bluuebunny/crossref_metadata_2025_split huggingface dataset:
Urinary Hepcidin in Thalassemic Syndromes.
Authors: Nemeth Elizabeta , Origa Raffaella , Ganz Tomas , Galanello Renzo 
Published: April 2020

Abstract
Hepcidin, a 25 amino-acid peptide hormone synthesized in the liver, is the key regulator of iron homeostasis. Hepcidin inhibits intestinal iron absorption, recycling of iron in the macrophages and mobilization of iron from hepatic stores. Hepcidin expression is induced by iron loading and inflammation and is suppressed by anemia and hypoxia, but the relative influences of these modifiers are not well understood. Thalassemia syndromes represent a clinical setting where hepcidin is regulated by opposing influences of ineffective erythropoiesis and elevated iron load. We evaluated urinary hepcidin levels in 10 thalassemia intermedia (TI) patients who had no or very few transfusions (less than 5, and all completed more than 15 years ago), and 11 thalassemia major (TM) patients who were regularly transfused and iron chelated. All patients had beta-zero thalassemia (beta 39C→G non-sense mutation). When compared to the unrelated controls, urinary hepcidin was decreased in TI and increased in TM [median (interquartile range) in ng hepcidin/mg creatinine: controls 44 (27–66); TI 6 (5–9); TM 218 (116–470); all comparisons p&lt;0.001 by One Way ANOVA with Dunn’s]. However, assessment of the hepcidin-to-ferritin ratio, an index of the appropriateness of hepcidin expression relative to the degree of iron loading, showed that the ratio was low in both thalassemia syndromes when compared to controls. The result suggests that even in TM patients, hepcidin is inappropriately low relative to the patients’ iron load. Importantly, in TM when measured over 1 week, hepcidin levels decreased in correlation with the patients’ rapidly decreasing Hb levels. In considering all the thalassemia patients together, urinary hepcidin levels correlated positively with serum ferritin and hemoglobin, and negatively with sTfR and serum erythropoetin. Multivariate analysis showed the strongest correlation with sTfR (r2=0.83).
The results indicate that in TI, the strong erythropoietic drive is the main regulator of hepcidin. The resulting hepcidin deficiency may be the cause of the increased iron absorption in TI. In TM, transfusions partially relieve the erythropoetic drive and increase the iron loading of macrophages thus raising hepcidin levels above those seen in TI. In the future, therapeutic use of hepcidin could restore normal iron homeostasis in some thalassemics, especially those not requiring transfusions.
Erythrocyte Adenylate Kinase Deficiency: First In-Depth Biochemical Characterization of the Y164C Mutant Enzyme Causing Hemolytic Anemia.
Authors: Abrusci Patrizia , Chiarelli Laurent R. , Fermo Elisa , Galizzi Alessandro , Zanella Alberto , Valentini Giovanna 
Published: April 2020

