Adult Erythrocytes Research Articles

MicroRNAs as Quality Assessment Tool in Stored Packed Red Blood Cell in Blood Banks.

Micro-ribonucleic acids are control gene expression in cells. They represent the changed cellular states that occur can be employed as biomarkers. Red blood cells alter biochemically and morphologically while they are being stored, which could be detrimental to transfusion. The effect of storage on the erythrocyte transcriptome is not mostly investigated. Because adult erythrocytes lack a nucleus, it has long been assumed that they lack deoxyribonucleic acid and ribonucleic acid. On the other hand, erythrocytes contain a diverse range of ribonucleic acids, of which micro-ribonucleic acids are key component. Changes in this micro-ribonucleic acid protect cells from death and adenine triphosphate depletion, and they are linked to specific storage lesions. As a result, changes in micro-ribonucleic acid in stored erythrocytes may be used as a marker to assess the quality and safety of stored erythrocytes. Therefore, this review ams to review the role of microRNA in stored packed red blood cells as quality indicator. Google Scholar, PubMed, Scopus, and Z-libraries are used for searching articles and books. The article included in this paper was written in the English language and had the full article. During long storage of RBCs, miR-16-2-3p, miR-1260a, miR-1260b, miR-4443, miR-4695-3p, miR-5100, let-7b, miR-16, miRNA-1246, MiR-31-5p, miR-203a, miR-654-3p, miR-769-3p, miR-4454, miR-451a and miR-125b- 5p are up regulated. However, miR-96, miR-150, miR-196a, miR-197, miR-381 and miR-1245a are down regulated after long storage of RBCs. The changes of this microRNAs are linked to red blood cell lesions. Therefore, micro-ribonucleic acids are the potential quality indicator in stored packed red blood cells in the blood bank. Particularly, micro-ribonucleic acid-96 is the most suitable biomarker for monitoring red blood cell quality in stored packed red blood units.

Использование поляризационно-интерференционной микроскопии для определения оптических свойств фетальных эритроцитов в тканях

Objective: to evaluate the possibility of using polarization interference microscopy for morphological diagnostics of fetal erythrocytes in tissues. Materials and methods. Blood smears used during the diagnostic and treatment process and histological sections prepared from autopsy material were used. During the diagnosis, a polarization-interference microscope PZO BIOLAR PI with a microphotometric nozzle UPI was used to determine the optical density and the measure of light transmission in erythrocytes. The method of polarization-interference microscopy contributed to the study of optical properties in individual erythrocytes. The optical properties of erythrocytes containing fetal-type hemoglobin and erythrocytes with adult-type hemoglobin were compared. The difference in the composition of the structure of erythrocytes determined the difference in light transmission and refraction. Based on the results of light transmission, the optical density of fetal and adult erythrocytes was calculated. In the conducted study of clinical blood smears, in neonatal patients, 60 measurements of the light transmission coefficient and 60 similar studies were performed in patients of the comparison group - persons older than 18 years. In the study of autopsy material, 120 measurements of the erythrocyte light transmission coefficient were carried out in placental tissue samples (chorionic villi and interstitial space). Results. The optical properties of erythrocytes were determined both in blood smears and in placental tissue preparations. Erythrocytes in blood smears with HbF had an increased light transmission coefficient in relation to the optical properties of erythrocytes with HbA in the control group. During the study of placental tissues, it was revealed that the indicators of the optical density of maternal erythrocytes with HbA in the erythrocytes of the interstitial space differed in higher values of indicators compared with the optical density of fetal erythrocytes with HbF in the vessels of chorionic villi. Conclusion. The determination of differences in the physico-optical properties of erythrocytes with fetal and adult hemoglobins makes it possible to determine the affiliation of erythrocytes both in blood smears and in the interstitial space of the placenta.

