Red Blood Cell Membrane Proteins Research Articles

Human red blood cells express the RNA sensor TLR7

Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.

Human red blood cells express the RNA sensor TLR7

Red blood cells (RBCs) express the nucleic acid-binding toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-binding TLRs is unknown. Here we show that human RBCs express the RNA sensor TLR7. TLR7 is present on the red cell membrane and is associated with the RBC membrane protein Band 3. In patients with SARS-CoV2-associated sepsis, TLR7-Band 3 interactions in the RBC membrane are increased when compared with healthy controls. In vitro, RBCs bind synthetic ssRNA and RNA from ssRNA viruses. Thus, RBCs may serve as a previously unrecognized sink for exogenous RNA, expanding the repertoire of non-gas exchanging functions performed by RBCs.

Ginseng total saponin improves red blood cell oxidative stress injury by regulating tyrosine phosphorylation and glycolysis in red blood cells

BackgroundOxidative stress is the main cause of many diseases, but because of its complex pathogenic factors, there is no clear method for treating it. Ginseng total saponin (GTS) an important active ingredients in Panax ginseng C.A. Mey (PG) and has potential therapeutic ability for oxidative stress due to various causes. However, the molecular mechanism of GTS in the treating oxidative stress damage in red blood cells (RBCs) is still unclear. PurposeThis study aimed to examine the protective effect of GTS on RBCs under oxidative stress damage and to determine its potential mechanism. MethodsThe oxidative stress models of rat RBCs induced by hydrogen peroxide (H2O2) and exhaustive swimming in vivo and in vitro was used. We determined the cell morphology, oxygen carrying capacity, apoptosis, antioxidant capacity, and energy metabolism of RBCs. The effect of tyrosine phosphorylation (pTyr) of Band 3 protein on RBCs glycolysis was also examined. ResultsGTS reduced the hemolysis of RBCs induced by H2O2 at the lowest concentration. Moreover, GTS effectively improved the morphology, enhanced the oxygen carrying capacity, and increased antioxidant enzyme activity, adenosine triphosphate (ATP) levels, and adenosine triphosphatase (ATPase) activity in RBCs. GTS also promoted the expression of membrane proteins in RBCs, inhibited pTyr of Band 3 protein, and further improved glycolysis, restoring the morphological structure and physiological function of RBCs. ConclusionsThis study shows, that GTS can protect RBCs from oxidative stress damage by improving RBCs morphology and physiological function. Changes in pTyr expression and its related pTyr regulatory enzymes before and after GTS treatment suggest that Band 3 protein is the main target of GTS in the treating endogenous and exogenous oxidative stress. Moreover, GTS can enhance the glycolytic ability of RBCs by inhibiting pTyr of Band 3 protein, thereby restoring the function of RBCs.

#2505 PTEN ablation in osteocytes exacerbates anemia in mice with subtotal nephrectomy

Abstract Background and Aims The identification of novel roles for bone as a principal endocrine organ has grown in recent decades, being now recognized for its contribution to the regulation of numerous systemic functions. In previous studies, we observed that 5/6Nx mice with osteocytic deletion of phosphatase and tensin homolog (PTEN), exhibited higher bone volume in 5/6Nx mice, consistent with studies under normal renal conditions. However, we also noted a decrease in blood hemoglobin levels, prompting further exploration. This study aims to elucidate the causative actions by which specific PTEN deletion in osteocytes influences red blood cell (RBC) homeostasis and to identify its possible involvement in anemia development. Method To achieve this goal, we used mice with targeted PTEN deletion in osteocytes (PTEN Ocy-cKO) by crossing DMP1-Cre ± and PTENflox/flox mice, subjected to subtotal nephrectomy (5/6Nx) at 2 months of age. Two weeks post-surgery, bone and blood samples were collected for plasma biochemistry, blood count, RBC membrane integrity, and preliminary bone+bone marrow proteomic analysis. Cre-negative mice and animals with normal renal function were included as controls. Results Our findings revealed that PTEN Ocy-cKO mice, under both normal and reduced renal function conditions, showed higher bone volume than corresponding controls. In terms of blood homeostasis, PTEN Ocy-cKO mice with normal renal function showed splenomegaly and a lower number of circulating RBCs. Notably, PTEN Ocy-cKO mice undergoing 5/6Nx experienced a profound decrease in RBC number in only two weeks, independent of renal erythropoietin expression. Further exploration ruled out bone marrow failure as a cause, as the erythroid progenitor (CD45-CD71+Ter119+) population remained similar in both groups. Extramedullary hematopoiesis was observed in the spleen, in addition to increased circulating reticulocyte number in the PTEN Ocy-cKO mice. Finally, altered RBC membrane protein patterns in PTEN Ocy-cKO mice suggested reduced mechanical/osmotic resistance. Preliminary proteomic analysis identified downregulation of Slc4a1 (Band3) and Phosphofructokinase (Pfkm), whose deficiencies have been previously linked to anemia. Conclusion In conclusion, our results propose that osteocytes contribute to RBC homeostasis through a pathway downstream of PTEN, and open a new avenue for understanding anemia in chronic kidney disease and a potential target for future treatments.

