bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intron Retention Mechanisms That Regulate SF3B1 and Mitoferrin Gene Expression during Late Erythropoiesis
Authors: Parra Marilyn , Yeo Gene , Conboy John G. 
Published: November 2019

Abstract
Intron retention (IR) regulates hundreds of erythroid genes in a differentiation stage-specific manner during terminal erythropoiesis. Regulated genes include highly expressed RNA binding proteins (RBPs) such as SF3B1, as well as iron transporters (e.g., mitoferrins 1 and 2), and cytoskeletal proteins (e.g., alpha spectrin). Selected IR transcripts are relatively abundant; 25-50% of the above-mentioned transcripts can be polyadenylated, retained in the nucleus, and efficiently spliced at all but one or two introns, thus limiting the amount of translatable cytoplasmic mRNA. We are studying novel IR regulatory mechanisms involving both splice site strength and deeper intronic elements, using model introns that are differentially regulated in human erythroblasts. SF3B1, a key pre-RNA splicing factor implicated in MDS, exhibits dynamic regulation of intron 4, with low IR in proerythroblasts and high IR in mature orthochromatic erythroblasts. Intron 4 sequences include three ultraconserved elements that encode cryptic exons we term 4a, 4b, and 4c, two of which (4a and 4c) encode premature termination codons (PTCs) expected to induce nonsense-mediated decay (NMD) if spliced into SF3B1 transcripts. We hypothesize that these PTC exons can have an additional function as inefficiently spliced decoy(s), that is, their splice sites may interact with splice sites at the boundaries of intron 4 to form non-productive complexes that do not permit efficient splicing but instead prevent excision of the intron. This concept represents an extension of decoy models previously proposed by others to explain selected exon skipping events, and is supported by several recent findings: eCLIP (enhanced cross-linking and immunoprecipitation) data indicate strong binding of 3' splice site factors U2AF1 and U2AF2 at these exons; low levels of exon 4a and 4c splicing can be seen in NMD-inhibited cells; and deletion of exon 4c from minigene splicing reporters decreases IR. Besides SF3B1, we identified numerous other dynamically-regulated IR events encompassing cryptic PTC exons that bind 3' splice site factors. In contrast to the dynamic regulation of IR in SF3B1, IR in SLC25A37 (mitoferrin-1) and SLC25A28 (mitoferrin-2) is stably maintained at a high level throughout terminal erythropoiesis. Baseline IR levels for stably retained introns correlate with splice site strength, but are also influenced by deeper intronic elements. For example, SLC25A28 intron 2 contains intronic elements that appear to reduce IR, since blocking them with antisense morpholinos leads to substantially increased IR levels. These studies demonstrate that IR in major erythroid genes is regulated by sequences within the retained introns that can either increase or decrease retention, suggesting that multiple IR pathways are employed during terminal erythropoiesis to regulate gene expression.

Disclosures
No relevant conflicts of interest to declare.
Argonaute 2 Silencing of Band 3 in Early Reticulocytes
Authors: Evgenia Bloch M , Branch Donald R. 
Published: November 2019

Abstract
Post-transcriptional gene silencing by RNA-induced silencing complex (RISC) in erythropoiesis is a rapidly evolving field of study. Disruption of microRNA (miRNA) synthesis has been shown to hinder erythropoiesis and has been linked to disease. The presence of miRNA in circulating RBCs has recently been reported; however, clear understanding of their role and effectors has yet to be determined.
In this study, freshly drawn RBC were density separated on discontinuous percoll gradients. The top two fractions, fraction 1 (F1) representative of the top layer containing the youngest, reticulocyte-like cells, and a second fraction with greater density and non-reticulocyte properties (F2), were isolated. Cell populations were analyzed to compare RBC protein levels using western blotting (WB). qRT-PCR was used to evaluate band 3 RNA. Argonaute (AGO2) RNA immunoprecipitation (RIP), was used to ascertain RISC mediated transcriptional repression of band 3 using qRT-PCR.
Band 3 protein expression was elevated in the denser F2 compared to F1, in freshly drawn normal RBCs. RNA levels of band 3 complemented protein levels, as increased band 3 RNA was observed in F2. RIP experiments revealed elevated post-transcriptional control of band 3 through increased association of its RNA with AGO2 in F1 compared to F2 thereby, correlating with greatly reduced, sometimes undetectable protein levels of band 3 in F1.
We confirm the presence of AGO2 in circulating RBCs. We report, for the first time, the post-transcriptional control of band 3, an integral RBC transmembrane protein, in circulating reticulocyte-like cells. These results suggest a possible subpopulation of reticulocytes, a pro-reticulocyte, with ongoing control of band 3 expression following enucleation, and a potential role of miRNA-RISC in band 3 pathology.