ETO2 Recruitment of NuRD to the γ-Globin Locus Involves Multivalent Interaction with GATAD2A

Increasing the expression of fetal hemoglobin ameliorates the symptoms of sickle cell disease and β-thalassemia. Therefore, blocking fetal hemoglobin silencing in adult erythrocytes represents a potent strategy for treating these diseases. Extensive research over the past five decades have uncovered critical transcription factors involved in silencing fetal hemoglobin, including Bcl11A and Zbtb7A (LRF), among others. The Nucleosome Remodeling and Deacetylase (NuRD) complex represents a central co-regulatory factor recruited by these proteins and DNA methylation to silence gene expression. Hence, NuRD represents a potential target for blocking silencing by multiple factors simultaneously, thereby leading to robust induction of fetal hemoglobin. We previously have shown that disrupting the MBD2-NuRD complex induces high levels of fetal hemoglobin in both the HUDEP-2 tissue culture cell line and primary patient-derived bone marrow stem cells [Yu et al. (2019) Haematologica, 104(12), 2361-2371]. Recent work by the Dean lab [Guo et al. (2020). Nucleic Acids Research, 48(18), 10226-10240] added to the list of factors that bind and recruit NuRD to the globin locus. They showed that ETO2, a well-established factor in erythroid differentiation, binds directly to NuRD, which required the tetramerization domain of ETO2. However, the specific contacts driving this interaction remain unknown. Based on previous work by the Bushweller [Liu et al. (2007) Cancer Cell, 11(6), 483-497] and Vermeulen [Spruijt et al. (2016) Cell Reports, 17(3), 783-798] groups, we hypothesized that ETO2 binds directly to the GATAD2A component of NuRD through the Nervy Homology Region-4 (NHR4) domain of ETO2 and multiple polyproline-leucine (PPPL) motifs in GATAD2A. Here we use NMR analyses to show that the ETO2-NHR4 domain binds each of four polyproline-leucine motifs in GATAD2A. We measure binding by chemical shift analysis which reveals a weak affinity (KD ~200-300 μM) for the individual peptides. We then show that the ETO2-NHR3 domain contributes to weak homo-oligomerization (dimer-tetramer formation) by gel filtration analysis. Importantly, multivalent interaction between the PPPLx4 region of GATAD2A and a construct including both the NHR3 and NHR4 domains of ETO2 leads to a dramatic increase in binding affinity (KD ~ 4 μM) as measured by isothermal titration calorimetry. Furthermore, using an in-cell bioluminescent resonance energy transfer assay (NanoBRET), we show that including the NHR2 tetramerization domain augments association between these proteins. Mutating key residues in either GATAD2A-PPPLx4 or ETO2-NHR4 domains disrupts this association in cells. Finally, we show that enforced expression of a peptide from GATAD2A containing the four polyproline-leucine motifs consistently induces a low-level of fetal hemoglobin mRNA expression in K-562 cells. Together, these findings establish that the ETO2-NHR4 domain plays a central role in recruitment of NuRD for gene silencing. Tetramerization of the NHR2 and NHR3 domains leads to multivalent association with GATAD2A, which dramatically increases binding affinity. This result has mechanistic implications for fetal hemoglobin silencing and erythroid differentiation, potentially leading to a novel therapeutic target for treating β-hemoglobinopathies. Furthermore, gene rearrangements involving the ETO family of proteins drive the development of acute myeloid leukemia. Our studies provide an explanation for how the stoichiometry of the fusion proteins may dictate which co-regulatory complexes are brought to a specific site and suggest a strategy for selectively targeting these complexes.

Cell landscape of larval and adult Xenopus laevis at single-cell resolution

The rapid development of high-throughput single-cell RNA sequencing technology offers a good opportunity to dissect cell heterogeneity of animals. A large number of organism-wide single-cell atlases have been constructed for vertebrates such as Homo sapiens, Macaca fascicularis, Mus musculus and Danio rerio. However, an intermediate taxon that links mammals to vertebrates of more ancient origin is still lacking. Here, we construct the first Xenopus cell landscape to date, including larval and adult organs. Common cell lineage-specific transcription factors have been identified in vertebrates, including fish, amphibians and mammals. The comparison of larval and adult erythrocytes identifies stage-specific hemoglobin subtypes, as well as a common type of cluster containing both larval and adult hemoglobin, mainly at NF59. In addition, cell lineages originating from all three layers exhibits both antigen processing and presentation during metamorphosis, indicating a common regulatory mechanism during metamorphosis. Overall, our study provides a large-scale resource for research on Xenopus metamorphosis and adult organs.

Animal trait variation at the within-individual level: erythrocyte size variation and malaria infection in a tropical lizard.

