EhVps35, a retromer component, is a key factor in secretion, motility, and tissue invasion by Entamoeba histolytica.

In humans and Drosophila melanogaster, the functional convergence of the endosomal sorting complex required for transport (ESCRT) machinery that is in charge of selecting ubiquitinated proteins for sorting into multivesicular bodies, and the retromer, that is the complex responsible for protein recycling to the plasma membrane and Golgi apparatus. ESCRT and retromer complexes are codependent for protein sorting recycling, degradation, and secretion. In this article, we studied the EhVps35 C isoform (referred to as EhVps35), that is the central member of the Entamoeba histolytica retromer, and its relation with the ESCRT machinery during sorting and protein recycling events and their involvement virulence. Our findings revealed that EhVps35 interacts with at least 300 proteins that participate in multiple cellular processes. Laser confocal and transmission electronic microscopy images, as well as secretion assays, revealed that EhVps35 is secreted in vesicles together with EhVps23 and EhADH (both ESCRT machinery proteins). In addition, immunoprecipitation, immunofluorescence, and molecular docking assays revealed the relationship among EhVps35 and other ESCRT machinery proteins. Red blood cell stimulus increased EhVps35 secretion, and the knockdown of the Ehvps35 gene in trophozoites reduced their capacity to migrate and invade tissues. This also impacts the cellular localization of ubiquitin, EhVps23 (ESCRT-I), and EhVps32 (ESCRT-III) proteins, strongly suggesting their functional relationship. Our results, taken together, give evidence that EhVps35 is a key factor in E. histolytica virulence mechanisms and that it, together with the ESCRT machinery components and other regulatory proteins, is involved in vesicle trafficking, secretion, migration, and cell proliferation.

Effect of a thiohydantoin salt derived from l-Arginine on Leishmania amazonensis and infected cells: Insights from biological effects to molecular docking interactions.

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.

Impact of nanosilver surface electronic distributions on serum protein interactions and hemocompatibility.

The translation of silver-based nanotechnology 'from bench to bedside' requires a deep understanding of the molecular aspects of its biological action, which remains controversial at low concentrations and non-spherical morphologies. Here, we present a hemocompatibility approach based on the effect of the distinctive electronic charge distribution in silver nanoparticles (nanosilver) on blood components. According to spectroscopic, volumetric, microscopic, dynamic light scattering measurements, pro-coagulant activity tests, and cellular inspection, we determine that at extremely low nanosilver concentrations (0.125-2.5µg ml-1), there is a relevant interaction effect on the serum albumin and red blood cells (RBCs). This explanation has its origin in the surface charge distribution of nanosilver particles and their electron-mediated energy transfer mechanism. Prism-shaped nanoparticles, with anisotropic charge distributions, act at the surface level, generating a compaction of the native protein molecule. In contrast, the spherical nanosilver particle, by exhibiting isotropic surface charge, generates a polar environment comparable to the solvent. Both morphologies induce aggregation at NPs/bovine serum albumin ≈ 0.044 molar ratio values without altering the coagulation cascade tests; however, the spherical-shaped nanosilver exerts a negative impact on RBCs. Overall, our results suggest that the electron distributions of nanosilver particles, even at extremely low concentrations, are a critical factor influencing the molecular structure of blood proteins' and RBCs' membranes. Isotropic forms of nanosilver should be considered with caution, as they are not always the least harmful.

In vitro Effect of Cannabidiol on Red Blood Cells: Implication in Long-Lasting Pathology Treatment.

