in vivo properties of human iPSC-RBCs. iPSC lines were produced from human peripheral blood mononuclear cells by transient expression of plasmids containing OCT4, SOX2, MYC, KLF4, and BCL-XL genes. Human iPSC-RBCs were generated in culture supplemented with human platelet lysate, and were CD34- CD235a+ CD233+ CD49dlow CD71low ; about 13% of iPSC-RBCs were enucleated before transfusion. Systemic administration of clodronate liposomes (CL) and cobra venom factor (CVF) to NOD scid gamma (NSG) mice markedly promoted the circulatory survival of human iPSC-RBCs following transfusion. While iPSC-RBCs progressively decreased with time, 90% of circulating iPSC-RBCs were enucleated 1 day after transfusion (CD235a+ CD233+ CD49d- CD71- ). Surprisingly, human iPSC-RBCs reappeared in the peripheral circulation at 3 weeks after transfusion at levels more than 8-fold higher than at 1 h after transfusion. Moreover, a substantial portion of the transfused nucleated iPSC-RBCs preferentially homed to the bone marrow, and were detectable at 24 days after transfusion. These results suggest that nucleated human iPSC-derived cells that homed to the bone marrow of NSG mice retained the capability to complete differentiation into enucleated erythrocytes and egress the bone marrow into peripheral blood. The results offer a new model using human peripheral blood-derived iPSC and CL/CVF-treated NSG mice to investigate the development and circulation of human erythroid cells in vivo.

Imaging flow cytometry data analysis using convolutional neural network for quantitative investigation of phagocytosis.

Macrophages play an important role in the adaptive immune system. Their ability to neutralize cellular targets through Fc receptor-mediated phagocytosis has relied upon immunotherapy that has become of particular interest for the treatment of cancer and autoimmune diseases. A detailed investigation of phagocytosis is the key to the improvement of the therapeutic efficiency of existing medications and the creation of new ones. A promising method for studying the process is imaging flow cytometry (IFC) that acquires thousands of cell images per second in up to 12 optical channels and allows multiparametric fluorescent and morphological analysis of samples in the flow. However, conventional IFC data analysis approaches are based on a highly subjective manual choice of masks and other processing parameters that can lead to the loss of valuable information embedded in the original image. Here, we show the application of a Faster region-based convolutional neural network (CNN) for accurate quantitative analysis of phagocytosis using imaging flow cytometry data. Phagocytosis of erythrocytes by peritoneal macrophages was chosen as a model system. CNN performed automatic high-throughput processing of datasets and demonstrated impressive results in the identification and classification of macrophages and erythrocytes, despite the variety of shapes, sizes, intensities, and textures of cells in images. The developed procedure allows determining the number of phagocytosed cells, disregarding cases with a low probability of correct classification. We believe that CNN-based approaches will enable powerful in-depth investigation of a wide range of biological processes and will reveal the intricate nature of heterogeneous objects in images, leading to completely new capabilities in diagnostics and therapy.

DRAMP 3.0: an enhanced comprehensive data repository of antimicrobial peptides.

Stapled antimicrobial peptides are an emerging class of artificial cyclic peptide molecules which have antimicrobial activity and potent structure stability. We previously published the Data Repository of Antimicrobial Peptides (DRAMP) as a manually annotated and open-access database of antimicrobial peptides (AMPs). In the update of version 3.0, special emphasis was placed on the new development of stapled AMPs, and a subclass of specific AMPs was added to store information on these special chemically modified AMPs. To help design low toxicity AMPs, we also added the cytotoxicity property of AMPs, as well as the expansion of newly discovered AMP data. At present, DRAMP has been expanded and contains 22259 entries (2360 newly added), consisting of 5891 general entries, 16110 patent entries, 77 clinical entries and 181 stapled AMPs. A total of 263 entries have predicted structures, and more than 300 general entries have links to experimentally determined structures in the Protein Data Bank. The update also covers new annotations, statistics, categories, functions and download links. DRAMP is available online at http://dramp.cpu-bioinfor.org/.