High levels of within-individual variation (WIV) in reiterative components in plants such as leaves, flowers, and fruits have been shown to increase individual fitness by multiple mechanisms including mediating interactions with natural enemies. This relationship between WIV and fitness has been studied almost exclusively in plant systems. While animals do not exhibit conspicuous reiterative components, they have traits that can vary at the individual level such as erythrocyte size. It is currently unknown if WIV in animals can influence individual fitness by mediating the outcome of interactions with natural enemies as it has been shown in plants. To address this issue, we tested for a relationship between WIV in erythrocyte size, hemoparasite infection status, and body condition (a proxy for fitness) in a Caribbean anole lizard. We quantified the coefficient of variation of adult erythrocytes size in $n = 95$ infected and $n = 107$ non-infected lizards. We found higher degrees of erythrocyte size variation in infected lizards than in non-infected individuals. However, we found no significant relationship between infection status or erythrocyte size variation, and lizard body condition. These results suggest that higher WIV in erythrocyte size in infected lizards is not necessarily adaptive but likely a consequence of the host response to infection. Many hemoparasites destroy their host cells as part of their life cycle. To compensate, the host lizard may respond by increasing production of erythrocytes resulting in higher WIV. Our results emphasize the need to better understand the role of within-animal variation as a neglected driver or consequence of ecological and evolutionary interactions.

Cord and peripheral blood erythrocyte analysis by scanning electron microscopy and flow cytometry.

Human umbilical cord blood is rich in hematopoietic cells. We aimed to focus on the morphological, biochemical, membrane protein profile and surface protein expression differences of erythrocytes, isolated from cord and adult peripheral blood using techniques such as high-resolution scanning electron microscopy (SEM), gel electrophoresis (SDS-PAGE) and flow cytometry. Adult peripheral blood was collected from consenting adults, and umbilical cord blood was procured from consenting mothers, post-delivery at Medical College, Kolkata. We emphasized on cord and adult peripheral blood erythrocytes' morphological variations using SEM images and protein expression by flow cytometric analysis. Some conventional biochemical analyses such as osmotic fragility of the cell membrane, haemoglobin co-oxidation study and lipid peroxidation assay were done for supporting evidence along with membrane protein content using gel electrophoresis. Our SEM images indicated clear morphological variations in cord erythrocyte with a higher degree of cellular deformities and difference in membrane texture. Flow cytometric analysis of cord erythrocyte showed a significant difference in CD235a expression than adults. We observed an overexpression of GLUT1 and decreased expression of Band 3 in cord erythrocyte membrane. Our results also showed cord erythrocytes have low osmotic fragility, a slower rate of co-oxidation of cord haemoglobin and a lesser lipid peroxidation level than that of adults. Cord blood erythrocytes have deeper indentations leading to higher flexibility, more oxygen-carrying capacity and less osmotic fragility in comparison with adult erythrocytes. The expression of CD235a and Band 4.5 (GLUT 1) was significantly higher in cord erythrocytes than peripheral adult erythrocytes.

Life-long sports engagement enhances adult erythrocyte adenylate energetics

Regular physical activity reduces age-related metabolic and functional decline. The energy stored in adenine nucleotides (ATP, ADP, and AMP) is essential to enable multiple vital functions of erythrocytes and body tissues. Our study aimed to predict the rate of age-related changes in erythrocyte adenylate energetics in athletes and untrained controls. The erythrocyte concentration of adenylates was measured in 68 elite endurance runners (EN, 20–81 years), 58 elite sprinters (SP, 21–90 years), and 62 untrained individuals (CO, 20–68 years). Resting concentrations of ATP, total adenine nucleotide pool, and ADP/AMP ratio were lowest in the CO group and highest in the SP group. The concentration of erythrocyte ADP and AMP was lowest in the EN group and highest in the CO group. In all studied groups, we found a significant increase in the concentration of most erythrocyte adenylate metabolites with age. For ADP and AMP, the trend was also significant but decreasing. Our study strongly suggests that lifelong sports and physical activity participation supports erythrocyte energetics preservation. Although the direction and the predicted rates of change are similar regardless of the training status, the concentrations of particular metabolites are more advantageous in highly trained athletes than in less active controls. Of the two analyzed types of physical training, sprint-oriented training seems to be more efficient in enhancing erythrocyte metabolism throughout adulthood and old age than endurance training.

