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Hemoglobin E (HbE), a common variant in Southeast Asian populations, results from a G to A substitution at codon 26 of the HBB gene, causing abnormal Hb and mild β-thalassemia-like symptoms. Here, we derived an induced pluripotent stem cell (iPSC) line, named MUi033-A, from a male homozygous for HbE. The iPSC line demonstrates a normal karyotype and embryonic stem cell-like properties including pluripotency gene expression, and tri-lineage differentiation potential. This iPSC resource holds the potential for investigating gene therapy targeting HbE mutation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hemoglobin Constant Spring (HbCS) is unstable hemoglobin resulting from a nucleotide substitution at the termination codon of the HBA2 gene (c.427 T > C). The homozygous state for HbCS is non-transfusion dependent in adults. Nevertheless, severe anemia is often observed in fetuses. Here, human induced pluripotent stem cell line MUi034-A was generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) derived from a 14-year-old female with homozygous HbCS who had a history of severe anemia and hydrops during fetal period. The MUi034-A cell line represented embryonic-like characteristics as they expressed specific pluripotency markers, differentiated into the three germ layers, and retained normal karyotyping., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: This study aimed to investigate the changes in gut microbiota in iron-overload thalassemia and the roles of an iron chelator on gut dysbiosis/inflammation, and metabolites, including short-chain fatty acids (SCFAs) and trimethylamine N-oxide (TMAO)., Main Methods: Adult male C57BL/6 mice both wild-type (WT: n = 15) and heterozygous β-thalassemia (BKO: n = 15) were fed on either a normal (ND: n = 5/group) or a high‑iron diet for four months (HFe: n = 10/group). HFe-treated WT and HFe-treated BKO groups were further subdivided into two subgroups and each subgroup given either vehicle (n = 5/subgroup) or deferiprone (n = 5/subgroup) during the last month. Gut microbiota profiles, gut barrier characteristics, levels of proinflammatory cytokines, and plasma SCFAs and TMAO were determined at the end of the study., Key Findings: HFe-fed WT mice showed distinct gut microbiota profiles from those of ND-fed WT mice, whereas HFe-fed BKO mice showed slightly different gut microbiota profiles from ND-fed BKO. Gut inflammation and barrier disruption were found only in HFe-fed BKO mice, however, an increase in plasma TMAO levels and decreased levels of SCFAs were observed in both WT and BKO mice with HFe-feeding. Treatment with deferiprone, gut dysbiosis and disturbance of metabolites were attenuated in HFe-fed WT mice, but not in HFe-fed BKO mice. Increased Verrucomicrobia and Ruminococcaceae were associated with the beneficial effects of deferiprone., Significance: Iron-overload leads to gut dysbiosis/inflammation and disturbance of metabolites, and deferiprone alleviates those conditions more effectively in WT than in those that are thalassemic., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Microorganisms produce siderophores in order to scavenge iron from the environment and this study focuses on the characterization of the two siderophores secreted by Bacillus megaterium. The general biological properties and pharmacokinetics following oral application of these compounds are reported., Methods: Under optimized culture conditions, the siderophores were harvested, purified by chromatography and identified using LC-MS and NMR. Two dihydroxamate siderophores were isolated, schizokinen (MW = 420) and schizokinen imide (MW = 402)., Results: Both compounds demonstrate strong antioxidant activity and were found to be relatively nontoxic to both human hepatocellular carcinoma (Huh7) and peripheral blood mononuclear cells. The siderophores possess a strong affinity for iron(III) and decrease the levels of the labile iron pool (LIP) in iron-loaded cells in a concentration-dependent manner. Schizokinen, was detected as both the free siderophore and the iron complex in the plasma and urine of rats after oral gavage., Conclusions: However, the bioavailability was low and thus schizokinen, like deferoxamine, has no potential as an orally active iron chelator for the treatment of systemic iron overload., General Significance: By virtue of the high affinity of schizokinen for tribasic metals, this siderophore does have considerable potential for the chelation of gallium(III) and the development of clinical diagnostic reagents., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: We previously demonstrated that iron-overload in non-thalassemic rats induced neurotoxicity and cognitive decline. However, the effect of iron-overload on the brain of thalassemic condition has never been investigated. An iron chelator (deferiprone) provides neuroprotective effects against metal toxicity. Furthermore, a T-type calcium channels blocker (efonidipine) effectively attenuates cardiac dysfunction in thalassemic mice with iron-overload. However, the effects of both drugs on brain of iron-overload thalassemia has not been determined. We hypothesize that iron-overload induces neurotoxicity in Thalassemic and wild-type mice, and not only deferiprone, but also efonidipine, provides neuroprotection against iron-overload condition., Main Methods: Mice from