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.

Current Status of Molecular Diagnosis of Hereditary Hemolytic Anemia in Korea.

Hereditary hemolytic anemia (HHA) is considered a group of rare hematological diseases in Korea, primarily because of its unique ethnic characteristics and diagnostic challenges. Recently, the prevalence of HHA has increased in Korea, reflecting the increasing number of international marriages and increased awareness of the disease. In particular, the diagnosis of red blood cell (RBC) enzymopathy experienced a resurgence, given the advances in diagnostic techniques. In 2007, the RBC Disorder Working Party of the Korean Society of Hematology developed the Korean Standard Operating Procedure for the Diagnosis of Hereditary Hemolytic Anemia, which has been continuously updated since then. The latest Korean clinical practice guidelines for diagnosing HHA recommends performing next-generation sequencing as a preliminary step before analyzing RBC membrane proteins and enzymes. Recent breakthroughs in molecular genetic testing methods, particularly next-generation sequencing, are proving critical in identifying and providing insight into cases of HHA with previously unknown diagnoses. These innovative molecular genetic testing methods have now become important tools for the management and care planning of patients with HHA. This review aims to provide a comprehensive overview of recent advances in molecular genetic testing for the diagnosis of HHA, with particular emphasis on the Korean context.

Radiofrequency dielectric spectroscopy study: Effects of pH, hydrogen bond donors and acceptors on the attachment of spectrin skeleton to the lipid membrane of erythrocytes.

Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, βsp (1.4 MHz) and γ1sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the βsp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the βsp and γ1sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the βsp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1sp relaxation.

Novel Biomarkers for Assessing Clinical Severity in Hereditary Spherocytosis - Application of Routine and Advanced Diagnostic Tests