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Updated Results of a Phase 1/2 Clinical Study of Zinc Finger Nuclease-Mediated Editing of BCL11A in Autologous Hematopoietic Stem Cells for Transfusion-Dependent Beta Thalassemia

Telomere shortening is associated with corticosterone stress response in adult barn swallows.

When vertebrates face stressful events, the hypothalamic–pituitary–adrenal (HPA) axis is activated, generating a rapid increase in circulating glucocorticoid (GC) stress hormones followed by a return to baseline levels. However, repeated activation of HPA axis may lead to increase in oxidative stress. One target of oxidative stress is telomeres, nucleoprotein complexes at the end of chromosomes that shorten at each cell division. The susceptibility of telomeres to oxidizing molecules has led to the hypothesis that increased GC levels boost telomere shortening, but studies on this link are scanty. We studied if, in barn swallows Hirundo rustica, changes in adult erythrocyte telomere length between 2 consecutive breeding seasons are related to corticosterone (CORT) (the main avian GC) stress response induced by a standard capture-restraint protocol. Within-individual telomere length did not significantly change between consecutive breeding seasons. Second-year individuals showed the highest increase in circulating CORT concentrations following restraint. Moreover, we found a decline in female stress response along the breeding season. In addition, telomere shortening covaried with the stress response: a delayed activation of the negative feedback loop terminating the stress response was associated with greater telomere attrition. Hence, among-individual variation in stress response may affect telomere dynamics.

Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease

BackgroundSickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising α- and γ-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of γ-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF.MethodsWe enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage–specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment.ResultsAs of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.3%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period.ConclusionsThis study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk–benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656)

Cellular Basis of Embryonic Hematopoiesis and Its Implications in Prenatal Erythropoiesis.

Primitive erythrocytes are the first hematopoietic cells observed during ontogeny and are produced specifically in the yolk sac. Primitive erythrocytes express distinct hemoglobins compared with adult erythrocytes and circulate in the blood in the nucleated form. Hematopoietic stem cells produce adult-type (so-called definitive) erythrocytes. However, hematopoietic stem cells do not appear until the late embryonic/early fetal stage. Recent studies have shown that diverse types of hematopoietic progenitors are present in the yolk sac as well as primitive erythroblasts. Multipotent hematopoietic progenitors that arose in the yolk sac before hematopoietic stem cells emerged likely fill the gap between primitive erythropoiesis and hematopoietic stem-cell-originated definitive erythropoiesis and hematopoiesis. In this review, we discuss the cellular origin of primitive erythropoiesis in the yolk sac and definitive hematopoiesis in the fetal liver. We also describe mechanisms for developmental switches that occur during embryonic and fetal erythropoiesis and hematopoiesis, particularly focusing on recent studies performed in mice.

Targeting Transcriptional Co-Activator PGC-1a for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is the most common inherited human hematologic disease, which causes hemolytic anemia, pain, disability, progressive multi-organ damage and early mortality. Clinical studies have shown that increased synthesis of fetal hemoglobin (HbF) in sickled erythroid cells leads to diminished severity of many clinical features of SCD. Therefore, therapeutic agents that can increase HbF production will be of benefit to SCD patients. Hydroxyurea (HU) is a FDA-approved therapeutic for treatment of SCD, but not all patients respond favorably or adequately. Therefore, other methods of targeting HbF are highly desired, particularly those that act by different mechanisms that might be used in combination with HU or alone (for those who do not tolerate HU). Transcriptional co-activator PGC-1α has recently been showed to play a crucial role in globin gene regulation and erythropoiesis by our group. Loss of function in the PGC-1α knock out mice significantly reduced the expression of murine embryonic εy- and βh1-globin genes; both of them are homologues of the human ε- and γ-globin genes. Forced overexpression of PGC-1α in vitroby adenovirus infection in bone marrow cells from SCD mice resulted in significantly increased human γ- and murine εy- and βh1-globin genes. These results directly validate a role for PGC-1α in human γ-globin induction and provide significant support for the hypothesis that modulating PGC-1α activity, or the signaling pathways that it regulates, in SCD patients may be an effective approach that could therapeutically benefit individuals with SCD. Here we report the effects of PGC-1α in inducing HbF in human primary erythoid progenitor CD34+cells. We infected CD34+cells with lentivirus carrying PGC-1α and found that over-expression of PGC-1α resulted in significantly increased F-cells in the culture, from 1.6% up to 41.1%. We further discovered several PGC-1α agonists that are highly selective for PGC-1α. We found that one of the PGC-1α agonists, Compound SR-18292, induces PGC-1α mRNA expression in a dose dependent manner (2.6-fold at 1 μM, 5.7-fold at 10 μM) in cultured CD34+cells without effecting cell viability. Consistent with the increase of PGC-1α levels, the percentage of F-cells increased from 2.76% (vehicle control) to 32.1% when cells exposed to 1 µM SR-18292, which is comparable to that of 25 µM HU (32.2%). The mean fluorescence intensity of F-cells in SR-18292 treated cells was higher than in controls, suggesting that the treatment not only increased the number of F-cells but also increased the concentration of HbF in F-cells. The increased levels of PGC-1α reactivates the expression of fetal γ-globin genes resulting in significant HbF accumulation in adult erythrocytes, suggesting that the transcriptional co-activator PGC-1α comprises a new molecular target for possible therapeutic intervention in treating SCD. Disclosures No relevant conflicts of interest to declare.

