Biology subjects

Stephen, L.

Publications and source records attributed to Stephen, L..

5 recordsLinked to original sources

bioRxiv2026

Investigating the significance of iron levels in influencing megakaryocytic commitment in megakaryocyte-erythroid progenitors

AimThe present study investigated the significance of iron in regulating megakaryopoiesis, by a diet-based intervention in an in-vivo model. MethodsMale C57BL/6 mice, aged 4-5 weeks were fed on varying iron diets. Following sacrifice, blood samples collected in EDTA tubes were used to analyse haematological parameters, and iron content of liver and spleen was assessed by biochemical analyses. Megakaryocyte-erythroid progenitors (MEPs) were isolated from bone marrow by magnetic bead-based selection. RNA isolated from bone marrow cells and MEPs were used for gene expression analyses, and RNA Sequencing to identify differentially expressed genes (DEGs) and associated pathways. ResultsMice fed on an iron-deficient diet had reduced hepatic iron content after 5 weeks (p < 0.01), while both the hepatic and spleen iron content increased after 3 weeks in mice on an iron-rich diet (p < 0.05) and developed iron overloading. Hb and RBC counts increased (p < 0.05) in iron-rich mice and decreased in iron-deficient mice (p < 0.05), which also showed elevated platelet counts (p < 0.01). This may be explained by increased expression of Gata1, Tal1 (p < 0.01) Mds1 and Pdpk1 (p < 0.05) in bone marrow cells from iron-deficient mice. MEPs isolated from these mice showed elevated expression of genes associated with megakaryocytic differentiation, platelet functions, and genes encoding TGF-{beta}R1 and Smad 2,3 and 4. ConclusionsIron deficiency may activate TGF-{beta} signalling and downstream Smad-mediated transcriptional programs within MEPs. This may promote a shift in lineage commitment towards megakaryopoiesis through elevated expression of megakaryopoiesis related genes.

molecular biology↗

bioRxiv2026

CD31+ T-cells express greater VEGF-A and CXCR4 levels than CD31- counterparts with VEGF-A expression exacerbated with advancing age

CD31+ T-cells reportedly possess angiogenic properties. These cells have recently been termed angiogenic T-cells (TANG). Advancing age is associated with altered circulating T-cell phenotypes, including TANG, and reduced angiogenesis. We examined various TANG subsets (CD3+, CD4+, CD8+), and their VEGF-A intracellular content in young (n=16, 18-30 years) and older (n=16, 50-65 years) male adults using flow cytometry. Cardiorespiratory fitness ([V]O2max) was quantified in all participants using a graded cycling ergometry test to volitional exhaustion. Resting blood samples were collected to measure circulating IL-6 and cytomegalovirus serostatus. CD31+ T-cells (TANG) contained more VEGF-A than CD31- T-cells (CD31+: 9374 {+/-} 8587 AU vs CD31-: 8722 {+/-} 8149 AU, p = 0.021) which was also exhibited in CD4+ and CD8+ subsets. Older adults possessed fewer CD4+ TANG cells as a proportion of total CD4+ T-cells than younger adults (young: 35 {+/-} 11%; older: 24 {+/-} 9%, p = 0.004), and CD3+ and CD4+ TANG subsets from older adults exhibited higher VEGF-A levels than younger adults (CD3+CD31+: young: 6081 {+/-} 4001 AU; older: 13426 {+/-} 10945 AU, p = 0.019; CD4+CD31+: young: 6373 {+/-} 3972 AU; older: 15660 {+/-} 12829 AU, p = 0.011). TANG cells were not associated with circulating IL-6, and TANG VEGF-A content was not associated with[V] O2max. Advancing age is associated with a pathological TANG phenotype, which may contribute to age-related inflammation and warrants further investigation as a potential therapeutic target.

immunology↗

bioRxiv2026

TNAP and PHOSPHO1 function synergistically to afford critical control over the mineralisation of the postnatal murine skeleton