BACKGROUND: Cannabidiol (CBD) is the principal non-hallucinogenic compound of Cannabis plants with high clinical interest because CBD has been described as having anti-inflammatory, analgesic and anticonvulsant properties. CBD is considered a multitarget compound as it can interact with a wide range of targets, explaining their multiplicity of effects. Some clinical studies have indicated certain side effects of CBD, including somnolence, anemia and diarrhea, while the elevation of transaminases is considered as an exclusion criterion from the trial. Since the red blood cells (RBCs) are a source of transaminase, we assayed in vitro effect on RBCs stability. METHODS: We performed in vitro experiments with RBCs obtained from human peripheral blood with normal hematological parameters exposed to CBD in the range of therapeutic uses. We evaluated RBCs morphological changes, membrane fragility and hemoglobin release as a reflection of hemolysis. RESULTS: CBD induced an increase in the hemoglobin release (3.27 µg/106 RBC), without altered RBC osmotic fragility. When RBCs suspensions were incubated with CBD the initial number of elements (RBCs + vesicles) was increased up to 65% after 20 min and returned to basal level after 40 min of incubation. In the first 20 min, the accounts of elements were enriched in the smaller vesicles that disappeared after the remaining 20 minutes. CONCLUSION: These results suggest that CBD affects the indemnity of erythrocytes in vitro, inducing the formation of hemolytic vesicles that can provide the basis for the development of anemia, transaminase elevation and underlying tissular iron overload in patients chronically treated with CBD.

Highly Defined Induced Pluripotent Stem Cell Lines Mimic Donor Red Blood Cell Antigen Profiles for Therapeutic and Diagnostic Use.

Our group generated two induced pluripotent stem cell (iPSC) lines for in vitro red blood cell (RBC) production from blood donors with extensively known erythrocyte antigen profiles. One line was intended to give rise to RBCs for transfusions in patients with sickle cell disease (SCD), while the other was developed to create RBC panel reagents. Two blood donors were selected based on their RBC phenotypes, further complemented by high-throughput DNA array analysis to obtain a more comprehensive erythrocyte antigen profile. Enriched erythroblast populations from the donors' peripheral blood mononuclear cells were reprogrammed into iPSCs using nonintegrative plasmid vectors. The iPSC lines were characterized and subsequently subjected to hematopoietic differentiation. iPSC PB02 and iPSC PB12 demonstrated in vitro and in vivo iPSC features and retained the genotype of each blood donor's RBC antigen profile. Colony-forming cell assays confirmed that iPSC PB02 and iPSC PB12 generated hematopoietic progenitors. These two iPSC lines were generated with defined erythrocyte antigen profiles, self-renewal capacity, and hematopoietic differentiation potential. With improvements in hematopoietic differentiation, these cells could potentially be more efficiently differentiated into RBCs in the future. They could serve as a complementary approach for obtaining donor-independent RBCs and addressing specific demands for blood transfusions.

Iron incorporation in red blood cells of pediatric sickle cell anemia: a stable isotope pilot investigation.

BACKGROUND/OBJECTIVES: Sickle cell anemia (SCA) is marked by hypoxia, inflammation, and secondary iron overload (IO), which potentially modulate hepcidin, the pivotal hormone governing iron homeostasis. The aim was to evaluate the iron incorporation in red blood cells (RBC) in SCA pediatric patients, considering the presence or absence of IO. SUBJECTS/METHODS: SCA children (n = 12; SCAtotal) ingested an oral stable iron isotope (57Fe) and iron incorporation in RBC was measured after 14 days. Patients with ≥1000 ng/mL serum ferritin were considered to present IO (SCAio+; n = 4) while the others were classified as being without IO (SCAio-; n = 8). Liver iron concentration (LIC) was determined by Magnetic Resonance Imaging (MRI) T2* method. RESULTS: The SCAio+ group had lower iron incorporation (mean ± SD: 0.166 ± 0.04 mg; 3.33 ± 0.757%) than SCAio- patients (0.746 ± 0.303 mg; 14.9 ± 6.05%) (p = 0.024). Hepcidin was not different between groups. Iron incorporation was inversely associated with serum ferritin level (SCAtotal group: r = -0.775, p = 0.041; SCAio- group: r = -0.982; p = 0.018) and sickle hemoglobin (HbS) presented positive correlation with iron incorporation (r = 0.991; p = 0.009) in SCAio- group. LIC was positively associated with ferritin (SCAtotal: r = 0.921; p = 0.026) and C reactive protein (SCAio+: r = 0.999; p = 0.020). CONCLUSION: SCAio+ group had lower iron incorporation in RBC than SCAio- group, suggesting that they may not need to reduce their intake of iron-rich food, as usually recommended. Conversely, a high percentage of HbS may indirectly exacerbate hypoxia and seems to increase iron incorporation in RBC. TRIAL REGISTRATION: This trial was registered at www.ensaiosclinicos.gov.br . Identifier RBR-4b7v8pt.

