PUM1 Mediates the Posttranscriptional Regulation of Human Fetal Hemoglobin

The fetal-to-adult hemoglobin switching at about the time of birth involves a shift in expression from γ-globin to β-globin in erythroid cells. Effective re-expression of fetal γ-globin can ameliorate sickle cell anemia and β-thalassemia. Despite the physiological and clinical relevance of this switch, its posttranscriptional regulation is poorly understood. Here, we identify Pumilo 1 (PUM1), an RNA-binding protein with no previously reported functions in erythropoiesis, as a direct posttranscriptional regulator of β-globin switching. PUM1, whose expression is regulated by the erythroid master transcription factor erythroid Krüppel-like factor (EKLF/KLF1), peaks during erythroid differentiation, binds γ-globin messenger RNA (mRNA), and reduces γ-globin (HBG1) mRNA stability and translational efficiency, which culminates in reduced γ-globin protein levels. Knockdown of PUM1 leads to a robust increase in fetal hemoglobin (∼22% HbF) without affecting β-globin levels in human erythroid cells. Importantly, targeting PUM1 does not limit the progression of erythropoiesis, which provides a potentially safe and effective treatment strategy for sickle cell anemia and β-thalassemia. In support of this idea, we report elevated levels of HbF in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain (p.(His1090Profs∗16); c.3267_3270delTCAC), which suggests that PUM1-mediated posttranscriptional regulation is a critical player during human hemoglobin switching

Multispectral Imaging for Microchip Electrophoresis Enables Point-of-Care Newborn Hemoglobin Variant Screening

Hemoglobin (Hb) disorders affect nearly 7% of the world\u27s population. Globally, around 400,000 babies are born annually with sickle cell disease (SCD), primarily in sub-Saharan Africa where morbidity and mortality rates are high. Screening, early diagnosis, and monitoring are not widely accessible due to technical challenges and cost. We hypothesized that multispectral imaging will allow sensitive hemoglobin variant identification in existing affordable paper-based Hb electrophoresis. To test this hypothesis, we developed the first integrated point-of-care multispectral Hb variant test: Gazelle-Multispectral. Here, we evaluated the accuracy of Gazelle-Multispectral for Hb variant newborn screening in 265 newborns with known hemoglobin variants including hemoglobin A (Hb A), hemoglobin F (Hb F), hemoglobin S (Hb S) and hemoglobin C (Hb C). Gazelle-Multispectral detected levels of Hb A, Hb F, Hb S, and Hb C/E/A2, demonstrated high correlations with the results reported by laboratory gold standard high performance liquid chromatography (HPLC) at Pearson Correlation Coefficient = 0.97, 0.97, 0.93, and 0.95. Gazelle-Multispectral demonstrated accuracy of 96.8% in subjects of 0–3 days, and 96.9% in newborns. The ability to obtain accurate results on newborn samples suggest that Gazelle-Multispectral can be suitable for large-scale newborn screening and for diagnosis of SCD in low resource settings

Catch Bonds in Sickle Cell Disease: Shear-Enhanced Adhesion of Red Blood Cells to Laminin

Could the phenomenon of catch bonding—force-strengthened cellular adhesion—play a role in sickle cell disease, where abnormal red blood cell (RBC) adhesion obstructs blood flow? Here we investigate the dynamics of sickle RBCs adhering to a surface functionalized with the protein laminin (a component of the extracellular matrix around blood vessels) under physiologically relevant micro-scale flow. First, using total internal reflectance microscopy we characterize the spatial fluctuations of the RBC membrane above the laminin surface before detachment. The complex dynamics we observe suggest the possibility of catch bonding, where the mean detachment time of the cell from the surface initially increases to a maximum and then decreases as a function of shear force. We next conduct a series of shear-induced detachment experiments on blood samples from 25 sickle cell disease patients, quantifying the number and duration of adhered cells under both sudden force jumps and linear force ramps. The experiments reveal that a subset of patients does indeed exhibit catch bonding. By fitting the data to a theoretical model of the bond dynamics, we can extract the mean bond lifetime versus force for each patient. The results show a striking heterogeneity among patients, both in terms of the qualitative behavior (whether or not there is catch bonding) and in the magnitudes of the lifetimes. Patients with large bond lifetimes at physiological forces are more likely to have certain adverse clinical features, like a diagnosis of pulmonary arterial hypertension and intracardiac shunts. By introducing an in vitro platform for fully characterizing RBC-laminin adhesion dynamics, our approach could contribute to the development of patient-specific anti-adhesive therapies for sickle cell disease. The experimental setup is also easily generalizable to studying adhesion dynamics in other cell types, for example leukocytes or cancer cells, and can incorporate disease-relevant environmental conditions like oxygen deprivation

OcclusionChip: A Functional Microcapillary Occlusion Assay Complementary to Ektacytometry for Detection of Small-Fraction Red Blood Cells with Abnormal Deformability

Red blood cell (RBC) deformability is a valuable hemorheological biomarker that can be used to assess the clinical status and response to therapy of individuals with sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, which uses shear or osmolar stress. However, ektacytometry is a population based measurement that does not detect small-fractions of abnormal RBCs. A single cell-based, functional RBC deformability assay would complement ektacytometry and provide additional information. Here, we tested the relative merits of the OcclusionChip, which measures RBC deformability by microcapillary occlusion, and ektacytometry. We tested samples containing glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in Occlusion Index (OI). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions. Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT. These results suggest that the single cell-based OcclusionChip enables detection of sickle hemoglobin (HbS)-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population based ektacytometry assays, and providing additional sensitivity and capacity to detect modest abnormalities in red cell function or small populations of abnormal red cells