Pulsation Activates Mechanosensitive Piezo1 to Form Long-Term Hematopoietic Stem Cells

The temporal and spatial origin and development of long-term, self-renewing hematopoietic stem cells (LT-HSC) remain a mystery. The first set of definitive HSCs is born from the hemogenic endothelial cells residing in the ventral wall of the dorsal aorta (DA) of the aorta-gonad-mesonephros region during embryonic development. Blood flow- and shear-stress-mediated nitric oxide-induced vasodilation are responsible for the endothelial-to-HSC transition (EHT). However, it remains unknown why the ventral wall, and not the dorsal wall, of the DA is the restricted site of the EHT when blood flows through the entire DA and exerts shear stress on both the ventral and dorsal sides of the DA. Using single-particle tracking and fast Fourier Transform analyses of pulsating blood vessels, we demonstrate that the circumferential strain in the ventral wall, and not dorsal wall, is concurrent with and responsible for the magnitude, the site, and timing of the HSC formation. We extended our findings by developing a bioreactor to establish the functional link between pulsation in the blood vessels and HSC formation. Using serial transplant, limiting dilution, and serial replating assays, we found that pulsation mediated circumferential stretching of hemogenic endothelial cells or Piezo1 activation (Yoda1) yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using delayed-type hypersensitivity assay, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our CUT&RUN CHIP-Sequencing & MASSArray methylation analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. To analyze the conserved role of PIEZO1-mediated mechanosensitive mechanisms in human hematopoiesis, we employed directed differentiation of constitutive RUNX1-mCherry human induced pluripotent stem cells (iPSCs) to hemogenic endothelial cells. We found that Yoda1-mediated PIEZO1 activation stimulated human endothelial-to-hematopoietic transition. In conclusion, pulsation-mediated circumferential strain activates Piezo1 to stimulate the endothelial-to-HSC transition via the induction of Dnmt3b expression. This leads to the formation of long-term self-renewing HSCs, which can engraft and reconstitute to multi-lineage, adult blood upon serial transplantations. Our identification of a novel biomechanical cue unravels the physiological mystery in HSC formation in the ventral wall of the DA. We also establish its cross-talk with mechanosensitive and epigenetic mechanisms to produce functional, long-term HSCs that reconstitute to form normal adult blood. This yields the therapeutic promise of developing transgene-free LT-HSC-based cellular therapies for the treatment of human blood disorders. Disclosures No relevant conflicts of interest to declare.

Neonatal clinical blood sampling led to major blood loss and was associated with bronchopulmonary dysplasia.