APEX2-based proximity proteomic analysis identifies candidate interactors for Plasmodium falciparum knob-associated histidine-rich protein in infected erythrocytes.

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Unraveling DEHP influence on hemoglobin S polymerization in sickle cell disease: Ex vivo, in vitro and in silico analysis.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the ß-globin chain and patients are frequently exposed to several blood transfusions in order to maintain physiological function. Transfusion blood bags are composed of PVC and phthalates (as DEHP) are often introduced to the material in order to confer malleability. In this sense, DEHP can easily elute to the blood and cause harmful effects. This study aimed to unravel DEHP effect on SCD patient's hemoglobin function. We found that HbS polymerization using whole erythrocytes is decreased by DEHP in ex vivo experiments and this effect might be mediated by the DEHP-VAL6 interaction, evaluated in silico. Isolated HbS exhibited less polymerization at low DEHP concentrations and increased polymerization rate at higher concentration. When analyzing the propensity to aggregate, HbS is more inclined to aggregate when compared to HbA due to the residue 6 mutation. Circular dichroism showed characteristic hemoglobin peaks for oxygenated HbS that are lost when oxygen is sequestered, and DEHP at higher concentration mildly recovers a peak close to the second hemoglobin one. Finally, by transmission electron microscopy we demonstrated that high DEHP concentration increased polymer formation with a more organized structure. These findings show for the first-time the beneficial effect of low-dose DEHP on HbS polymerization.

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

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

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

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

Effect of octreotide on oxidative stress in the erythrocyte and kidney tissue in adriamycin-induced experimental nephrotic syndrome model.

INTRODUCTION: Nephrotic syndrome (NS) is one of the reasons of end-stage kidney disease, and elucidating the pathogenesis and offer new treatment options is important. Oxidative stress might trigger pathogenesis systemically or isolated in the kidneys. Octreotide (OCT) has beneficial antioxidant effects. We aimed to investigate the source of oxidative stress and the effect of OCT on experimental NS model. METHODS: Twenty-four non-uremic Wistar albino rats were divided into 3 groups. Control group, 2 mL saline intramuscular (im); NS group, adriamycin 5 mg/kg intravenous (iv); NS treatment group, adriamycin 5 mg/kg (iv) and OCT 200 mcg/kg (im) were administered at baseline (Day 0). At the end of 21 days, creatinine and protein levels were measured in 24-hour urine samples. Erythrocyte and renal catalase (CAT) and thiobarbituric acid reactive substance (TBARS) were measured. Renal histology was also evaluated. RESULTS: There was no significant difference among the 3 groups in terms of CAT and TBARS in erythrocytes. Renal CAT level was lowest in NS group, and significantly lower than the control group. In treatment group, CAT level significantly increased compared with NS group. In terms of renal histology, tubular and interstitial evaluations were similar in all groups. Glomerular score was significantly higher in NS group compared with control group and it was significantly decreased in treatment group compared to NS group. CONCLUSIONS: Oxidative stress in NS might be due to the decrease in antioxidant protection mechanism in kidney. Octreotide improves antioxidant levels and histology in renal tissue and might be a treatment option.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Novel bioanalytical strategy using isotope pattern deconvolution and ICP-QMS for the study of iron incorporation in erythrocytes: An insight to better assessment.

Iron is an essential element for human life and its nutritional status in the human body is directly linked to human health. More than 1015 atoms of iron per second are necessary for the maintenance of haemoglobin formation. To predict iron bioavailability three approaches are normally employed: (a) faecal recovery; (b) plasma appearance; and (c) erythrocyte incorporation (the most used). Isotope Pattern Deconvolution (IPD) is a mathematical tool that allows the isolation of distinct isotope signatures from mixtures of natural abundance and enriched tracers. In this work we propose a novel strategy to assess erythrocyte iron incorporation, based on the use of an iron stable isotope (57Fe) and the IPD concept. This strategy allows direct calculation of the exogenous concentration of 57Fe incorporated into RBCs after supplementation. In this way, to determine the mass of iron incorporated into erythrocytes, the unique prediction that must be made is the blood volume, estimate to reproduce the natural dilution of the tracer (57Fe) in the blood. This novel bioanalytical approach was applied for the measurements of iron incorporation and further iron absorption studies in humans, using a group of twelve healthy participants, that should be further evaluated for the assessment of other chemical elements that could be of health concerns and directly impact society.