both wild-type (WT) and β-thalassemic type (HT) groups were assigned to be fed with a standard-diet or high-iron diet containing 0.2% ferrocene/kg of diet (HFe) for 4 months consecutively. After three months of HFe, 75-mg/kg/d deferiprone or 4-mg/kg/d efonidipine were administered to the HFe-fed WT and HT mice for 1 month., Key Findings: HFe consumption caused an equal impact on circulating iron-overload, oxidative stress, and inflammation in WT and HT mice. Brain iron-overload and iron-mediated neurotoxicity, such as oxidative stress, inflammation, glial activation, mitochondrial dysfunction, and Alzheimer's like pathologies, were observed to an equal degree in HFe fed WT and HT mice. These pathological conditions were mitigated by both deferiprone and efonidipine., Significance: These findings indicate that iron-overload itself caused neurotoxicity, and T-type calcium channels may play a role in this condition., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Inhaled sodium nitrite has been reported to decrease pulmonary artery pressure in hemoglobin E/β-thalassemia (HbE/β-thal) patients with pulmonary hypertension. This study investigated the pharmacokinetics and pharmacodynamics of inhaled nebulized sodium nitrite in 10 healthy subjects and 8 HbE/β-thal patients with high estimated pulmonary artery pressure. Nitrite pharmacokinetics, fraction exhaled nitric oxide (FE NO ), estimated right ventricular systolic pressure (eRVSP) measured by echocardiography, and platelet activation were determined. Nebulized sodium nitrite at doses used in this study (37.5 and 75 mg for healthy subjects and 15 mg for HbE/β-thal patients) was well tolerated and did not cause changes in methemoglobin levels and systemic blood pressure. Absorption of inhaled nitrite was rapid with the absolute bioavailability of 18%. In whole blood, nitrite exhibited the dose-independent pharmacokinetics with clearance (CL) of 1.5 l/h/kg, volume of distribution (V d ) of 1.3 l/kg and half-life (t 1/2 ) of 0.6 h. CL and V d of nitrite was higher in red blood cells (RBC) than whole blood and plasma. HbE/β-thal patients had lower nitrite CL and longer t 1/2 in RBC than healthy subjects. FE NO increased immediately after inhalation. Following nitrite inhalation, eRVSP remained unchanged but platelet activation was suppressed as evidenced by inhibition of adenosine diphosphate (ADP)-induced P-selectin expression and increase in phosphorylated vasodilator-stimulated phosphoprotein (P-VASP Ser239 ) in platelets. There were no changes in markers of oxidative and nitrosative stress after inhalation. Our results support further development of inhaled nebulized sodium nitrite for treatment of pulmonary hypertension in β-thalassemia., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The morbidity and mortality in thalassemia patients are predominantly caused by iron overload cardiomyopathy (IOC). Iron-induced cardiac intracellular Ca 2+ ([Ca 2+ ] i ) dysregulation is among the core pathophysiological processes in IOC-related heart failure. Although cardioprotective roles of deferiprone (DFP) and N-acetylcysteine (NAC) have been reported, their effect on cardiac [Ca 2+ ] i transients and Ca 2+ -regulatory protein expression in thalassemic mice is unknown. In the present study, iron overload condition was induced in wild-type (WT) and heterozygous β-thalassemic (HT) mice by a high-iron diet. The iron-overloaded mice subsequently received a vehicle, DFP, NAC, or DFP plus NAC co-therapy. In both WT and HT iron-overloaded mice, DFP and NAC had similar efficacy in decreasing plasma non-transferrin-bound iron, decreasing cardiac iron concentration (CIC) and relieving systolic dysfunction. DFP plus NAC co-therapy, however, was better than the monotherapy in reducing CIC and restoring cardiac [Ca 2+ ] i transient amplitude and rising rate. All regimens produced no change in cardiac Ca 2+ -regulatory protein expression. We provided the first evidence regarding the synergistic effect of combined iron chelator-antioxidant therapy on cardiac [Ca 2+ ] i homeostasis in iron-overloaded thalassemic mice, with consistent improvement of cardiac contractility., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Brain iron overload is chronic and slow progressing and plays an important role in the pathogenesis of neurodegenerative disorders. Magnetic resonance imaging (MRI) is a useful noninvasive tool for determining liver iron content, but it has not been proven to be adequate for evaluating brain iron overload. We evaluated the usefulness of MRI-derived parameters to determine brain iron concentration in β-thalassemic mice and the effects of the membrane permeable iron chelator, deferiprone. Sixteen β-thalassemic mice underwent 1.5T MRI of the brain that included a multiecho T2*-weighted sequence. Brain T2* values ranged from 28 to 31ms for thalassemic mice. For the iron overloaded thalassemic mice, brain T2* values decreased, ranging from 8 to 12ms, which correlated with the iron overload status of the animals. In addition, brain T2* values increased in the group with the treatment of deferiprone, ranging from 18 to 24ms. Our results may be useful to understand brain pathology in iron overload. Moreover, data could lead to an earlier diagnosis, assist in following disease progression, and demonstrate the benefits of iron chelation therapy., (Copyright © 2019. Published by Elsevier Inc.)