Abstract
Adenylate kinases (AK, EC 2.7.4.3) are monomeric enzymes that catalyze the reversible transfer of the γ-phosphate group from a phosphate donor (normally ATP) to a phosphate acceptor (normally AMP) releasing two molecules of nucleoside diphosphates (ADP). Therefore, the AK enzymes play a crucial role in the synthesis of nucleotides that are required for a variety of cellular metabolic processes. At present, six isoforms of AK with different tissue localization and substrate specificity have been characterized in man. AK1 is a cytosolic enzyme, mainly expressed in skeletal muscle, brain and erythrocytes. Adenylate kinase deficiency is a rare red blood cell enzymopathy causing hemolysis; the genetic disorder is transmitted as an autosomal recessive trait. To date 7 different mutations have been reported. To analyze the mutations at the protein level, we have undertaken the expression, purification and enzymological characterization of the human AK1 in the wild-type and Y164C mutant form. The cDNA encoding the AK1 was obtained from a blood sample of a healthy donor, with normal AK1 activity. Maximal expression (about 100–120 mg of AK1 per liter of E.coli culture) was obtained after 6 hours of induction with 0.5 mM IPTG at 37°C. The recombinant enzyme was purified to homogeneity following a simple procedure. The specific activity was greater than 2000 U/mg. AK1 exhibited a relatively high stability (T50 after 10 min of incubation, 53°C). Moreover, Mg-ATP protected enzyme activity from heat inactivation (T50 about 20 degrees higher). The catalytic efficiency values towards ATP and AMP were essentially identical and similar to that of the authentic enzyme. The Y164C mutation has been found in an Italian patient with congenital hemolytic anemia and undetectable erythrocyte AK1 activity. By site directed-mutagenesis we have produced the Y164C mutant protein and we have performed the first in-depth functional analysis of AK1 causing hemolytic anemia. The mutant enzyme essentially exhibited Km values unchanged, but drastic reductions of the catalytic efficiency towards both substrates (2 orders of magnitude). The Y164C mutant displayed little alterations in the thermostability properties. However, Mg-ATP failed to protect the enzyme activity from heat inactivation. Our data strongly suggest that the absence of AK1 activity observed in the patient homozygous for the Y164C mutation is due to inability of the enzyme to undergo conformational changes essential for the catalytic cycle.
The Allantois and Chorion, Isolated before Chorio-Allantoic Fusion, Have Hematopoietic Potential.
Authors: Zeigler Brandon M. , Daisuke Sugiyama , Guo Y. , Karen Downs M. , Nancy Speck A. 
Published: April 2020

Abstract
The chorio-allantoic placenta provides for the exchange of nutrients, gas, and waste between the fetus and mother. The murine placenta is also enriched for long term repopulating hematopoietic stem cells, and is therefore a stem cell niche (Gekas et al. Dev. Cell8, 365, 2005 and Ottersbach et al. Dev. Cell8, 377, 2005). However, it is not known if the placenta is colonized by hematopoietic stem cells that originate from other sites in the conceptus, or whether the placenta itself is also a site of hematopoietic cell emergence. The placenta is formed through the fusion of the allantois, a mesodermal extension from the primitive streak, with the chorionic plate, which is comprised of extraembryonic ectoderm and mesoderm. Here we show that the allantois and chorion, isolated before chorio-allantoic fusion and prior to the establishment of circulation, contain hematopoietic potential. Both allantois and chorion explants produced CD45 positive cells, some of which also expressed Mac-1 and Gr-1, when cultured on OP9 stromal cells in the presence of cytokines that promote myeloid cell differentiation. Cultures of allantois and chorion explants in the presence of lymphoid promoting cytokines induced B220 and CD19 positive B cells. Spatial and temporal analysis of Runx1, a marker for definitive hematopoiesis, showed Runx1 is not expressed in either the allantois or the chorion prior to fusion, but is expressed at the base of the allantois and at the chorio-allantoic junction following fusion. Both allantoic explant cultures, and cultures of whole embryos from which allantoises were removed in order to prohibit chorio-allantoic fusion showed that Runx1 expression in neither tissue is dependent upon fusion. We conclude that the allantois and chorion have intrinsic hematopoietic potential prior to the establishment of circulation. Our results furthermore suggest that the placenta is both a niche and a source of hematopoietic cells.
BMP4 Favors Hematopoietic Differentiation from Murine Embryonic Stem Cells and Acts by Activation of the cdx-hox Pathway.
Authors: Lengerke Claudia , Wang Yuan , Yates Frank , Maouche-Chretien Leila , Daley George Q. 
Published: April 2020