Background: Hereditary spherocytosis (HS) is caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeletal and (trans)membrane proteins. This ultimately leads to instability of the RBC membrane and subsequent membrane loss, resulting in the transformation of the biconcave RBC into a poorly deformable spherocyte, which is susceptible to premature splenic clearance. The diagnosis of HS has substantially improved over the last years and new advanced techniques have been established, including osmotic gradient ektacytometry, eosin-5'-maleimide (EMA) binding test and next-generation sequencing (NGS). However, there is little knowledge on the applicability of these diagnostic outcome parameters as biomarkers for clinical severity in HS. Aim: To explore possible correlations between established and novel diagnostic outcome parameters and clinical severity in a well-defined cohort of HS patients. Methods: This monocenter retrospective cohort study involves patients diagnosed with HS upon referral to our department between 2012 and 2023. Clinical characteristics, routine and advanced diagnostic laboratory parameters, and genotype were evaluated. Clinical severity was classified as previously described, based on parameters such as hemoglobin, reticulocyte percentage and bilirubin 1. The following laboratory tests were included: routine hematological parameters (Cel-Dyn Sapphire or Alinity hq), EMA binding test, osmotic fragility test (OFT) and osmotic gradient ektacytometry (osmoscan, Lorrca MaxSis). The latter technique was also explored for other parameters than the commonly used EI max, O hyper and O min. NGS gene panel analysis was used for molecular diagnosis. Differences between groups were determined using either Chi-squared test, unpaired t-test or Mann-Whitney U test and correlations using point-biserial, Pearson's or Spearman's test via GraphPad Prism. Results: Eighty-two non-splenectomized patients were included. Thirty-five of these (42.7%) were classified as mild, and forty-seven as moderate-severe (57.3%)). Demographic characteristics nor genotype differed significantly, albeit SPTB variants occurred more frequently in the moderate-severe group (15 vs. 4 in the mild group). Expectedly, both reticulocyte count and EI max correlated with clinical severity. RBC distribution width (RDW) correlated moderately, suggesting greater RBC heterogeneity (r=0.606, p<0.0001) is associated with a more severe clinical phenotype. Similarly, EMA binding test results correlated with clinical severity (r=0.390, p<0.001), indicating more pronounced membrane loss in clinically more severe patients. We further evaluated three novel osmoscan parameters ( Figure 1): EI O290, the elongation index (EI) at physiologic isotonicity, which correlated with clinical severity (r=-0.500, p<0.0001), and also with EI max (r=0.856, p<0.0001). The O min-width and O max-width, defined as the difference in osmolality between the intersection points of y=EI min or EI max +/- 0.05 and the osmoscan curve. Thisassesses the effect of changes in osmolality on EI min or EI max, respectively. O min-width correlated with RDW (r-0.439, p<0.0001), OFT results (N=30, r=0.671, p<0.0001) and clinical severity (r=0.390, p<0.001). O max-width correlated with clinical severity (r=-0.375, p<0.001) and strongly with percentage of hyperchromic cells (N=76, r=-0.751, p<0.0001), yet weakly with RDW (-0.273, p<0.05). All significant correlations with clinical severity are displayed in Figure 2. Conclusion: In this study, a number of routine and advanceddiagnostic laboratory parameters were found to correlate with clinical severity in HS. O min-width and O max-width are novel osmoscan-derived biomarkers that correlated with RDW, hyperchromic cells (O max-width), OFT results (O min-width) and clinical severity. EI O290, another novel osmoscan-derived biomarker, also correlated with clinical severity, although not stronger than EI max and AUC. The biomarkers identified in this study, together with patient-reported outcome measures (PROMs), could be used to revise the previously established severity classification system and thereby aid in stratifying clinical severity and subsequent clinical decision-making.

Red Blood Cells from Individuals with Lesch-Nyhan Syndrome: Multi-Omics Insights into a Novel S162N Mutation Causing Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency.

Lesch-Nyhan syndrome (LN) is an is an X-linked recessive inborn error of metabolism that arises from a deficiency of purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disease manifests severely, causing intellectual deficits and other neural abnormalities, hypercoagulability, uncontrolled self-injury, and gout. While allopurinol is used to alleviate gout, other symptoms are less understood, impeding treatment. Herein, we present a high-throughput multi-omics analysis of red blood cells (RBCs) from three pediatric siblings carrying a novel S162N HPRT1 mutation. RBCs from both parents-the mother, a heterozygous carrier, and the father, a clinically healthy control-were also analyzed. Global metabolite analysis of LN RBCs shows accumulation of glycolytic intermediates upstream of pyruvate kinase, unsaturated fatty acids, and long chain acylcarnitines. Similarly, highly unsaturated phosphatidylcholines are also elevated in LN RBCs, while free choline is decreased. Intracellular iron, zinc, selenium, and potassium are also decreased in LN RBCs. Global proteomics documented changes in RBC membrane proteins, hemoglobin, redox homeostasis proteins, and the enrichment of coagulation proteins. These changes were accompanied by elevation in protein glutamine deamidation and methylation in the LN children and carrier mother. Treatment with allopurinol incompletely reversed the observed phenotypes in the two older siblings currently on this treatment. This unique data set provides novel opportunities for investigations aimed at potential therapies for LN-associated sequelae.

Characterization of anti-erythrocyte and anti-platelet antibodies in hemolytic anemia and thrombocytopenia induced by Plasmodium spp. and Babesiaspp. infection in mice.