Although disturbance of cardiac Ca 2+ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca 2+ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca 2+ transients and Ca 2+ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca 2+ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca 2+ transients including decreased intracellular Ca 2+ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca 2+ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Repair of a splicing defect of β-globin pre-mRNA harboring hemoglobin E (HbE) mutation was successfully accomplished in erythroid cells from patients with β-thalassemia/HbE disorder by a synthetic splice-switching oligonucleotide (SSO). However, its application is limited by short-term effectiveness and requirement of lifelong periodic administration of SSO, especially for chronic diseases like thalassemias. Here, we engineered lentiviral vectors that stably express U7 small nuclear RNA (U7 snRNA) carrying the splice-switching sequence of the SSO that restores correct splicing of β E -globin pre-mRNA and achieves a long-term therapeutic effect. Using a two-step tiling approach, we systematically screened U7 snRNAs carrying splice-switching SSO sequences targeted to the cryptic 5' splice site created by HbE mutation. We tested this approach and identified the most responsive element for mediating splicing correction in engineered U7 snRNAs in HeLa-β E cell model cell line. Remarkably, the U7 snRNA lentiviral vector (U7 βE4+1) targeted to this region effectively restored the correctly-spliced β E -globin mRNA for at least 5 months. Moreover, the effects of the U7 βE4+1 snRNA lentiviral vector were also evident as upregulation of the correctly-spliced β E -globin mRNA in erythroid progenitor cells from β-thalassemia/HbE patients treated with the vector, which led to improvements of pathologies in erythroid progenitor cells from thalassemia patients. These results suggest that the splicing correction of β E -globin pre-mRNA by the engineered U7 snRNA lentiviral vector provides a promising, long-term treatment for β-thalassemia/HbE., (Copyright © 2018 Elsevier Inc. All rights reserved.)

β-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbb th3/+ β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbb th3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbb th3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbb th3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6G hi Ly6C low ) appeared as band form cells, and a striking expansion of immature (Ly6G low Ly6C low ) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbb th3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbb th3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions., (© 2017 by The American Society of Hematology.)