Disclosures
No relevant conflicts of interest to declare.
MASL1 Is a Critical Modifier of Hepcidin Expression Via Smad1/5/8 Phosphorylation in the Bone Morphogenetic Protein (BMP) Signaling Pathway
Authors: Kumkhaek Chutima , LaChance Christian , Aerbajinai Wulin , Zhu Jianqiong , Rodgers Griffin P. 
Published: November 2019

Abstract
Iron is essential for hemoglobin synthesis during terminal erythropoiesis. Hepcidin is the main regulator of iron homeostasis and is repressed by erythropoiesis. Although several candidates have been proposed to act as hepcidin inhibitors and erythroid regulators such as growth differentiation factor 15 (GDF15), twisted gastrulation BMP signaling modulator 1 (TWSG1) or erythroferrone (ERFE), their role in hepcidin repression during erythropoiesis is still unclear. We previously demonstrated that malignant fibrous histiocytoma-amplified sequence 1 (MASL1) is important for terminal erythropoiesis. In addition, down-regulation of MASL1 expression in macrophages strongly enhances IL-6 production following LPS or poly IC stimulation. Therefore, we hypothesized that MASL1 could directly (or indirectly) influence hepcidin expression. We found that endogenous MASL1 expression was significantly decreased in CD34+ cells treated with IL-6 compared with EPO-treated CD34+ cells at day 3 (0.35±0.05 fold vs 2.34±0.13 fold, P=0.002) and day 7 (1.03±0.75 fold vs 205.31±10.83 fold, P=0.001) of differentiation. In contrast, endogenous hepcidin expression was markedly increased in CD34+ cells treated with IL-6 compared with EPO-treated CD34+ cells. Interestingly, an increased hepcidin expression was detected in MASL1-knockdown CD34+ cells at day 3 of EPO-induced differentiation when compared with mock (47.47±23.49 fold vs 2.10±2.46 fold, P=0.029) or control lentiviral vector (47.47±23.49 fold vs 2.54±1.29 fold, P=0.029). Of note, ERFE, GDF15 and TWSG1 expression were decreased in MASL1-knockdown CD34+ cells at day 3 of EPO-induced differentiation. In addition, endogenous MASL1 expression is down-regulation after LPS treatment in PMA-induced THP1 cells but IL-6 is enhanced in MASL1-knockdown-PMA-induced THP1 cells after LPS treatment. In human hepatic cells (Huh-7), we found a significant decrease in hepcidin expression in MASL1-overexpressed Huh-7 cells after BMP2 (5.85±0.32 fold vs 9.98±0.97 fold, P=0.028) or BMP6 (10.07±1.35 fold vs 20.3±0.75 fold, P=0.007) stimulation for 24 hrs. Moreover, up-regulation of MASL1 enhanced the phosphorylation of Erk1/2 proteins while inhibited the phosphorylation of Smad1/5/8 proteins in Huh-7 cells after BMP2 or BMP6 stimulation for 1 hr that consequently affect in down-regulation of hepcidin expression. Strikingly, MASL1 overexpressed-Huh7 cells showed moderately decreased nuclear localization of phospho-Smad1/5/8 after BMP2 or BMP6 treatment. Taken together, these data demonstrate that MASL1 is a critical modifier of hepcidin expression potentially via additional mechanisms related to erythropoiesis and body iron homeostasis. Further clarification of these pathways may be the useful in developing novel treatment of anemias or iron disorders.