Malaria and Babesiosis are acute zoonotic disease that caused by infection with the parasite in the phylum Apicomplexa. Severe anemia and thrombocytopenia are the most common hematological complication of malaria and babesiosis. However, the mechanisms involved have not been elucidated, and only a few researches focus on the possible role of anti-erythrocyte and anti-platelet antibodies. In this study, the Plasmodium yoelii, P. chabaudi, Babesia microti and B. rodhaini infected SCID and ICR mice. The parasitemia, survival rate, platelet count, anti-platelet antibodies, and the level of IFN-γ and interleukin (IL) -10 was tested after infection. Furthermore, the P. yoelii, P. chabaudi, B. rodhaini and B. microti infected ICR mice were treated with artesunate and diminaze, the development of the anti-erythrocyte and anti-platelet antibodies in chronic stage were examined. At last, the murine red blood cell and platelet membrane proteins probed with auto-antibodies induced by P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection were characterized by proteomic analysis. The high anti-platelet and anti-erythrocyte antibodies were detected in ICR mice after P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection. Actin of murine erythrocyte and platelet is a common auto-antigen in Plasmodium and Babesia spp. infected mice. Our findings indicate that anti-erythrocyte and anti-platelet autoantibodies contribute to thrombocytopenia and anemia associated with Plasmodium spp. and Babesia spp. infection. This study will help to understand the mechanisms of malaria and babesiosis-related thrombocytopenia and hemolytic anemia.

Establishment of a flow cytometry screening method for patients with glucose transporter 1 deficiency syndrome

ObjectiveWe assessed the usefulness of flow cytometry as a functional assay to measure glucose transporter 1 (GLUT1) levels on the surface of red blood cells (RBCs) from Japanese patients with glucose transporter 1 deficiency syndrome (Glut1DS). MethodsWe recruited 13 genetically confirmed Glut1DS patients with a solute carrier family 2 member 1 (SLC2A1) mutation (eight missense, one frameshift, two nonsense, and two deletion) and one clinically suspected Glut1DS-like patient without an SLC2A1 mutation, and collected whole blood with informed consent. We stained pelleted RBCs (1 μL) from the patients with a Glut1.RBD ligand and anti-glycophorin A antibody, which recognizes a human RBC membrane protein, and analyzed the cells using flow cytometry. ResultsRelative GLUT1 levels quantified by flow cytometry in 11 of 13 patients with definite Glut1DS were 90% below those of healthy controls. Relative GLUT1 levels were not reduced in two of 13 Glut1DS patients who had a missense mutation and no intellectual disability and one Glut1DS-like patient without an SLC2A1 mutation. Relative GLUT1 levels were significantly reduced in Glut1DS patients with an SLC2A1 mutation, more severe intellectual disability, and spasticity. ConclusionsThis method to detect GLUT1 levels on RBCs is simple and appears to be an appropriate screening assay to identify severe Glut1DS patients in the early stage before the development of irreversible neurologic damage caused by chronic hypoglycorrhachia.

Preparation and Structural Analysis of a Sialo-Oligosaccharide from Glycophorin in Carp Red Blood Cell (RBC) Membranes

Glycophorins (GPs) in the red blood cell (RBC) membranes of carp (Cyprinus carpio L.) exhibit bacteriostatic activity against various gram-negative and gram-positive bacteria. This antibacterial property is attributed to a sialo-oligosaccharide. For a structural analysis of this O-linked sialo-oligosaccharide, it is necessary to prepare and purify the oligosaccharide from carp GPs. To prepare sialo-oligosaccharides, RBC membrane proteins were solubilized, and anion exchange column chromatography was performed to obtain the GPs. The GP yield from carp RBC membranes was ca.100% as total sialic acid. Then, released oligosaccharides were obtained from GP preparation by β-elimination. Second, the resulting oligosaccharide fraction was separated into two sialo-oligosaccharide components (P-1 and P-2) using the same anion exchange column. The oligosaccharide preparations that contained high amounts of NaCl were desalted with an activated charcoal column combined with ammonium bicarbonate in acetonitrile as an eluent. Using NMR to analyse the structure of the antimicrobial P-1, we determined the alignment of each proton and C1 carbon. To determine the linkage sites, we performed GC-MS using the permethylated P-1. Lastly, we determined that the structure of P-1 was NeuGcα2→6 (Fucα1→4) (Glcα1→3) Galβ1→4GalNAc-ol.