In the presence of red blood cells (RBCs), nitrite inhibits platelets through its conversion to nitric oxide (NO) by the reductase activity of partially deoxygenated hemoglobin. Inhaled sodium nitrite is being investigated as a therapy for pulmonary hypertension. Here, we measured platelet aggregation, P-selectin expression, platelet-leukocyte aggregates and phosphorylated vasodilator-stimulated phosphoprotein (P-VASP Ser239 ) following sodium nitrite inhalation in healthy subjects. In vitro incubation of nitrite with deoxygenated whole blood showed an increase in P-VASP Ser239 , which was inhibited by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. Immediately and 60 min after nitrite inhalation, P-VASP Ser239 increased in platelets. Platelet aggregation, P-selectin expression, platelet-monocyte and platelet-lymphocyte aggregates decreased after inhalation. In conclusion, sodium nitrite administered to healthy subjects by inhalation can inhibit platelet activation and increase P-VASP Ser239 in platelets. Platelet inhibition by nitrite administration may be useful in disorders associated with platelet hyperactivity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The thalassemias are a group of genetic disorders characterized by a deficiency in the synthesis of globin chains. In this study the MUi009-A human induced pluripotent stem cell line was successfully generated from peripheral blood CD34+ haematopoietic progenitors of a 32year old male who had coinherited a homozygous β°-thalassemia mutation at codon 41/42 (-TCTT) and a heterozygous α-thalassemia 4.2 deletion. The MUi009-A cell line exhibited embryonic stem cell characteristics with consistent pluripotency marker expression and the capability of differentiating into the three germ layers. The cell line may provide a tool for drug testing and gene therapy studies., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Hemoglobin Constant Spring (HbCS, HBA2: c.427T>C) is a common nondeletional α-thalassemia resulting from a nucleotide substitution at the termination codon of the HBA2 gene. Homozygosity for HbCS is characterized with mild anemia, jaundice, and splenomegaly. In this study, the human induced pluripotent stem cell line MUi017-A was successfully generated from peripheral blood CD34+ hematopoietic progenitors of a 52year old female with homozygous HbCS. The MUi017-A cell line exhibited embryonic stem cell characteristics with consistent expression of specific pluripotency markers and the capability of differentiating into the three germ layers. The cell line may be used for the disease modeling., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Heart failure related to cardiac siderosis remains a major cause of death in transfusion dependent anaemias. Replacement fibrosis has been reported as causative of heart failure in siderotic cardiomyopathy in historical reports, but these findings do not accord with the reversible nature of siderotic heart failure achievable with intensive iron chelation., Methods: Ten whole human hearts (9 beta-thalassemia major, 1 sideroblastic anaemia) were examined for iron loading and fibrosis (replacement and interstitial). Five had died from heart failure, 4 had cardiac transplantation for heart failure, and 1 had no heart failure (death from a stroke). Heart samples iron content was measured using atomic emission spectroscopy. Interstitial fibrosis was quantified by computer using picrosirius red (PSR) staining and expressed as collagen volume fraction (CVF) with normal value for left ventricle <3%., Results: The 9 hearts affected by heart failure had severe iron loading with very low T2* of 5.0 ± 2.0 ms (iron concentration 8.5 ± 7.0 mg/g dw) and diffuse granular myocardial iron deposition. In none of the 10 hearts was significant macroscopic replacement fibrosis present. In only 2 hearts was interstitial fibrosis present, but with low CVF: in one patient with no cardiac siderosis (death by stroke, CVF 5.9%) and in a heart failure patient (CVF 2%). In the remaining 8 patients, no interstitial fibrosis was seen despite all having severe cardiac siderosis and heart failure (CVF 1.86% ±0.87%)., Conclusion: Replacement cardiac fibrosis was not seen in the 9 post-mortem hearts from patients with severe cardiac siderosis and heart failure leading to death or transplantation, which contrasts markedly to historical reports. Minor interstitial fibrosis was also unusual and very limited in extent. These findings accord with the potential for reversibility of heart failure seen in iron overload cardiomyopathy., Trial Registration: ClinicalTrials.gov Identifier: NCT00520559.

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of β-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in β-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in β-thalassemia trait carriers., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Nontransferrin-bound iron (NTBI) is a heterogeneously speciated plasma iron, typically detectable when transferrin saturation (TfSat) exceeds 75%. Here, we examine factors affecting NTBI levels by a recently discovered direct chelator-based (CP851) fluorescent bead-linked flow-cytometric assay (bead-NTBI), compared with the established indirect nitrilotriacetate (NTA) assay in 122 iron-overloaded patients, including 64 on recent iron chelation therapy and 13 healthy volunteers. Both methods correlated (r = 0.57, P < 0.0001) but with low agreement, attributable to 2 major factors: (1) the NTA method, unlike the bead method, is highly dependent on TfSat, with NTBI under-estimation at low TfSat and over-estimation once Tf is saturated, (2) the bead method detects <3-fold higher values than the NTA assay in patients on recent deferiprone-containing chelation due to greater detection of chelate complexes but lower values for patients on deferasirox. The optimal timing of sample collection relative to chelation dosing requires further study. Patients with splenectomy, high-storage iron, and