AimStudies indicate that reduced foetal haemoglobin levels are related to increased neonatal morbidity rates. This study investigated the relationships between sampling‐related blood loss and adult blood transfusions administered during postnatal days 1‐14 and the development of severe neonatal morbidities in extremely preterm infants born before 28 weeks of gestation.MethodsThe medical files of 149 extremely preterm infants born at two university hospitals in Sweden from 2013 to 2018 were investigated.ResultsBlood sampling resulted in a 58% depletion of the endogenous blood volume postnatal days 1‐14 (median 40.4 mL/kg, interquartile range 23.9‐53.3 mL/kg) and correlated with the adult erythrocyte transfusion volume (rS = 0.870, P < .001). Sampling‐related blood loss on postnatal days 1‐7, adjusted for gestational age at birth and birth weight standard deviation score, was associated with the development of bronchopulmonary dysplasia (BPD) (odds ratio by a 10‐unit increase 2.4, 95% confidence interval 1.1‐5.4) (P = .03). No associations were found between blood sampling and intraventricular haemorrhage or necrotising enterocolitis in the full statistical model. The largest proportion of sampling‐related blood was used for blood gas analyses (48.7%).ConclusionDiagnostic blood sampling led to major endogenous blood loss replaced with adult blood components and was associated with the development of BPD.

2028 - PULSATION IN BLOOD VESSELS STIMULATES MECHANOSENSITIVE AND EPIGENETIC MECHANISMS TO PROMOTE THE BIRTH OF HEMATOPOIETIC STEM CELLS

During fetal development, a subset of endothelial cells changes their fate to become HSCs in the aorta-gonad-mesonephros (AGM) region. However, the mechanisms regulating the endothelial-to-hematopoietic transition (EHT) remain largely unknown. Our machine learning and confocal imaging demonstrate that the rhythmic contraction in ventral part of dorsal aorta is concurrent with EHT events. We also found that pulsation-mediated cyclical contraction stretches AGM in circumferential direction. Using serial transplant, limiting dilution, and replating assays, we found that the circumferential stretching of hemogenic endothelial cells or Piezo1 activation yields 3-times higher amounts of Long Term (LT)-HSC formation; which reconstitute to normal multi-lineage adult blood. Using vaccine-challenge, adult globin expression, MPO enzyme activity, immunoglobulins, and T-cell receptor rearrangement analyses, we found that circumferential stretching or Piezo1 activation-derived HSCs reconstitute to functional T and B cells, adult erythrocytes, and myeloid cells. Our Piezo1fl/flxScl-Cre conditional knockout, gene-silencing, & confocal imaging further demonstrate that circumferential stretching of blood vessels activates Piezo1; which enhances epigenetic regulator Dnmt3b expression to stimulate the EHT. Our Cut&Run & MassArray analyses demonstrate that Dnmt3b suppresses endothelial genes during EHT. In conclusion, we found that pulsation-mediated circumferential stretching of AGM stimulates the EHT by Piezo1 activation and Dnmt3b over-expression. This leads to the formation of long-term self-renewing HSCs engrafting upon serial transplantations and reconstituting normal adult blood. To our knowledge, this is the first report demonstrating how biomechanical forces utilize epigenetic machinery to promote cell fate transition as well as a bioreactor to develop and expand LT-HSCs.

Reduced Plasmodium Falciparum Growth in Erythrocytes Is Associated with Fetal Hemoglobin Expression