Blood cells and some hematological parameters of red drum (Linnaeus, 1766) in Vietnam / Células sanguíneas e alguns parâmetros hematológicos do tambor vermelho (Linnaeus, 1766) no Vietnã

This research focuses on hematological characteristics, erythrocyte morphology and some biochemical parameters of red drum Sciaenops ocellatus (Perciformes: Sciaenidae), cultured in natural water environment in areas nea river mouth (L1), estuaries (L2) in Ha Tinh province and coastal areas (L3) in Nha Trang city, Khanh Hoa province of Vietnam. A total of 18 speciments were examined in research, six in each location. Blood was drawn from the tail vein, using a microscope to research morphology and automated gauges to determine blood biochemical parameters. Analysis of blood samples showed that the rate of red drum’s erythrocyte morphology disorders in all three locations was quite high. The two main types of disorders were nuclear deformity and nuclear-matter distribution. Changes in erythrocyte size, shape and nuclear were related to salt concentration at culture locations. Blood hemoglobin content was stable in all three regions. Other hematological parameters such as the number of erythrocytes, blood biochemical parameters (glucose, SGOT, SGPT, urea, creatine, plasma iron, albumin, and protein) have differences among the locations, which showed the different reactions of the same species with different environmental conditions.

Esta pesquisa se concentra nas características hematológicas, na morfologia eritrocitária e em alguns parâmetros bioquímicos do tambor vermelho Sciaenops ocellatus (Perciformes: Sciaenidae), cultivado em ambiente aquático natural em áreas próximas à foz do rio (L1), em estuários (L2) na província de Ha Tinh e em áreas costeiras (L3) na cidade de Nha Trang, província de Khanh Hoa do Vietnã. Um total de 18 espécimes foi examinado na pesquisa, seis em cada local. O sangue foi coletado da veia da cauda, usando um microscópio para pesquisar a morfologia e medidores automatizados para determinar os parâmetros bioquímicos do sangue. A análise das amostras de sangue mostrou que a taxa de distúrbios morfológicos dos eritrócitos do tambor vermelho em todos os três locais era bastante alta. Os dois principais tipos de distúrbios eram deformidade nuclear e distribuição de matéria nuclear. Mudanças no tamanho, na forma e no núcleo dos eritrócitos foram relacionadas à concentração de sal nos locais de cultivo. O conteúdo de hemoglobina no sangue era estável em todas as três regiões. Outros parâmetros hematológicos, como número de eritrócitos, parâmetros bioquímicos sanguíneos (glicose, SGOT, SGPT, ureia, creatina, ferro plasmático, albumina e proteína), apresentam diferenças entre os locais, o que evidencia diferentes reações de uma mesma espécie em diferentes ambientes e condições.

Hypochromic red cells as predictors of anemia in patients undergoing hemodialysis: an observational retrospective study.

The percentage of hypochromic red blood cells (%HRC) estimates the availability of iron by evaluating the degree of hemoglobinization. We investigated whether %HRC was a predictor of anemia in patients undergoing hemodialysis. We recruited 142 patients undergoing routine hemodialysis between 2017 and 2019. Delta hemoglobin level (ΔHb)1mo-baseline was calculated as the difference between the hemoglobin level at 1 month after study enrollment and that at the time of study enrollment. Development of anemia was defined as hemoglobin level ≤ 15% of baseline. The median %HRC was 3.1%. There was a significant negative correlation between (ΔHb)1mo- baseline and %HRC (r = - 0.63, P < 0.001). The incidence of anemia was significantly higher in patients with %HRC > 3.1% than in those with %HRC ≤ 3.1%. In the multivariate logistic regression analysis, %HRC was significantly related to the development of anemia (odds ratio 2.57, 95% confidence interval [CI] 1.72-3.85, P < 0.001). The best cutoff value for %HRC to predict the development of anemia was 4.3%, with a sensitivity and specificity of 67.74 (95% CI, 54.7-79.1) and 97.50 (95% CI, 91.3- 99.7), respectively. %HRC is an independent predictor of anemia in patients undergoing hemodialysis. %HRC ≤ 4.3% is an early marker to consider changing the anemia treatment.

EpoR stimulates rapid cycling and larger red cells during mouse and human erythropoiesis.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor-/- mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.

A single-cell resolved cell-cell communication model explains lineage commitment in hematopoiesis.

Cells do not make fate decisions independently. Arguably, every cell-fate decision occurs in response to environmental signals. In many cases, cell-cell communication alters the dynamics of the internal gene regulatory network of a cell to initiate cell-fate transitions, yet models rarely take this into account. Here, we have developed a multiscale perspective to study the granulocyte-monocyte versus megakaryocyte-erythrocyte fate decisions. This transition is dictated by the GATA1-PU.1 network: a classical example of a bistable cell-fate system. We show that, for a wide range of cell communication topologies, even subtle changes in signaling can have pronounced effects on cell-fate decisions. We go on to show how cell-cell coupling through signaling can spontaneously break the symmetry of a homogenous cell population. Noise, both intrinsic and extrinsic, shapes the decision landscape profoundly, and affects the transcriptional dynamics underlying this important hematopoietic cell-fate decision-making system. This article has an associated 'The people behind the papers' interview.