increased erythropoiesis had greater discrepancy between assays, consistent with differential access by both methods to the NTBI pools associated with these clinical variables. The bead-NTBI assay has advantages over the NTA assay, being less dependent on TfSat, hence of less tendency for false-negative or false-positive values at low and high TfSat, respectively., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: Although iron-overload conditions can be found in β-thalassemic patients, resulting in cellular damage, particularly in the liver, the mechanism for this iron-mediated hepatic injury specifically in β-thalassemic (HT) mice is unclear. This study aimed to investigate the roles of L-type calcium channels (LTCC), T-type calcium channels (TTCC) and divalent metal transporter1 (DMT1) in iron-mediated hepatic injury in HT mice., Main Methods: Iron chelator deferoxamine (DFO), LTCC blocker, TTCC blocker and DMT1 blocker were used to determine the roles of these channels regarding liver iron accumulation, apoptosis and iron regulatory protein expression in HT mice., Key Findings: TTCC and DMT1 blockers and DFO decreased liver iron and malondialdehyde (MDA) in HT mice indicating their antioxidant effects, whereas LTCC blocker produced no decrease in liver iron or MDA. However, only DFO decreased liver apoptosis through the reduced Bax/Bcl-2 ratio in wild type (WT) mice. The levels of iron regulatory hormone hepcidin were markedly higher in HT mice even before iron loading while ferroportin levels did not alter. Each of the pharmacological interventions increased ferroportin protein back to normal levels only in WT while HT mice showed no difference., Significance: Thalassemic mice have different hepcidin/ferroportin and apoptotic protein expression as a defense mechanism to iron-overload compared with those in WT mice. DFO was the most effective intervention in preventing liver apoptosis under iron-overload conditions in WT but did not have the same effect in HT mice., (Copyright © 2016 Elsevier Inc. All rights reserved.)

α(0)-Thalassemia occurs from a deletion of 2 linked α-globin genes and interaction of these defective genes leads to hemoglobin (Hb) Bart's hydrops fetalis, the most severe and lethal thalassemia syndrome. Identification of α(0)-thalassemia carriers is thus essential for the prevention and control program. An immunochromatographic (IC) strip test was developed for rapid screening of α(0)-thalassemia by testing for Hb Bart's in the blood samples using a specific monoclonal antibody against Hb Bart's. To evaluate its sensitivity and specificity, the IC strip test was assessed in a cohort with various thalassemia genotypes from 4 different laboratories in Thailand and Australia. The result showed 97% sensitivity in α-thalassemia carriers with 2 α-globin genes deletion and Hb H disease. This is, in particular, the useful rapid screening test for regions where β-thalassemia and homozygous Hb E are also common. Similar hematologic and Hb data make it impossible to address the concomitant inheritance of α(0)-thalassemia in these samples without polymerase chain reaction (PCR)-based techniques, leading to misdiagnosis of the risk of having Hb Bart's hydrops fetalis. However, α-globin genotyping should be carried out in samples with positive IC strip as positive reactivity was also observed in homozygous α(+)-thalassemia carriers who have 2 trans α-globin gene deletions. These results indicate that in combination with red blood cell indices, the IC strip test could rule out mass populations for further α(0)-thalassemia detection by PCR-based analysis. The Alpha Thal IC strip also has the potential to replace testing for Hb H inclusion bodies, as it appears to be more sensitive, specific, and less labor intensive., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The assessment of myocardial iron using T2* cardiovascular magnetic resonance (CMR) has been validated and calibrated, and is in clinical use. However, there is very limited data assessing the relaxation parameters T1 and T2 for measurement of human myocardial iron., Methods: Twelve hearts were examined from transfusion-dependent patients: 11 with end-stage heart failure, either following death (n=7) or cardiac transplantation (n=4), and 1 heart from a patient who died from a stroke with no cardiac iron loading. Ex-vivo R1 and R2 measurements (R1=1/T1 and R2=1/T2) at 1.5 Tesla were compared with myocardial iron concentration measured using inductively coupled plasma atomic emission spectroscopy., Results: From a single myocardial slice in formalin which was repeatedly examined, a modest decrease in T2 was observed with time, from mean (± SD) 23.7 ± 0.93 ms at baseline (13 days after death and formalin fixation) to 18.5 ± 1.41 ms at day 566 (p<0.001). Raw T2 values were therefore adjusted to correct for this fall over time. Myocardial R2 was correlated with iron concentration [Fe] (R2 0.566, p<0.001), but the correlation was stronger between LnR2 and Ln[Fe] (R2 0.790, p<0.001). The relation was [Fe] = 5081•(T2)-2.22 between T2 (ms) and myocardial iron (mg/g dry weight). Analysis of T1 proved challenging with a dichotomous distribution of T1, with very short T1 (mean 72.3 ± 25.8 ms) that was independent of iron concentration in all hearts stored in formalin for greater than 12 months. In the remaining hearts stored for <10 weeks prior to scanning, LnR1 and iron concentration were correlated but with marked scatter (R2 0.517, p<0.001). A linear relationship was present between T1 and T2 in the hearts stored for a short period (R2 0.657, p<0.001)., Conclusion: Myocardial T2 correlates well with myocardial iron concentration, which raises the possibility that T2 may provide additive information to T2* for patients with myocardial siderosis. However, ex-vivo T1 measurements are less reliable due to the severe chemical effects of formalin on T1 shortening, and therefore T1 calibration may only be practical from in-vivo human studies.