Abstract
Cdx4 and cdx1, members of the caudal family of homeodomain-containing transcriptional regulators, are important for specifying the hematopoietic fate of mesoderm in the zebrafish. We have shown that the cdx4 gene plays a role in enhancing hematopoietic fate during in vitro differentiation of murine ESCs (Davidson et al., Nature 2003). Cdx4 induces hox genes, and genetic modification of mESCs with a combination of cdx4 and hoxb4 promotes long-term engraftment of ESC-derived HSCs in lethally irradiated primary and secondary mice (Wang et al, submitted). While cdx1 is known to be a direct target of signaling by the embryonic morphogens fgf, wnt3a, and retinoids, morphogens acting upstream of cdx4 have not yet been defined. Our goal is to determine optimal morphogen conditions for hematopoietic commitment from murine embryonic stem cells by evaluating activation of the cdx-hox pathway. We have developed quantitative RT-PCR assays for the cdx genes (cdx4, cdx1 and cdx2) and multiple hox genes as well as markers specific to hematopoietic stem cells and lineages. We have used these assays, together with a reporter line engineered to express GFP from the brachury locus (Fehling et al., Development 2003), to characterize the conditions for mesodermal induction and hematopoietic fate specification following addition of morphogens to differentiating cultures of ES cells under serum-free conditions. Among all morphogens tested (BMP4, activin, nodal, wnt3a, wnt5a, sonic hedgehog, indian hedgehog, retinoic acid), only BMP4 has been found to strongly induce CDX4 gene expression within the developing embryoid bodies, while addition of the BMP4 inhibitor noggin to serum suppressed CDX4 expression. Addition of BMP4 significantly increases the number of emerging CD41+ and CD45+ cells, the precursors of definitive hematopoietic stem cells. We are currently analyzing the functional changes following BMP4 exposure, and correlating hematopoietic maturation with changes in the Hox gene expression pattern. Analysis of the cdx-hox gene pathway provides a means of otpimizing induction of hematopoietic fate by application of embryonic morphogens.
Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.
Authors: Rupon Jeremy W. , Wang ShouZhen , Gaensler Karin , Lloyd Joyce , Ginder Gordon D. 
Published: April 2020

Abstract
The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.
Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.
Authors: Olivier Emmanuel N. , Qiu Caihong , Bouhassira Eric E. 
Published: April 2020

Abstract
Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.
High Throughput Screen To Identify Small Molecules That Differentially Regulate Expression of the Globin Genes.
Authors: Ebert Benjamin L. , Mak Raymond , Pretz Jennifer L. , Peck David , Haggerty Stephen , Tolliday Nicola , Golub Todd R. 
Published: April 2020

Abstract
Several lines of evidence indicate that the pharmacological activation of fetal hemoglobin is an effective therapy for sickle cell anemia and beta thalassemia, but novel treatments for these diseases are needed. We developed and validated a high throughput assay to detect differential regulation of the globin genes and utilized this assay in a small molecule screen to identify novel compounds that increase the relative expression of gamma globin. In our assay, transcripts for the alpha, beta, delta, epsilon, gamma, theta, and zeta globin genes are amplified by multiplexed ligation-mediated PCR. Labeled amplicons are captured on different fluorescent microspheres using molecular barcodes, and the relative abundance of labeled amplicons is detected by high speed flow cytometry. To recapitulate the activity of compounds in the bone marrow of patients as accurately as possible, the screen was performed using primary human erythroid progenitor cells cultured in vitro. The assay was adapted to 384-well format with robotic liquid handling. In validation studies, the assay detected the expected increases in globin gene expression during erythroid differentiation, increased gamma globin expression in umbilical cord blood progenitor cells, and increased gamma globin expression in cells treated with known inducers of fetal hemoglobin including hydroxyurea and sodium butyrate. We screened a library of 1040 known bioactive compounds, 75% of which are FDA approved drugs, and a library of 600 compounds produced by diversity oriented synthesis that have been shown to inhibit histone deacetylase (HDAC) activity. In the screen, we rediscovered previously identified globin gene regulators, further validating our globin assay. For example, corticosteroids, known activators of fetal hemoglobin, increased the relative expression of gamma globin. Thyroid hormone specifically increased expression of delta globin, consistent with clinical observations that hemoglobin A2 levels are increased in hyperthyroidism and decreased in hypothyroidism. We identified ten novel compounds from the diversity oriented synthesis library that powerfully induce expression of the gamma globin gene relative to beta globin. Moreover, HDAC inhibition reversed the ontogeny of globin gene expression, coordinately increasing expression of fetal and embryonic relative to the adult globin genes. Relative to beta globin gene expression, gamma and epsilon globin were induced while delta globin was unaffected by HDAC inhibitors; relative to alpha globin expression, zeta globin was increased and theta globin was unaffected. The identification of compounds that differentially regulate globin gene expression may provide lead compounds for the development of novel therapies for sickle cell disease and beta thalassemia and may help elucidate the molecular events underlying switching of the globin genes during normal development.
Genomic Organization, Chromatin Structure, and Transcriptional Regulation of the Murine Alpha Hemoglobin Stabilizing Protein (AHSP) Gene.
Authors: Dore Louis C. , Vakoc Christopher R. , Blobel Gerd A. , Hardison Ross C. , Bodine David M. , Gallagher Patrick G. , Weiss Mitchell J. 
Published: April 2020