Disclosures
No relevant conflicts of interest to declare.
An HPFH Mutation in the γ-Globin Gene Promoter of Humanized Cooley's Anemia Mice Rescues Them from Perinatal Lethality and Renders Them Transfusion Independent
Authors: Fontenard Suean Daimia , Huo Yongliang , Liu Shanrun , Lockhart Jonathan , Berlett Michael , Ryan Thomas M 
Published: November 2019

Abstract
Cooley's Anemia (CA), β-thalassemia major, is a genetic disease caused by an impairment in β-globin protein synthesis. The resulting excess in α-globin chains causes the premature destruction of erythroid cells (ineffective erythropoiesis), anemia, and if left untreated, death within the first years of life. Several mutations in the promoters of the fetal γ-globin genes have been identified which impair the silencing of the fetal genes in adulthood, a condition termed hereditary persistence of fetal hemoglobin (HPFH). The amount of fetal hemoglobin (Hb F) expressed and the distribution within the RBC population (pancellular vs heterocellular), varies widely with different HPFH mutations. It has also been observed that thalassemia patients who co-inherit HPFH mutations that express higher levels of Hb F have milder disease symptoms. The purpose of this study is to determine whether the incorporation of a non-deletional HPFH mutation into the promoter of the human g-globin gene in a humanized mouse model of CA can rescue the animals from their perinatal lethality. Heterozygous humanized HPFH -175 mice express pancellular, high-levels of Hb F into adulthood. Homozygous HPFH -175 CA mice are rescued from their perinatal lethality, surviving solely on 100% human Hb F and are transfusion independent for life. This result is significant because it demonstrates that introduction of an HPFH mutation into the γ-globin gene promoter by gene editing may be a viable therapeutic option for CA patients in the future.

Disclosures
No relevant conflicts of interest to declare.
Hemoglobin S Screening Using the « Sickle Scan » - Biomedomics System. the Necker-Enfants Malades Hospital Experience
Authors: MINE Louis , Nguyen-Khoa Thao , Allaf Birch , Ribeil Jean-Antoine , Remus Christelle , Gauthereau Valérie , Enouz Sarah , KIM Jason S. , Yang Xiaoxi , Gluckman Eliane , Munnich Arnold , Cavazzana Marina , Girot Robert 
Published: November 2019

Abstract

Context: The principle of Sickle Scan (Biomedomics, Inc.) is a rapid, point-of-care qualitative lateral flow immuno-assay kit for the identification of sickle cell conditions of Hb A, S and C. Sickle Scan was specifically developed to allow for the identification of sickle cell trait Hb AS, heterozygotes AC, and Hb SS, Hb SC and Sβ° patients. Other sickle cell conditions as SD, SE, SO-Arab, S Lepore,… cannot be identified using Sickle Scan system. The test must be done using venous blood or capillary blood (fresh or dried blood spots).
Patients and methods: Two hundred and fifty patient samples (143 adults and 107 newborns) were analyzed. All tests were performed according the manufacturer's recommendations in one laboratory by 2 observers. The reference tests used for comparison were HPLC (NBS Variant - Bio-Rad) and capillary electrophoresis (Capillarys 3 - Sebia).
Results:
Comments: In adult patients, the 2 observers concordantly detected the presence of Hb A, Hb S and Hb C. There were 4 differences of interpretation between them (no Hb A in a AS patient and no Hb A in 3 SS transfused patients). The percentage of Hb A in these 3 last patients was respectively 13.6%, 17.7% and 18%. There was no false positivity neither in the patient heterozygous AE nor in the patient SD. No false negativity occurred for Hb S and C. In newborns, the accuracy of the test was excellent for the identification of the phenotypes FA, FAS, FAC, FS (SS / Sβ°). The lowest detected values of Hb S and Hb C in FAS and FAC newborns were respectively 2.4 and 3.4 %. We observed an inconstant cross-reactivity of the antibody anti Hb S with the hemoglobins E and D, in respectively 3/25 FAE phenotype and 6/26 in the FAD phenotype. There was no cross reaction with hemoglobin Bart's and O-Arab.
In FAS newborns the mean and extreme values of the percentage of Hb A were m=8.2 (2.6-15.5) and no difficulties occurred for the identification of these low percentages of Hb A. This observation is different from those made in adult patients for which one observer did not find Hb A in transfused patients with highest values of Hb A comprised between 13.6 and 18.
Conclusions: In this series of adult and newborn patients, the Sickle Scan appeared as an accurate method for the identification of AS, AC, SS/Sβ° and SC phenotypes. False positive tests were observed in some patients with hemoglobin E or D but no false negative results were found as regards the identification of Hb S and Hb C.
Table Table.