Anti-inflammatory potential of green synthesized silver nanoparticles of triphala extract

The present study was to investigate the in vitro anti-inflammatory activity of the hydroalcoholic extract of triphala and its green synthesized silver nanoparticles using the human red blood cells membrane stabilization method and protein denaturation method and the results were compared with standard diclofenac sodium. The presence of flavonoids, phenolics, alkaloids, carbohydrates, and glycosides were found in phytochemical screening. The plant extract and the silver nanoparticles showed positive anti-inflammatory activity through HRBC membrane stabilization and inhibition of protein denaturation in a concentration-dependent manner. The study was concluded that the hydroalcoholic extract and SNPs of triphala exhibited potent anti-inflammatory agent due to the presence of important chemical constituents such as polyphenols and flavonoids and this anti-inflammatory properties help in the prevention of many chronic disorders.

A novel splicing mutation of ANK1 is associated with phenotypic heterogeneity of hereditary spherocytosis in a Chinese family

Hereditary spherocytosis (HS) is a common hematological genetic disorder that results in anemia, jaundice and splenomegaly. It is caused by mutations in the ANK1, SPTA, SPTB, SLC4A1 and EPB42 genes, which encode red blood cell membrane and skeletal proteins. Patients show high heterogeneity in phenotype and genotype and the genotype-phenotype correlation still requires clarification. Here, a novel splicing mutation (ANK1: c.4391-2 A>C) was identified by whole-exome sequencing (WES) and Sanger sequencing in a Chinese boy who exhibited a moderately severe HS phenotype. However, his father exhibited a mild phenotype, despite carrying the same HS-causing mutation. The function of the mutant ANK1 protein was analyzed by both bioinformatics and experimental analysis. The mutant protein (p.N1463Kfs*4) showed a different 3D-structure and altered subcellular localization, when compared with the wild-type ANK1 protein. These changes disrupted the normal cell membrane structure and resulted in spheroidized red blood cells. Amplification of cDNA from the son and his father revealed a difference in expression of the abnormal transcript produced by the splicing mutation. We proposed that the lower expression of the mutant allele may have contributed to the relatively mild symptoms of the father. Our study verified ANK1 c. c.4391-2 A>C as a novel pathogenic mutation that causes HS. We have also provided new insights into the interpretation of phenotypic variability within families, which could greatly improve the clinical diagnosis and genetic counseling of HS.

Assessment of Correlation Analysis, Phytochemical Profile, and Biological Activities of Endemic Scorzonera Species from Turkey.

Scorzonera species belong to the Asteraceae family comprising more than 25000 species. The present study aimed to examine the phytochemical profiles and biological activities of S. sandrasica Hartvig et Strid, S. coriacea A. Duran&Aksoy, and S. ahmet-duranii Makbul&Coskuncelebi which are endemic species to Turkey. Flavonoids such as hyperoside, isoquercitrin, rutin, isoorientin, orientin, 7-O-methyl isoorientin, luteolin-7-O-β-glycoside, apigenin-7-O-β-glucoside, vitexin, isovitexin as well as caffeoylquinic acid derivatives including chlorogenic acid, 4,5-O-dicaffeoylquinic acid, and 1,5-O-dicaffeoylquinic acid contents were analyzed to clarify phytochemical content of the extracts. Aerial parts of the investigated extracts were determined as contain flavonoids in high amounts. Chlorogenic acid and its derivatives were detected in all investigated species, in varying amounts, both in the roots and aerial parts. S. coriacea aerial parts contained the highest total phenolic and flavonoids. The strongest inhibitory activities on ABTS and DPPH radicals were also observed with S. coriacea aerial parts by 8.07±0.28 and 13.94±0.53 μg/ml of IC50 values, respectively. Total phenolic contents of the extracts were significantly correlated with DPPH (r=-0.9842, p=0.0004) and ABTS free radical scavenging (r=-0.9870, p=0.0003) and total antioxidant capacity (r=0.8173, p=0.0470), as well as total flavonoid contents (r=0.8820, p=0.0201). S. sandrasica aerial parts and S. ahmet-duranii roots exhibited the greatest red blood cell membrane protection and protein denaturation inhibition, respectively. From the phytochemical point of view, all the selected species were analyzed for the first time.