Invasion-inhibitory peptides chosen by natural selection analysis as an antimalarial strategy.

Plasmodium vivax's biological complexity has restricted in vitro culture development for characterising antigens involved in erythrocyte invasion and their immunological relevance. The murine model is proposed as a suitable alternative in the search for therapeutic candidates since Plasmodium yoelii uses homologous proteins for its invasion. The AMA-1 protein is essential for parasite invasion of erythrocytes as it is considered an important target for infection control. This study has focused on functional PyAMA-1 peptides involved in host-pathogen interaction; the protein is located in regions under negative selection as determined by bioinformatics analysis. It was found that pyama1 has two highly conserved regions amongst species (>70%) under negative selection. Fourteen synthetic peptides spanning both conserved regions were evaluated; 5 PyAMA-1 peptides having high specific binding (HABP) to murine erythrocytes were identified. The parasite's invasion inhibition capability was analysed through in vitro assays, suggesting that peptides 42681 (43-ENTERSIKLINPWDKYMEKY-62), 42903 (206-RYSSNDANNENQPFSFTPEK-225) and 42904 (221-FTPEKIENYKDLSYLTKNLR-240) had greater than 50% inhibition profile and restricted P. yoelii intra-erythrocyte development. This work proposes that the screening of conserved HABPs under negative selective pressure might be good candidates for developing a synthetic anti-malarial vaccine since they share functionally-relevant characteristics, such as interspecies conservation, specific RBC binding profile, invasion and parasite development inhibition capability, and the predicted B-epitopes within were recognised by sera obtained from experimentally-infected mice.

Peroxiredoxins in erythrocytes: far beyond the antioxidant role.

The red blood cells (RBCs) are essential to transport oxygen (O2) and nutrients throughout the human body. Changes in the structure or functioning of the erythrocytes can lead to several deficiencies, such as hemolytic anemias, in which an increase in reactive oxidative species generation is involved in the pathophysiological process, playing a significant role in the severity of several clinical manifestations. There are important lines of defense against the damage caused by oxidizing molecules. Among the antioxidant molecules, the enzyme peroxiredoxin (Prx) has the higher decomposition power of hydrogen peroxide, especially in RBCs, standing out because of its abundance. This review aimed to present the recent findings that broke some paradigms regarding the three isoforms of Prxs found in RBC (Prx1, Prx2, and Prx6), showing that in addition to their antioxidant activity, these enzymes may have supplementary roles in transducing peroxide signals, as molecular chaperones, protecting from membrane damage, and maintenance of iron homeostasis, thus contributing to the overall survival of human RBCs, roles that seen to be disrupted in hemolytic anemia conditions.

RNA polymerase II pausing regulates chromatin organization in erythrocytes.

Chicken erythrocytes are nucleated cells often considered to be transcriptionally inactive, although the epigenetic changes and chromatin remodeling that would mediate transcriptional repression and the extent of gene silencing during avian terminal erythroid differentiation are not fully understood. Here, we characterize the changes in gene expression, chromatin accessibility, genome organization and chromatin nuclear disposition during the terminal stages of erythropoiesis in chicken and uncover complex chromatin reorganization at different genomic scales. We observe a robust decrease in transcription in erythrocytes, but a set of genes maintains their expression, including genes involved in RNA polymerase II (Pol II) promoter-proximal pausing. Erythrocytes exhibit a reoriented nuclear architecture, with accessible chromatin positioned towards the nuclear periphery together with the paused RNA Pol II. In erythrocytes, chromatin domains are partially lost genome-wide, except at minidomains retained around paused promoters. Our results suggest that promoter-proximal pausing of RNA Pol II contributes to the transcriptional regulation of the erythroid genome and highlight the role of RNA polymerase in the maintenance of local chromatin organization.