Disclosures
Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.
Mathematical Modeling of White Blood Cell Population Dynamics for Diagnosis and Prognosis
Authors: Chaudhury Anwesha , Sohani Aliyah R , Higgins John M. 
Published: November 2019

Abstract
The complete blood count (CBC) is one of the most commonly ordered clinical tests because it provides a snapshot of the current state of the hematologic system in a patient, including an estimate of the number of currently circulating white blood cells (WBC). This simple assessment of patient WBC count is valuable in the screening and monitoring of a very wide range of conditions, from infection to malignancy to autoimmunity. Many routine WBC counts are measured by sophisticated automated hematology analyzers that rely on the principles of flow cytometry and characterize the morphology of thousands of individual WBCs in the process of providing a simple WBC count. These single-WBC measurements collected at high-throughput are usually discarded, but the probability distributions they represent provide a glimpse into the dynamics of the circulating WBC population, potentially enabling clinicians not only to estimate current numbers of circulating WBCs but also to infer whether the numbers of WBC subtypes (neutrophils, lymphocytes, and monocytes) are likely to increase or to decrease in the subsequent days and weeks.
We utilize this otherwise discarded single-WBC information in combination with semi-mechanistic mathematical models of neutrophil, lymphocyte, and monocyte population dynamics to infer patient-specific rates of neutrophil, lymphocyte, and monocyte flux (Figure 1). We characterize differences in average flux for patients with different classes of disease (infection, hematologic malignancy, and cardiac ischemia) and show how the data collected in a simple CBC and differential may be helpful in the early diagnosis and prognosis for a number of important diseases. For instance, we assess the diagnostic efficiency of inferred WBC dynamics to help identify (1) patients with initially negative cardiac troponins who are most likely to have subsequent positive levels, and (2) patients with newly discovered leukocytosis who are more likely to have an underlying malignant hematologic process. In a retrospective study of 73 patients with mild to moderate leukocytosis (11 x103/uL < WBC < 20 x103/uL) of unknown etiology, this approach showed promise in helping to identify patients who were diagnosed with a hematologic malignancy in the subsequent 6 months (Figure 2).
Figure 2: Nuclear maturation distributions of neutrophils (a) and lymphocytes (b) were derived from raw CBC measurements for patients found to have moderately elevated WBC counts (> 11x103/uL and < 20 x103/uL) before an underlying disease process had been identified. A statistically-significant fraction of patients with a subsequent new diagnosis of a hematologic malignancy could be identified based on either their single-neutrophil or single-lymphocyte characteristics at the time of the CBC up to 6 months prior to diagnosis, with multivariate discriminants showing even better performance.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Calcium Independent Events Trigger VWF-Dependent Shear-Induced Procoagulant Platelet Formation
Authors: Kholmukhamedov Andaleb , An Ann , Liu Fang , Janecke Rae , Jobe Shawn M 
Published: November 2019

Abstract
Elevated shear stress initiates platelet phosphatidylserine (PSer) externalization. Key intracellular mechanisms mediating the initiation of PSer externalization in apoptotic (Bax/Bak) and agonist-stimulated (sustained cytosolic calcium and cyclophilin D-mediated mPTP formation) platelets have been identified. In contrast, little is known about the intracellular processes mediating shear-induced procoagulant platelet (SiPP) formation.
To investigate the intracellular mechanisms regulating SiPP formation, either human or murine platelets were sheared on a plate-cone viscometer for 3 min at 10,000 s-1 in various conditions as indicated. Platelets were differentially labeled and evaluated by flow cytometry, and platelets with PSer externalization were defined as SiPPs.
In shear conditions engagement of VWF with GPIb-a induces unfolding of the membrane proximal GPIb-a mechanosensing domain. That this triggering mechanism is essential for SiPP formation was supported by the marked inhibition of SiPP formation in the absence of VWF, in VWF with deletion of the GPIbα-binding A1 domain, and in the presence of antibodies blocking the interaction of GPIb-IX and VWF. Calcium-dependent processes play an essential role in agonist-initiated PSer externalization. However, extracellular Ca2+ is not required for SiPP formation. Inhibition of Ca2+ entry, removal of extracellular Ca2+ and chelation of intracellular Ca2+, all had no effect on SiPP formation. Caspase activation mediates PSer externalization in the apoptotic platelet, but neither pharmacologic inhibition of caspases nor calpain inhibitors prevented SiPP formation. In contrast to the negligible roles of calcium- and caspase mediated events, SiPP formation is abrogated both in the absence of the mPTP potentiator cyclophilin D and in cyclosporine A-treated platelets. Furthermore, SiPP formation was closely correlated with mPTP formation as measured by loss of mitochondrial transmembrane potential. Together, these results identify a new mechanistic pathway of SiPP formation distinct from previously described initiating mechanisms for PSer externalization. Targeting of this distinct pathway may provide a novel pharmacologic strategy for the treatment of high shear hematopathologies, like arterial thrombosis.