Abstract
Alpha Hemoglobin Stabilizing Protein (AHSP, Eraf) is an abundant erythroid protein that binds and stabilizes alpha globin and alpha hemoglobin (Hb). In mice, loss of AHSP causes hemolytic anemia, with elevated levels of reactive oxygen species and Hb precipitation in erythrocytes. Loss of AHSP exacerbates beta thalassemia phenotypes in mice, presumably by enhancing the toxicity of excessive free alpha Hb. Based on these findings, AHSP is a candidate modifier gene for beta thalassemia in humans. No mutations in the AHSP coding region have been identified in patients to date. However, several groups reported an inverse correlation between beta thalassemia severity and erythroid AHSP expression levels, raising the possibility that AHSP is a quantitative trait modifier of beta thalassemia. To address this possibility, it is important to define the mechanisms that control expression of the AHSP gene. Transcripts of murine Ahsp are inducible by GATA-1. The goals of the current studies are to investigate the mechanisms of this induction and to define the DNA domain that regulates the locus. Using phylogenetic comparisons, we identified a hotspot for mammalian chromosomal rearrangement just downstream of the Ahsp gene. This hotspot is located at the end of a syntenic block of approximately 350 kb that is conserved in mammals and likely marks the 3′ end of the gene regulatory domain. We focused our initial functional studies on a 7 kb genomic region bounded at the 5′ (centromeric) end of Ahsp by the nearest adjacent gene, an EST expressed in multiple tissues, and at the 3′ (telomeric) end by the rearrangement hotspot. In transient transfection assays, the Ahsp promoter region conferred erythroid-specific expression to a linked reporter gene. In heterologous cells, GATA-1 transactivated the Ahsp promoter in a dose-dependent fashion. To examine GATA-1 binding and its subsequent effects on the Ahsp gene in vivo, we used G1E-ER4 cells, a GATA-1 null erythroblast line that undergoes terminal erythroid maturation after activation of an estradiol-inducible form of GATA-1. We made several findings with regards to the role of GATA-1 in Ahsp gene regulation. First, GATA-1 and its cofactor, Friend of GATA-1 (FOG-1), bind directly to the Ahsp locus at regions that contain conserved GATA consensus motifs and are predicted to be important erythroid regulatory elements by our bioinformatic studies. Second, GATA-1 induces epigenetic changes in chromatin structure that are associated with gene activation, including formation of a DNase I hypersensitive site, hyperacetylation of histones H3 and H4, and methylation of histone H3 lysine-4. Together, these findings begin to establish the DNA region and mechanisms that control Ahsp transcription, allowing for further studies to map the cis elements responsible for population variations in gene expression.