Emergence of uniform linearly-arranged micro-droplets entrapping DNA and living cells through water/water phase-separation.

Living cells maintain their lives through self-organization in an environment crowded with a rich variety of biological species. Recently, it was found that micro-droplets containing biomacromolecules, which vary widely in size, are generated accompanied by water/water phase-separation by simple mechanical mixing of an aqueous solution with binary polymers. Here, we report that cell-sized droplets of nearly the same size are generated as a linear array within a glass capillary upon the introduction of a binary polymer solution of polyethylene glycol (PEG) and dextran (DEX). Interestingly, when DNA molecules are added to the polymer solution, stable droplets entrapping DNA molecules are obtained. Similarly, living cells are entrapped spontaneously for the linearly-arranged cell-sized droplets. This simple method for generating micro-droplets entrapping DNA and also living cells is expected to stimulate further study on the self-construction of protocells and micro organoids.

Effects of osmolality and solutes on the morphology of red blood cells according to three-dimensional refractive index tomography.

Phosphate-buffered saline (PBS) and Alsever's solution (AS) are frequently used as media in blood-related studies, while 0.9% normal saline (NS) is frequently used in transfusion medicine. Despite the frequent use, the effects of these solutions on the shape and volume of red blood cells (RBCs) have not been reported. We collected blood samples from five healthy adults and used three-dimensional refractive index tomography to investigate the changes in the morphology of RBCs caused by changes in osmolality and solutes at the single-cell level. After diluting 2 µL of RBCs 200-fold with each solution (PBS, AS, and 0.9% NS), 40 randomly selected RBCs were microscopically observed. RBC shape was measured considering sphericity, which is a dimensionless quantity ranging from 0 (flat) to 1 (spherical). RBCs in plasma or AS showed a biconcave shape with a small sphericity, whereas those in 0.9% NS or PBS showed a spherical shape with a large sphericity. Moreover, we confirmed that sodium chloride alone could not elicit the biconcave shape of RBCs, which could be maintained only in the presence of an osmotic pressure-maintaining substance, such as glucose or mannitol. Although 0.9% NS solution is one of the most commonly used fluids in hematology and transfusion medicine, RBCs in 0.9% NS or PBS are not biconcave. Therefore, as the debate on the use of NS continues, future clinical studies or applications should consider the effect of glucose or mannitol on the shape of RBCs.

Plasma Hemoglobin and Red Blood Cell Mass Levels as Dynamic Prognostic Markers for Progression and Survival in Pancreatic Neuroendocrine Tumors.

There are scarce data on readily available markers enabling immediate risk stratification and personalized management in patients with advanced pancreatic neuroendocrine tumors. This study explores the association of red blood cells-related parameters as prognostic markers in patients harboring pancreatic neuroendocrine tumors. Retrospective analysis of a tertiary medical center database, acquiring data of patients with pancreatic neuroendocrine tumors including demographics, tumor-related parameters and consecutive imaging results, vital status at last follow-up, and red blood cells parameters at baseline, last follow-up, and dynamics (last/baseline ratio). Univariate and multivariable analyses were performed. Sixty-seven patients were identified (mean age at diagnosis of 63±11 years, 56.7% males). Patients with disease progression had lower hemoglobin, red blood cells mass values and hematocrit at the last evaluation (p<0.001 for all comparisons), with red blood cells mass level<3.9 m/µl and a 6% and 9% relative reduction in hemoglobin and hematocrit levels, respectively, associated with an increased risk for disease progression. Similarly, patients deceased during the study period had lower hemoglobin, red blood cells mass values and hematocrit (p<0.03 for all) than those alive, at last follow-up. Eleven percent reduction in hemoglobin level was noted indicating a higher mortality risk (p=0.04). Negative hemoglobin and hematocrit dynamics were independently associated with increased risk for disease progression (p=0.03 and 0.049, respectively). In conclusion, decrease in red blood cells mass, hemoglobin and/or hematocrit levels are all associated with poor prognosis in patients with pancreatic neuroendocrine tumors. We suggest utilizing these parameters as complementary follow-up prognostic markers to radiologic imaging in this patients population.