Introduction: The risk of clinical Plasmodium falciparum malaria is significantly low in the first few months of life. Much of this protection has been attributed to maternal antibodies transmitted to the infant in utero or via breast milk, however less severe disease has also been associated with fetal hemoglobin (HbF). Impaired P. falciparum growth in cord blood erythrocytes and reduced digestion of HbF protein by recombinant plasmepsin II, a hemoglobinase, has also been observed in vitro, while in vivo studies have demonstrated decreased P. falciparum growth in high HbF-containing human gamma (γ)-transgenic murine red cells. Though inefficiency of hemoglobinase digestion of HbF appears plausible given enhanced alpha (α)/γ relative to α/beta (β) dimer and tetramer stability, it has been difficult to assess such inefficiency in vitro given the dramatic effect of current protease inhibitors on parasite growth. Instead, more focus has drifted towards oxidative stress and poor cytoadherence as mechanisms underlying the protection conferred by HbF. To test the hypothesis that HbF affects malaria infection, we mapped P. falciparum growth in cord blood and heterozygote A/HPFH erythrocytes using precise DNA replication measurements throughout the parasite blood stage cell-cycle.Methods: Adult (AA), cord, and heterozygote Hereditary Persistence of Fetal Hemoglobin (A/HPFH) samples were collected within one week of each other and within 1 month of assay date. The P. falciparum 3D7 IG06 parasite line (a gift from Daniel Goldberg, Washington University School of Medicine, St. Louis) was used for growth assays. All growth assays were performed at 1% oxygen using synchronized schizonts obtained by magnet purification via the MACS system. Average number of nuclei per parasite was determined via flow cytometry using SYBR Mean Fluorescence Intensity (MFI) as previously described.Results: By 32 hours post invasion into cord blood, a definite decrease in P. falciparum DNA replication, a sensitive marker of parasite growth, was observed compared to growth in adult erythrocytes (Figure 1A). Compared to the complete growth defect observed in deoxygenated sickle cell trait (AS), the almost 40% reduction (p=0.0002) in the average number of nuclei in cord blood RBCs was modest, allowing for growth through schizogony. The growth inhibition in cord blood erythrocytes was not sensitive to changes in oxygen levels (data not shown). Ring/early trophozoite stage parasites were morphologically similar in both adult and cord blood erythrocytes (Figure 1B), with growth differences beginning to be apparent between 28 and 32 hours, coincident with the onset of hemoglobin digestion. To ensure that the growth defect observed was not due to cord blood erythrocyte factors unrelated to HbF, we examined heterozygous A/HPFH RBCs which are similar to adult AA erythrocytes, but with expression of HbF. Parasites in A/HPFH erythrocytes (with ~20-30% HbF), demonstrated a 25% growth reduction in the average number of nuclei at 36hpi, compared to those in adult AA RBCs (Figure 2A and 2B), indicating the growth inhibitory effect of HbF.Conclusion: Though less striking compared to the severe stalling observed in deoxygenated AS RBCs, growth of P. falciparum 3D7 IG06 in cord blood erythrocytes is clearly delayed compared to growth in adult AA erythrocytes. This growth defect is oxygen independent, evidence that a different mechanism of parasite growth inhibition is involved than that observed in AS RBCs. The consistent growth delay observed in heterozygote A/HPFH confirms that parasite growth deficiency also occurs at lower percentages of HbF than those found in cord blood presumably because α2/γ-β heterodimers are also resistant to digestion by P. falciparum hemoglobinases. Finally, these data suggest that treatment of sickle cell disease by increasing fetal hemoglobin might be safer in malaria zones than would be employment of direct anti-sickling agents. [Display omitted] DisclosuresHankins:Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy; Novartis: Research Funding.

3D organization of chicken genome demonstrates evolutionary conservation of topologically associated domains and highlights unique architecture of erythrocytes' chromatin.

How chromosomes are folded, spatially organized and regulated in three dimensions inside the cell nucleus are among the longest standing questions in cell biology. Genome-wide chromosome conformation capture (Hi-C) technique allowed identifying and characterizing spatial chromatin compartments in several mammalian species. Here, we present the first genome-wide analysis of chromatin interactions in chicken embryonic fibroblasts (CEF) and adult erythrocytes. We showed that genome of CEF is partitioned into topologically associated domains (TADs), distributed in accordance with gene density, transcriptional activity and CTCF-binding sites. In contrast to mammals, where all examined somatic cell types display relatively similar spatial organization of genome, chicken erythrocytes strongly differ from fibroblasts, showing pronounced A- and B- compartments, absence of typical TADs and formation of long-range chromatin interactions previously observed on mitotic chromosomes. Comparing mammalian and chicken genome architectures, we provide evidence highlighting evolutionary role of chicken TADs and their significance in genome activity and regulation.

Development of Erythroid Progenitors under Erythropoietin Stimulation in Xenopus laevis Larval Liver.

Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. β2-globin mRNA expression peaked at day 7 after the start of culture. Larval β2-globin was highly expressed in NF56-derived cells, while adult β2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3-4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.