Crohn's disease is an inflammatory bowel disease. Because of strong heritability, it is possible to deploy the pattern of DNA variations, such as single nucleotide polymorphisms (SNPs), to accurately predict the state of this disease. However, there are many possible SNP subsets, which make finding a best set of SNPs to achieve the highest prediction accuracy impossible in one patient's lifetime. In this paper, a new technique is proposed that relies on chromosomes of various lengths with significant order feature selection, a new cross-over approach, and new mutation operations. Our method can find a chromosome of appropriate length with useful features. The Crohn's disease data that were gathered from case-control association studies were used to demonstrate the effectiveness of our proposed algorithm. In terms of the prediction accuracy, the proposed SNP prediction framework outperformed previously proposed techniques, including the optimum random forest (ORF), the univariate marginal distribution algorithm and support vector machine (USVM), the complimentary greedy search-based prediction algorithm (CGSP), the combinatorial search-based prediction algorithm (CSP), and discretized network flow (DNF). The performance of our framework, when tested against this real data set with a 5-fold cross-validation, was 90.4% accuracy with 87.5% sensitivity and 92.2% specificity., (© 2013 Published by Elsevier Ltd. All rights reserved.)

Nitrite anion is bioactive nitric oxide (NO) species circulating in blood, and represents the NO bioavailability and endothelial function. In this study, we aimed to investigate the nitrite levels and the correlation with hemolysis and severity in β-thalassemia/hemoglobin E (β-thal/HbE). 38 Children (12.0±1.9 years of age) with a diagnosis of mild, moderate and severe β-thalassemia were enrolled in the study. The blood nitrite levels and potential plasma NO consumption were measured by the chemiluminescence method. The nitrite levels in whole blood and erythrocytes of the severe thalassemia subjects were lower than those of the control subjects. At day 7 after transfusion of packed erythrocytes, the nitrite levels in erythrocytes increased. The plasma hemoglobin and NO consumption increased in the severe thalassemia subjects. The nitrite levels in erythrocytes inversely correlated with plasma hemoglobin, lactate dehydrogenase activity, potential NO consumption, and lipid peroxidation. Our studies demonstrate the decreased NO bioavailability in thalassemia, which could result from endothelial dysfunction, the increased potential NO consumption in plasma by cell-free hemoglobin and oxidative stress., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aggregatability and oxidative damage of red blood cells (RBCs), platelet activation and increased amount of blood cells-derived microparticles (MPs) are thought to be the etiologies for the thrombotic risk in thalassemia, but with unclear mechanisms. Here we report cellular origins and increases in number, oxidative stress status, and procoagulant activity, as well as altered proteome of MPs isolated from β-thal/HbE patients. Flow cytometric analysis revealed that β-thal/HbE patients had significantly higher levels of phosphatidylserine (PS)-bearing MPs in platelet-free plasma (PFP) as compared to normal subjects. The high levels of MPs correlated with not only the increased procoagulant activity but also the increased platelet counts. Additionally, these PS-bearing MPs were originated mostly from platelets and RBCs, both of which had increased levels of reactive oxygen species. Proteome analysis of MPs by 2-DE followed by Q-TOF MS and MS/MS analyses identified 29 proteins with significantly altered levels in MPs derived from β-thal/HbE patients (e.g. the increased levels of peroxiredoxin 6, apolipoprotein E, cyclophilin A and heat shock protein 90). These findings suggest that the oxidative damage in platelets and RBCs potentially induces production of MPs with altered proteome that may, in turn, facilitate thromboembolic complications, which are commonly found in thalassemic patients. This article is part of a Special Issue entitled: Integrated omics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

alpha-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of alpha-globin protein. Couples who both carry the alpha-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the alpha-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for alpha-thalassemia diagnosis was developed by genotyping alpha-thalassemia-1, the Southeast Asian type (--(SEA)) and Thai type (--(THAI)) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the --(SEA) and --(THAI) deletions and two normal fragments, psizeta- and alpha2-globin gene. The primers were capable of distinguishing alpha-thalassemia 1 heterozygotes from alpha-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --(THAI), --(SEA), psizeta-globin, and alpha2-globin gene fragments were 79.9 +/- 0.2, 89.4 +/- 0.5, 92.8 +/- 0.2, and 85.0 +/- 0.2 degrees C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia.