Novel potential therapeutics to modify iron metabolism and red cell synthesis in diseases associated with defective erythropoiesis.

Under normal conditions, iron metabolism is carefully regulated to sustain normal cellular functions and the production of hemoglobin in erythroid cells. Perturbation to the erythropoiesis-iron metabolism axis can result in iron imbalances and cause anemia or organ toxicity. Various congenital and acquired diseases associated with abnormal red cell production are characterized by aberrant iron absorption. Several recent studies have shown that improvements in red blood cell production also ameliorate iron metabolism and vice versa. Many therapeutics are now under development with the potential to improve a variety of hematologic diseases, from ß-thalassemia and iron-refractory iron deficiency anemia to anemia of inflammation and polycythemia vera. This review summarizes selected mechanisms related to red cell production and iron metabolism and describes potential therapeutics and their current uses. We also consider the potential application of the discussed therapeutics on various diseases, alone or in combination. The vast repertoire of drugs under development offers new opportunities to improve the clinical care of patients suffering from congenital or acquired red blood cell disorders with limited or no treatment options.

Association between dietary polyunsaturated fatty acids and their concentration in blood plasma, red blood cell, and semen of dogs.

Background: In dogs, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) affect the fatty acid (FA) profile of blood plasma, erythrocyte membrane (EM), and semen, but their correlation has not yet been investigated. Aim: In this study, we evaluated the association between dietary PUFA and their profile in blood plasma, EM, and semen of dogs, with the possibility to predict the semen profile using the values of the three first. Methods: Twelve male dogs received the same standard commercial diet for 4 weeks. The FA profile was analyzed by gas chromatography in paired diet, blood (plasma and EM determinations), and semen samples. Data were analyzed with SAS Proc Corr version 9.4. Pearson´s correlation coefficient (significant if p < 0.05) was used to assess the association of dietary FA profiles with those in blood plasma, EM, and semen. Results: There was a positive correlation between dietary eicosapentaenoic acid (EPA) and blood plasma (r = 0.97), EM (r = 0.94) and semen (r = 0.92) EPA, and between dietary docosahexaenoic acid (DHA) and arachidonic acid (ARA) and semen DHA (r = 0.93) and ARA (r = 0.92), respectively. There was a negative correlation between dihomo-gamma-linolenic acid (DGLA) in the diet and EM DGLA (r = -0.94). Conclusion: The dietary EPA is correlated with blood plasma, EM, and semen EPA concentrations, and dietary DHA and ARA are associated with semen DHA and ARA concentrations in dogs. These findings suggest that dietary EPA, DHA, and ARA concentrations could be helpful to predictive markers for such concentrations in the semen of dogs.

Bosentan attenuates sickle cell disease erythrocyte HbS polymerization and impaired deformability induced by endothelin-1.

The effects of endothelin-1 (ET-1) on erythrocytes from sickle cell disease (SCD) patients have been described, but mechanisms of ET-1 regarding primary erythrocyte functions remain unknown. ET-1 is a vasoconstrictor peptide produced by endothelial cells, and the expression of ET-1 is increased in SCD. The present study used ex vivo experiments with sickle cell erythrocytes, ET-1, and bosentan, a dual antagonist of ETA and ETB receptors. We performed a hemoglobin S (HbS) polymerization assay with three concentrations of ET-1 (1, 20, and 50 pg/mL) and bosentan (100 nmol/L). ET-1 increased HbS polymerization at all concentrations, and this effect was suppressed by bosentan. For the deformability assay, red blood cells (RBCs) were incubated on a Sephacryl column with the same concentrations of ET-1 and bosentan. ET-1 decreased deformability, and this effect was reversed by bosentan. To observe erythrocyte adhesion, ET-1 and bosentan were incubated with RBCs in thrombospondin-coated 96-well plate, which demonstrated that ET-1 decreased adhesion but that bosentan enhanced adhesion. We also assessed erythrocyte apoptosis and observed decreased eryptosis induced by ET-1, and these effects were inhibited bosentan. Thus, these findings demonstrated that ET-1 modulates HbS polymerization, erythrocyte deformability, adhesion to thrombospondin, and eryptosis, and these effects were suppressed or enhanced by bosentan.