Patient age does not affect mefloquine concentrations in erythrocytes and plasma during the acute phase of falciparum malaria

ObjectiveTo evaluate whether patient age has a significant impact on mefloquine concentrations in the plasma and erythrocytes over the course of treatment for uncomplicated falciparum malaria. MethodsA total of 20 children aged between 8 and 11 years and 20 adult males aged between 22 and 41 years with uncomplicated falciparum malaria were enrolled in the study. Mefloquine was administered to patients in both age groups at a dose of 20mgkg−1. The steady-state drug concentrations were measured by reversed-phase high performance liquid chromatography. ResultsAll patients had an undetectable mefloquine concentration on day 0. In adults, the plasma mefloquine concentrations ranged from 770 to 2930ngmL−1 and the erythrocyte concentrations ranged from 2000 to 6030ngmL−1. In children, plasma mefloquine concentrations ranged from 881 to 3300ngmL−1 and erythrocyte concentrations ranged from 3000 to 4920ngmL−1. There was no significant correlation between mefloquine concentrations in the plasma and erythrocytes in either adults or children. ConclusionIn the present study, we observed no effect of patient age on the steady-state concentrations of mefloquine in the plasma and erythrocytes. We found that the mefloquine concentration in the erythrocytes was approximately 2.8-times higher than in the plasma. There were no significant correlations between mefloquine concentrations in the erythrocytes and plasma for either age group.

Prospective Evaluation of Fetal Haemoglobin Induction in Maternal Erythrocytes: A Preliminary Analysis of a Cohort of 345 Parturients

Introduction. The mechanisms of haemoglobin (Hb) subtype switch remain largely unknown even though it has a therapeutic potential (Bauer et al., 2012). In fact, reactivation of fetal haemoglobin (HbF) production in adult erythrocytes (F-cells) is protective in many Hb disorders. Pregnancy may represent the only physiological condition where HbF production increases transiently (Ibrahim et al, 2009; Yamada et al., 2012). Moreover, flow cytometry (FC), by intracellular staining using anti-HbF antibodies, has improved the evaluation of maternal F-cells (Corcoran et al., 2014; Chen et al., 2000). In this study, the objectives are: 1) to document HbF expression prospectively peri- and post-partum and confirm its maternal origin; 2) to assess potential causes of HbF modulation throughout pregnancy (i.e. β-hCG, EPO) and; 3) to identify gene differences between HbF-positive and HbF-negative pregnant women.Methods. In this observational, single institution, prospective study, 879 pregnant women were screened. A total of 345 participants were included in the study: the first consecutive 176 women with negative total HbF expression (<1%), and the first consecutive 169 women with positive total HbF expression (≥1%). Hb studies were conducted with high performance liquid chromatography (HPLC), and FC was performed if total HbF expression was ≥1%. Other assessments included DNA sampling, complete blood counts, β-hCG and EPO levels, and qualitative data (i.e. to account for other factors such as tobacco, progesterone use, etc.). Analyses were conducted at each trimester except DNA sampling, which was done during the first visit only. HbF-positive women had an additional post-partum visit to assess changes of HbF expression and account for hereditary persistence of HbF. Descriptive statistics and Pearson correlations were planned for this analysis.Results. At the time of analysis, 93 HbF-positive and 147 HbF-negative women had completed follow-up. Results revealed that, among all pregnant women screened, 22.07% (n = 194) had positive HbF expression. Of this percentage, 90.72% (n = 176) filled the inclusion criteria. Of all women with positive HbF expression during 1 or more of the first 3 visits, 72.04% showed a post-partum decrease of HbF below 1%. Furthermore, FC analyses in HbF-positive women confirmed the maternal origin of HbF in all cases and revealed an average of 13.28% (sd = 5.38%) F-cells when total HbF was ≥ 1%. Only modest correlations could be found so far between HbF levels and β-hCG (ρ = 0.25) or EPO (ρ = 0.07). Comparative DNA analyses of HbF-positive and HbF-negative women are currently underway and will be presented at the meeting.Conclusions. To our knowledge, this represents the largest prospective study evaluating HbF expression throughout pregnancy, as well as the first to evaluate post-partum HbF dynamics and the genetic background of HbF-negative and HbF-positive pregnant women. Preliminary data shows that: 1) a significant percentage of pregnant women have a transient elevation of HbF which cannot be explained by fetomaternal hemorrhage nor hereditary persistence of HbF; 2) the proportion of F-cells in women remains constant peri- and post-partum when HbF ≥ 1%; 3) a large proportion of maternal erythrocytes express HbF at low levels and; 4) HbF modulation during pregnancy is only modestly correlated to β-hCG and EPO. These data may lead to the study of a new paradigm of Hb subtype switch as well as HbF-induction therapeutics for the benefit of Hb disorders. DisclosuresNo relevant conflicts of interest to declare.