Erythropoiesis is regulated such that a sufficient number of mature erythrocytes is produced. Down-regulation of erythropoiesis causes various types of anemia. Although some anemia-related genes have been identified, there are several types of anemic disease for which the molecular mechanisms are yet unclear, suggesting that unidentified genes in addition to the classical cytokine pathways play important roles in anemia. To address this issue, a new animal model for anemia is required. We established a reversible anemic model in zebrafish by keeping fish at 17 degrees C, a low water temperature. In zebrafish kidney marrow, expression of several genes encoding hematopoietic transcription factors (Runx1, scl, c-myb and GATA-2) and particularly erythropoiesis-related factors (klfd, hbaa1, ba1, GATA-1, EPO, and EPOr) was down-regulated, whereas myelopoiesis-related factors (csf1a and csf3) was up-regulated in low temperature conditions. We propose that this zebrafish model is useful to identify novel genes for hematopoiesis, particularly erythropoiesis., (2010 Elsevier Inc. All rights reserved.)

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Basic diagnosis of hemoglobinopathies can be performed by analysis of erythrocyte indices as well as by the separation and quantification of the common hemoglobin (Hb) fractions Hb A(2), Hb S, Hb C, Hb D, Hb E, and Hb F. This study used an automatic capillary zone electrophoresis system to diagnose various types of hemoglobinopathies common in the Thai population. A total of 459 adults were recruited, which consisted of normal, various types of thalassemia carriers, and thalassemia patients with different genotypes. Hb types and quantification of all Hb components were determined by an automated capillary zone electrophoresis. The automatic capillary electrophoresis system can separate and quantitate Hbs A, F, E, A(2), Constant Spring (CS), H, and Bart's in a way that is comparable with other Hb analysis methods. Moreover, the Hb A(2) peak can be distinguished clearly from the Hb E peak in individuals who carry Hb E. The slightly increased levels of Hb A(2), 3.5% +/- 0.4%, which is shown in the carriers of Hb E, confirm that Hb E is the silent phenotype of beta(+)-thalassemia.

Background: Despite large populations carrying traits for thalassemia in countries implementing universal iron fortification, there are few data on the absorption and utilization of iron in these persons., Objective: We aimed to determine whether iron absorption or utilization (or both) in women heterozygous for beta-thalassemia, alpha-thalassemia 1, or hemoglobin E (HbE) differed from that in control subjects and compound HbE/beta-thalassemia heterozygotes., Design: In Thai women (n = 103), red blood cell indexes, iron status, non-transferrin-bound iron, and growth differentiation factor 15 were measured, and body iron was calculated. Fractional iron absorption was measured from meals fortified with isotopically labeled ((57)Fe) Fe sulfate, and iron utilization was measured by the infusion of ((58)Fe) Fe citrate., Results: Iron utilization was approximately 15% lower in alpha-thalassemia 1 or beta-thalassemia heterozygotes than in controls. When corrected for differences in serum ferritin, absorption was significantly higher in the alpha- and beta-thalassemia groups, but not the HbE heterozygotes, than in controls. HbE/beta-thalassemia compound heterozygotes had lower iron utilization and higher iron absorption and body iron than did controls. Nontransferrin-bound iron and growth differentiation factor 15 were higher in the compound heterozygotes, but not in the other groups, than in the controls., Conclusions: In alpha-thalassemia 1 and beta-thalassemia heterozygotes with ineffective erythropoesis, dietary iron absorption is not adequately down-regulated, despite a modest increase in body iron stores. In populations with a high prevalence of these traits, a program of iron fortification could include monitoring for possible iron excess and for iron deficiency.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.