Biomineralisation is essential for skeletal integrity, yet the synergistic roles of tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 in postnatal bone mineralisation remain poorly defined. To decipher this, we generated a novel murine model in which Alpl was deleted in Prx1- expressing cells (AlplPrx1/Prx1) in mice with a global Phospho1-/- deficiency to overcome the perinatal lethality that arises upon dual global deletion. Using a multi-modal approach to spatially phenotype the limbs of these animals, we reveal mice lacking both TNAP and PHOSPHO1 exhibit a distinct lack of mineralisation and altered anatomical structure at postnatal day 1 (PN1) and 3-weeks of age. Although viable, these mice did not thrive due to their reduced size, thus further investigations were conducted on mice with a heterozygous deletion of TNAP (Alplwt/Prx1;Phospho1-/-). Although smaller than wild-types at PN1 and 3 weeks old, these mice did not display the gross limb deformations observed in the homozygous animals and the single, functioning Alpl allele rescued the loss of biomineralisation observed following dual phosphatase deletion. At 6-weeks of age, compromised epiphyses and metaphyses were only seen in AlplPrx1/Prx1 animals. Further, we found that tibial geometry and porosity was significantly altered by Phospho1 deletion (Phospho1-/-), which was compounded in the Alplwt/Prx1;Phospho1-/- mice and linked to alterations in collagen configuration, matrix mineralisation and growth plate deformities. Together, our findings establish the mechanistic framework for TNAP and PHOSPHO1 in permissive biomineralisation, providing critical insights into this fundamental process. Significance StatementBiomineralisation is essential for skeletal development and is critically dependent on phosphatases that release inorganic phosphate for hydroxyapatite formation. Our study investigates the dual role of PHOSPHO1 and TNAP in this process, using a novel murine knockout model. Deletion of both enzymes results in complete loss of bone mineralisation, demonstrating their critical synergistic function. Further, we show that PHOSPHO1 and TNAP exhibit distinct, spatially-restricted functions in the tibia and thus enhances our understanding of the fundamentals processes underpinning biomineralisation. These findings also have clinical relevance as they have the potential to inform on treatment strategies for hypo- and hyper-mineralised pathologies.

developmental biology↗

bioRxiv2024

Exploring the link between extended red blood cell parameters and platelet indices in voluntary blood donors

BackgroundAs regular blood donors are prone to iron deficiency, importance of extended red blood cell (eRBC) parameters in identifying donors with depleted iron stores was investigated. Thrombocytosis has been well documented in patients affected with IDA. Thus, significance of eRBC parameters in identifying iron deficiency associated thrombocytosis was also examined in this cohort. MethodsBlood samples were collected in EDTA tubes from consenting donors for analyses of routine haematological and eRBC parameters. Serum samples were isolated for estimation of iron parameters. ResultsIron deficient donors had significantly altered eRBC parameters. Among them, Ret-He with a cut-off of [≥]32 pg had high AUC (0.822) and showed relatively high sensitivity & specificity in detecting iron deficiency. Combination of Ret-He with CCI increased sensitivity & specificity to 90.6% and 98.2%, in detection of donors affected with iron restricted erythropoiesis. This cohort had increased platelet counts, which showed significant association with Ret-He ({beta}= -0.373), RBC-He ({beta}= -0.384), CCI ({beta}= 0.384), Hypo-He ({beta}=0.494) and Micro-R ({beta}= 0.299). Elevated platelet counts also showed significant correlations with these eRBC parameters, which was absent in iron replete donors. ConclusionseRBC parameters are sensitive indicators of non-anaemic iron deficiency, which may be enhanced by combining them. Their significant association with elevated platelet counts in iron deficient donors, highlights their importance in reflecting iron deficiency associated thrombocytosis. Impact StatementThe present study discusses utility of eRBC parameters in detecting non-anaemic iron deficiency, in regular voluntary blood donors. Previous reports have investigated the importance of these parameters in identifying IDA in blood donors. The present study is the first one which indicates combining different eRBC parameters such as Ret-He and CCI increases their accuracy of detection. They were also significantly associated with elevated platelet counts in iron deficient donors, which was absent in iron replete individuals. This link between eRBC parameters and higher platelet counts in healthy donors affected with non-anaemic iron deficiency has not been reported before.

bioinformatics↗

bioRxiv2024

Investigating the influence of iron parameters in regulating platelet recovery in Apheresis Platelet Donors

IntroductionThe present study investigates changes in haematological parameters in the donor during apheresis and following donation. Effect of iron parameters in influencing platelet recovery was studied. MethodsBlood samples were collected in EDTA tubes for analyses of haematological parameters. Serum samples were isolated for estimation of iron parameters. ResultsHb, RBC count and Hct increased during apheresis, but no significant changes were observed after 48 hrs post donation. Platelet recovery rates increased from 78 % after 48 hours to > 100% on day 10 (p < 0.001). Ferritin, serum iron & Transferrin saturation initially increased and subsequently declined from day 7 post donation, which may influence platelet recovery. Recovery rates were also higher in donors with lower iron stores. ConclusionsChanges in haematological parameters during plateletpheresis are transient and optimal time required for platelet recovery is dependent on pre-donation counts. Recovery of platelet counts may be positively regulated by donor iron parameters.

molecular biology↗