A5050767877- Erythrocyte Function and Pathophysiology
- Blood properties and coagulation
- Blood groups and transfusion
- Hemoglobinopathies and Related Disorders
- Malaria Research and Control
- Pancreatic function and diabetes
- Complement system in diseases
- Phagocytosis and Immune Regulation
- Mosquito-borne diseases and control
- Neonatal Health and Biochemistry
- CRISPR and Genetic Engineering
- Signaling Pathways in Disease
- Lipid Membrane Structure and Behavior
- Ion Transport and Channel Regulation
- Pluripotent Stem Cells Research
- Hemoglobin structure and function
- Renal Diseases and Glomerulopathies
- Metabolism, Diabetes, and Cancer
- Caveolin-1 and cellular processes
- Hippo pathway signaling and YAP/TAZ
- Viral Infectious Diseases and Gene Expression in Insects
- Methane Hydrates and Related Phenomena
- Heat shock proteins research
- RNA Interference and Gene Delivery
- Blood disorders and treatments
University of Bristol
2015-2024
NHS Blood and Transplant
2014-2022
Bristol Institute for Transfusion Sciences
2014-2022
National Institute for Health Research
2016-2022
National Health Service
2018-2020
Southmead Hospital
2011
Abstract With increasing worldwide demand for safe blood, there is much interest in generating red blood cells vitro as an alternative clinical product. However, available methods generation of from adult and cord progenitors do not yet provide a sustainable supply, current systems using pluripotent stem generate viable cells. We have taken approach, immortalizing early erythroblasts stable line, which provides continuous supply The immortalized differentiate efficiently into mature,...
The study of human erythropoiesis in health and disease requires a robust culture system that consistently reliably generates large numbers immature erythroblasts can be induced to differentiate synchronously. We describe method modified from Leberbauer et al. (2005) obtain homogenous population peripheral blood mononuclear cells (PBMC) without prior purification CD34(+) cells. This pure expanded 4x10(8) 1x10(8) PBMC after 13-14 days culture. Upon synchronized differentiation, high levels...
The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade human erythrocyte. process invasion is nonetheless remarkably rapid. To date, mechanistic models have focused predominantly on parasite actomyosin motor contribution to energetics entry. Here, we conducted numerical analysis using dimensions for an archetypal merozoite predict respective contributions host-parasite interactions invasion, in particular role...
Significance The blood-stage malaria parasite, the merozoite, invades human red blood cell (RBC) using receptor–ligand interactions between parasite and host surface, yet function of these to invasion is not known. We have analyzed binding one key merozoite ligand, called erythrocyte-binding antigen 175 (EBA175), glycophorin A on RBC surface (the most dominant antigen) explored how this interaction affects biophysical properties cell. Using a combination imaging techniques, we demonstrate...
Report26 April 2018Open Access Transparent process Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing Joseph Hawksworth School Biochemistry, University Bristol, UK Bristol Institute for Transfusion Sciences, National Health Service Blood and Transplant (NHSBT), Search more papers by this author Timothy J Satchwell NIHR Research Unit, Marjolein Meinders Deborah E Daniels Fiona Regan Imperial College Healthcare NHS Trust, London, &...
CRISPR-based DNA editing technologies enable rapid and accessible genome engineering of eukaryotic cells. However, the delivery genetically encoded CRISPR components remains challenging sustained Cas9 expression correlates with higher off-target activities, which can be reduced via Cas9-protein delivery. Here we demonstrate that baculovirus, alongside its cargo, used to package deliver proteins human Using protein-loaded baculovirus (pBV), or base editors proteins, leading efficient in By...
Enucleation is the step in erythroid terminal differentiation when nucleus expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell (FACS) to obtain pure populations of produced vitro culture. Nano LC mass spectrometry was first used determine distribution profile obtained purified reticulocyte extruded populations. In general...
Abstract The invasive blood-stage malaria parasite – the merozoite induces rapid morphological changes to target erythrocyte during entry. However, evidence for active molecular in host cell that accompany invasion is lacking. Here, we use inhibition assays, resealing and high-definition imaging explore red responses invasion. We show although entry does not involve actin reorganisation, it require ATP complete process. Towards dissecting requirement, present an depth quantitative...
Despite the identification of high-incidence red cell antigen Era nearly 40 years ago, molecular background this antigen, together with other 2 members Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing individuals serologically defined alloantibodies identified several missense mutations within PIEZO1 gene, encoding amino acid substitutions extracellular domain Piezo1 mechanosensor ion channel. Confirmation as carrier molecule for antigens was...
Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and apparent double plasma membrane. Despite ubiquitous expression SEC23B, the effects mutations in this are confined to erythroid lineage basis specificity remains be defined. In addition, little known regarding stage at which disparate...
The process of maturation reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss cell surface area and volume, removal remnant organelles redundant proteins, highly selective membrane cytoskeletal remodeling. However, mechanisms underlie drive these maturational processes vivo are currently poorly understood and, at present, derived through vitro culture fail undergo final transition erythrocytes. Here, we...
Background Protein 4.2 deficiency caused by mutations in the EPB42 gene results hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage erythropoiesis these hallmarks protein arise a novel patient whether they cause disruption band 3 macrocomplex.Design Methods used immunoprecipitations detergent extractability assess strength associations within macrocomplex cytoskeleton erythrocytes. Patient erythroblasts were cultured from...
Band 3 is the most abundant protein in erythrocyte membrane and forms core of a major multiprotein complex. The absence band human erythrocytes has only been reported once, homozygous Coimbra patient. We used vitro culture erythroblasts derived from this patient, separately short hairpin RNA-mediated depletion 3, to investigate development 3-deficient specifically assess stability retention dependent proteins during terminal erythroid differentiation. Further, using lentiviral transduction...
Abstract Investigating the role that host erythrocyte proteins play in malaria infection is hampered by genetic intractability of this anucleate cell. Here we report reticulocytes derived through vitro differentiation an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development parasite Plasmodium falciparum . Using CRISPR-mediated gene knockout subsequent complementation, validate essential for receptor basigin P....
Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain highly specialized erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced protein 4.2 expression. We use vitro differentiation of erythroblasts transduced shRNAs targeting ANK1 generate reticulocytes novel ankyrin-R 'near null' human phenotype less than 5% normal ankyrin Using this model, we demonstrate absence negatively impacts reticulocyte...
Abstract The Glucose transporter 1 (GLUT1) is one of the most abundant proteins within erythrocyte membrane and required for glucose dehydroascorbic acid (Vitamin C precursor) transport. It widely recognized as a key protein red cell structure, function, metabolism. Previous reports highlighted importance GLUT1 activity these uniquely glycolysis-dependent cells, in particular increasing antioxidant capacity needed to avoid irreversible damage from oxidative stress humans. However, studies...
Plasmodium falciparum invasion of the red blood cell is reliant upon essential interaction PfRh5 with host receptor protein basigin. Basigin exists as part one or more multiprotein complexes, most notably through monocarboxylate transporter MCT1. However, potential requirement for basigin association MCT1 and wider role membrane context lateral associations during merozoite has not been established. Using genetically manipulated in vitro derived reticulocytes, we demonstrate ability to...
The central role of the multifunctional protein nephrin within macromolecular complex forming glomerular slit diaphragm is well established, but mechanisms linking to cytoskeleton and signaling pathways involved in maintaining integrity filter remain incompletely understood. Here, we report that interacts with bicarbonate/chloride transporter kidney anion exchanger 1 (kAE1), detected by yeast two-hybrid assay confirmed immunoprecipitation co-localization studies. We low-level expression kAE1...