Disclosures
No relevant conflicts of interest to declare.
Impaired GPVI Mediated Platelet Functional Responses and Signaling in CD45 Knockout Mice
Authors: Inamdar Vaishali , Kostyak John , Badolia Rachit , Dangelmaier Carol , Kim Soochong , Manne Bhanukanth , Kunapuli Satya P. 
Published: November 2019

Abstract
Background: CD45 is a Receptor Protein Tyrosine Phosphatase C (PTPRC) and regulates Src Family Kinases activation in Lymphocytes. Although it is known to be absent from the platelet surface, proteomics studies prove that the CD45 c-terminal catalytic domain is present in platelets. Thus the aim of this study is to identify presence of CD45 c-terminal domain in platelets and characterize the functional implications of CD45 deficiency in platelets using a global CD45 knockout mouse.
Results: Platelets from CD45-deficient mice displayed a selective impairment of aggregation and dense granule secretion mediated by the collagen receptor Glycoprotein VI. CD45 deficient mice show increased bleeding times, indicating an important role for CD45 in hemostasis. However, there was no difference observed in thrombus generation and thrombus stability using the ferric chloride-induced carotid artery injury model. Signaling downstream of the GPVI receptor, indicated by Src Family Kianse (SFK), Syk and Phospholipase C_2 (PLCg2) tyrosine phosphorylation, was also impaired. In order to establish the presence of CD45 in platelets we used an established primary antibody that recognizes the c-terminal domain of CD45. We observed that this antibody recognized a protein of approximately 65 kDa, which is the expected size of the c-terminal 1 and 2 domains of CD45, in wild type (WT) mice but not in knockout (KO)mice.
Conclusion: Thus we conclude that CD45 is expressed in platelets as a truncated form, possibly generated by proteolytic cleavage, and regulates GPVI signaling, through regulation of Src Family Kinase activation.
Figure Figure.

Disclosures
No relevant conflicts of interest to declare.
Selected Expression and Functional Importance of α4a-Tubulin in Platelet Biogenesis
Authors: Strassel Catherine , Hovasse Agnes , Moog Sylvie , Mageira Magda , Batzenschlager Morgane , Schaeffer-Reiss Christine , Kile Benjamin , Gachet Christian , Janke Carsten , Lanza Francois 
Published: November 2019

Abstract
Platelets are produced from mature megakaryocytes (MK) following a profound cellular reorganization. This includes the assembly of microtubules (MT) into a unique submenbranous coiled structure, the marginal band (MB). This process is thought to depend on a specific αβ-tubulin isotype repertoire. The MK-restricted-β1-tubulin, the predominant isoform of the MB, is already known to be important for platelet biogenesis but the implication of other isotypes is currently unknown.
Our goal was to establish the αβ-tubulin repertoire in platelets and during megakaryopoiesis and to evaluate the implication of selected isotypes in platelet formation.
To establish an exhaustive list of the tubulin isotypes, we used combination of RT PCR and proteomic analyses to quantify the expression of each isotype in human platelets and in human MK differentiated in culture from CD34+ hematopoietic progenitors. Information gained on the hierarchical combination of tubulin isoforms in the course of platelet biogenesis has been extended at the functional level to investigate both their role in marginal band formation and platelet functions
β6-, β5- and α1c-tubulin transcripts were already present in CD34+ cells and decreased during the final stages of megakaryopoiesis. On the other hand, β1-, α4A- and α8-tubulin transcripts were only observed later during MK differentiation and in platelets. Quantitative LC-SRM mass spectrometry confirmed the predominant expression of β1 and α4A-isotypes in platelets. A functional role of the newly identified α4a-tubulin was supported by the thrombocytopenia and enlarged platelets with a decreased number of MT coils (1-3) comprising less-acetylated tubulin in mice carrying a point mutation in tuba4a. Additionally, a tendency to increased responses to several agonists was observed in these platelets.
This study reveals new information on the evolution of the tubulin isotype repertoire in platelet formation pointing to a role of less-widely expressed α-isotypes.

Disclosures
No relevant conflicts of interest to declare.