Sickle Red Blood Cell-Derived Extracellular Vesicles Activate Endothelial Cells and Enhance Sickle Red Cell Adhesion Mediated by von Willebrand Factor

Endothelial activation and sickle red blood cell (RBC) adhesion are central to the pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles (REVs) are more abundant from SS RBCs compared with healthy RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to promote endothelial cell (EC) activation through cell signalling and transcriptional regulation at longer terms. However, the SS REV-mediated short-term non-transcriptional response of EC is unclear. Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 h. Compared with AA REVs, SS REVs promoted human pulmonary microvascular ECs (HPMEC) activation indicated by increased von Willebrand factor (VWF) expression. Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs exposed to SS REVs. This enhanced SS RBC adhesion was reduced by haeme binding protein haemopexin or VWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs. Consistent with these observations, haemin- or SS REV-induced microvascular stasis in SS mice with implanted dorsal skin-fold chambers that was inhibited by ADAMTS13. The adhesion induced by SS REVs was variable and was higher with SS RBCs from patients with increased markers of haemolysis (lactate dehydrogenase and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Our results emphasise the critical contribution made by REVs to the pathophysiology of SCD by triggering acute microvascular EC activation and abnormal RBC adhesion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using VWF as a potential target

Antithrombin-III Mitigates Thrombin-Mediated Endothelial Cell Contraction and Sickle Red Blood Cell Adhesion in Microscale Flow

Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin-III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient-derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin-activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule-1 (VCAM-1) mediated by thrombin. Our findings suggest that, by attenuating thrombin-mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD

Anticipatory Burden in Adult-Child Caregivers: A Concept Analysis

This study aims to analyze the concept of anticipatory burden in adult-child caregivers. A systematic literature review was performed using four databases, Pubmed, CINAHL, PsycINFO and Medline, with the keywords of “anticipatory burden” and “anticipated burden”. Simplified Wilson’s classic concept analysis modified by Walker and Avant was employed to identify the attributes, antecedents and consequences of anticipatory burden in the adult-child caregivers. Eighteen articles were analyzed. Attributes of anticipatory burden in adult-child caregivers were found to be: (1) subjective burden, (2) anticipation, (3) overestimation, (4) inability, and (5) family relationship. Antecedents were identified as: (1) potential care recipients, (2) caregiving willingness, and (3) a lack of resources. Consequences included: (1) prediction of caregiving willingness, (2) impacts on caregivers’ health, (3) intervention promotion, and (4) behavioral changes. As the adult-child caregiver is one of the main types of family caregivers for the fast-growing aging population, it is important to understand the attributes, antecedents, and consequences of their anticipatory burden. Based on the results of this study, resources such as intervention, policy, and counseling services are recommended to help adult-child caregivers lower their anticipatory burden and get better prepared for providing family care

Analysis and optimization of electrochemiluminescence periodic signal processing based on singular value decomposition

Electrochemiluminescence (ECL) detection is one of the most prevailing electrochromism approaches to test biotin and chemicals. As thorny ECL signals by impurities are devastative in judging the right substance and its concentration precisely, it is crucial to dispose the noise and process it into smooth curves without filtering essential biochemical information conveyed in the signal. This contribution investigates Singular Value Ratio (SVR) spectrum to extract periodic signals from every noise ECL signal. A novel improved method of rearranging periodic compositions from complex signals is proposed. Finally, actual ECL signal generated by Ru(bpy)32+ is analyzed and optimized to demonstrate that the improved technique is efficacious and feasible. Keywords: Electrochemiluminescence (ECL), Periodic signal extraction, Singular value decomposition (SVD), Singular value ratio (SVR

Red Blood Cell Adhesion to ICAM-1 is Mediated by Fibrinogen and is Associated with Right-to-Left Shunts in Sickle Cell Disease

Sickle cell disease (SCD), which afflicts 100 000 Americans, as well as millions worldwide, is associated with anemia, lifelong morbidity, and early mortality. Abnormal adhesion of sickle red blood cells (RBCs) to activated vascular endothelium may contribute acutely to the initiation of painful vaso-occlusive crises and chronically to endothelial damage in SCD. Sickle RBCs adhere to activated endothelium through several adhesion mechanisms. In this study, using whole blood from 17 people with heterozygous SCD (HbS variant) and 55 people with homozygous SCD (HbSS) analyzed in an in vitro microfluidic assay, we present evidence for the adhesion of sickle RBCs to immobilized recombinant intercellular adhesion molecule 1 (ICAM-1). We show that sickle RBC adhesion to ICAM-1 in vitro is associated with evidence of hemolysis in vivo, marked by elevated lactate dehydrogenase levels, reticulocytosis, and lower fetal hemoglobin levels. Further, RBC adhesion to ICAM-1 correlates with a history of intracardiac or intrapulmonary right-to-left shunts. Studies of potential ICAM-1 ligands on RBC membranes revealed that RBC–ICAM-1 interactions were mediated by fibrinogen bound to the RBC membrane. We describe, for the first time, RBC rolling behavior on ICAM-1 under high shear rates. Our results suggest that firm adhesion of sickle RBCs to ICAM-1 most likely occurs in postcapillary venules at low physiological shear rates, which is facilitated by initial rolling in high shear regions (eg, capillaries). Inhibition of RBC and ICAM-1 interactions may constitute a novel therapeutic target in SCD