Dynamic Palmitoylation of Red Cell Membrane Proteins Governs Susceptibility to Invasion by the Malaria Parasite, Plasmodium falciparum.

Phosphorylation and other post-translational modifications of red blood cell (RBC) proteins govern membrane function and have a role in the invasion of RBCs by the malaria parasite, Plasmodium falciparum. Furthermore, a percentage of RBC proteins are palmitoylated, although the functional consequences are unknown. We establish dynamic palmitoylation of 118 RBC membrane proteins using click chemistry and acyl biotin exchange (ABE)-coupled LC-MS/MS and characterize their involvement in controlling membrane organization and parasite invasion. RBCs were treated with a generic palmitoylation inhibitor, 2-bromopalmitate (2-BMP), and then analyzed using ABE-coupled LC-MS/MS. Only 42 of the 118 palmitoylated proteins detected were palmitoylated in the 2-BMP-treated sample, indicating that palmitoylation is dynamically regulated. Interestingly, membrane receptors such as semaphorin 7A, CR1, and ABCB6, which are known to be involved in merozoite interaction with RBCs and parasite invasion, were found to be dynamically palmitoylated, including the blood group antigen, Kell, whose antigenic abundance was significantly reduced following 2-BMP treatment. To investigate the involvement of Kell in merozoite invasion of RBCs, a specific antibody to its extracellular domain was used. The antibody targeting Kell inhibited merozoite invasion of RBCs by 50%, implying a role of Kell, a dynamically palmitoylated potent host-derived receptor, in parasite invasion. Furthermore, a significant reduction in merozoite contact with the RBC membrane and a consequent decrease in parasite invasion following 2-BMP treatment demonstrated that palmitoylation does indeed regulate RBC susceptibility to parasite invasion. Taken together, our findings revealed the dynamic palmitoylome of RBC membrane proteins and its role in P. falciparum invasion.

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), β-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.

Evolution of red blood cell membrane complement regulatory proteins and rheology in septic patients: An exploratory study.

BackgroundDuring sepsis, red blood cell (RBC) deformability is altered. Persistence of these alterations is associated with poor outcome. Activation of the complement system is enhanced during sepsis and RBCs are protected by membrane surface proteins like CD35, CD55 and CD59. In malaria characterized by severe anemia, a study reported links between the modifications of the expression of these RBCs membrane proteins and erythrophagocytosis. We studied the evolution of RBCs deformability and the expression of RBC membrane surface IgG and regulatory proteins in septic patients.MethodsBy flow cytometry technics, we measured at ICU admission and at day 3–5, the RBC membrane expression of IgG and complement proteins (CD35, 55, 59) in septic patients compared to RBCs from healthy volunteers. Results were expressed in percentage of RBCs positive for the protein. RBC shape was assessed using Pearson's second coefficient of dissymmetry (PCD) on the histogram obtained with a flow cytometer technique. A null value represents a perfect spherical shape. RBC deformability was determined using ektacytometry by the elongation index in relation to the shear stress (0.3–50 Pa) applied to the RBC membrane. A higher elongation index indicates greater RBC deformability.ResultsRBCs from 11 septic patients were compared to RBCs from 21 volunteers. At ICU admission, RBCs from septic patients were significantly more spherical and RBC deformability was significantly lower in septic patients for all shear stress ≥1.93 Pa. These alterations of shape and deformability persists at day 3–5. We observed a significant decrease at ICU admission only in CD35 expression on RBCs from septic patients. This low expression remained at day 3–5.ConclusionsWe observed in RBCs from septic patients a rapid decrease expression of CD35 membrane protein protecting against complement activation. These modifications associated with altered RBC deformability and shape could facilitate erythrophagocytosis, contributing to anemia observed in sepsis. Other studies with a large number of patients and assessment of erythrophagocytosis were needed to confirm these preliminary data.

In vivo and In vitro Anti-inflammatory and Free Radical Scavenging Activities of Methonolic Extract of Different Parts from Nauclea vanderguchtii De Wild (Rubiaceae)

Aims: Nauclea vanderguchtii (N. vanderguchtii) species belongs to the family Rubiaceae and it is widely used in traditional Cameroonian medicine against inflammatory diseases such as arthritis, rheumatism and gastric disorders. The present study was aimed to evaluate anti-inflammatory effect of a methanol extracts of leaves, stems, roots and barks with multiparametric analyses through in vitro assays and an in vivo model.
 Methodology: Leaves, stems, barks and roots of N. vanderguchtii were air-dried and a methanolic extract was further obtained. Red blood cell membrane stabilization and protein denaturation were carried out as screening assays for anti-inflammatory activity in vitro, following by Diphenyl picryl-hydrazyl (DPPH), 2,2’- azino-bis – (3 - ethylbenzothiazoline -6- sulfonique (ABTS) and Ferric Reducing Antioxidant Power ( FRAP) antioxidant activity. Furthermore, the anti-inflammatory capacity of leaves and stems methanolic extract was evaluated in vivo by carrageenan-induced oedema.
 Results: Each part of Nauclea vanderguchtii, effectively and significantly stabilized red blood cell membrane. The methanol leaves extract exhibited better effect (53.12%), followed by stems (50.55%), barks (50.98%) and roots (49.6%) compare to an ibuprofen (51.16%), a standard reference drug. Those extracts also inhibited the denaturation of the egg albumin (P ˂ .05; P ˂ .01). Methanol stem and leaves extracts from plant were the effective scavengers of ABTS - radical (95.92 ± 0.37%), DPPH - radical (91.12 ± 0.13%). FRAP of methanolic extract was concentration-dependent. Moreover, methanol leaves extract of Nauclea vanderguchtii, significantly (P<.01) prevented paw edema with the maxima 58.97% (200 mg / kg).
 Conclusion: This study shows that N. vanderguchtii extracts possess significant anti-inflammatory and antiradical activities. These activities are more pronounced in leaves than other parts of plant and justify the traditional use.

Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers.

Chemotherapy is a difficult treatment for cancer patients because of the low effective accumulation of chemo-drugs and their detrimental side effects. Nanoparticles have shown promise as a solution to these problems. However, the known differences in the porosity and vascularization of tumor vessels, and other factors, including the potential formation of a "protein crown," the short half-life time in circulation, and the low drug distribution, often limit their application. To address these problems, biomimetic nanoparticles coated with cell membranes have been developed and shown to have advantages such as prolonged circulation, high biocompatibility, and enhanced targeting abilities in drugs and nanoparticles, thus exhibiting good application prospects in cancer therapy for liver, lung, and melanoma cancers. Accordingly, we designed a PH-sensitive biomimetic nanodrug delivery system with a delicate "core-shell" structure based on red blood cell membranes. Briefly, core nanoparticles were synthesized by the self-assembly of natural amphoteric polymers, including hydrophilic carboxymethylcellulose sodium and hydrophobic stearic acid. For the shell structure, red blood cell membranes were modified using folic acid by a lipid tether (1,2-distearoyl-sn-glycero-3-phosphoethanolamine) to increase tumor-targeting ability, whereas polyethylene glycol was inserted to decrease lipid tether modification-induced potential sequestration by either the mononuclear phagocyte system or the reticuloendothelial system. Via a series of formulation optimizations, paclitaxel was packaged into the red blood membrane-based core-shell nanoparticles with an average size of 226.9±2.75nm and a negative Zeta potential of -14.5±0.3mV. More importantly, the examinations focusing on CD47, a representative red blood cell membrane protein, revealed not only the successful establishment of the membrane shell but also the right-side-out membrane orientation on our core-shell nanoparticles. Our nanodrug delivery system showed good biocompatibility and sensitivity to acidic tumor microenvironments while effectively prolonging the circulation time of paclitaxel and further enhancing its antitumor effects on epithelial malignancies, including liver, lung, and melanoma cancers. In particular, our nanodrug delivery system significantly alleviated paclitaxel-induced renal toxicity. Taken together, our findings highlight that the red blood membrane-based core-shell nanoparticle is a promising biomimetic nanodrug delivery system for functionally delivering chemotherapeutic drugs, and it has promise in clinical applications.