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Background: COVID-19 virus has caused the world's deadliest pandemic. Early April 2020, the Delhi Government made it compulsory for people to wear face masks while going outdoors to curb disease spread. Prolonged use of surgical masks during the pandemic has been reported to cause many adverse effects. Intermittent hypoxia has been shown to activate erythropoietin (EPO leading to increased hemoglobin mass., Aim: To analyze whether face mask induced intermittent hypoxia has any effect on the hemoglobin levels of healthy blood donors., Materials and Methods: We retrospectively analyzed donor data from 1st July 2019-31st December 2020 for hemoglobin distribution across hemoglobin ranges and donor deferral on basis of hemoglobin. Study population was divided into two cohorts Group 1- (1st July 2019-31 st March 2020): before implementation of mandatory face masks Group 2- (1st April 2020-31 st December 2020): after implementation of mandatory face masks RESULTS: Mean Hb of blood donors in Group 2 (15.01 ± 1.1 g/dl) was higher than Group1 (14.49 ± 1.15 g/dl), (p < 0.0001). 47.1 % group2 donors had Hb of 16.1-18 g/dl compared to group1 (38.4 %). 52.9 % group 2 donors had Hb between 12.5-15 g/dl compared to 61.6 % Group 1 (p < 0.05). Deferral due to anemia was lesser in group 2 compared to group 1 (p < 0.00001). Group 2 had significantly higher deferral due to high Hb (>18 gm/dl) was than Group 1 (p = 0.0039)., Conclusion: This study including 19504 blood donors spanning over one and a half year shows that prolonged use of face mask by blood donors may lead to intermittent hypoxia and consequent increase in hemoglobin mass., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Primitive electronic waste (e-waste) recycling is ongoing in Guiyu, so toxic heavy metals may continue to threaten the health of children in the area., Objective: This study primarily aimed to evaluate the effect of e-waste exposure on haemoglobin (Hb) synthesis in preschool children., Methods: Medical examinations were conducted with the permission of children's guardians and the approval of the Ethics Committee of the Medical College of Shantou University. This study recruited 224 children (aged 3-6 years, exposed group) who lived in Guiyu and 204 children (aged 3-6 years, control group) who lived in a town free of e-waste pollution. Blood levels of lead, Hb, ferritin, folate and vitamin B 12 were tested in all children. Furthermore, all children were assessed for thalassemia, and their parents were asked to fill in questionnaires., Results: There were no significant differences in the level of ferritin, folate, or vitamin B 12 between the exposed and control groups (P > 0.05). No children were identified as having thalassemia in all study participants. Blood lead level (BLL) and the risk of children with BLL ≥ 10 µg/dL in the exposed group were significantly higher than those in the control group (all P < 0.01). Three subgroups of each group were created according to BLL (Group A: < 5.0 µg/dL; Group B: 5.0-9.9 µg/dL; Group C: ≥ 10.0 µg/dL). Hb level decreased with elevated BLL in the exposed group (P = 0.03), but not in the control group (P = 0.14). Hb levels in group B and group C were also significantly lower in the exposed group than in the control group (Group B: 122.6 ± 9.5 g/L versus 125.8 ± 8.2 g/L, P = 0.01; Group C: 120.3 ± 7.3 g/L versus 123.6 ± 8.3 g/L, P = 0.03). In addition, the prevalence of anaemia associated with BLLs above 10 µg/dL and between 5.0 and 9.9 µg/dL were both significantly higher in the exposed group than in the control group (4.0% vs. 0.5%, 5.4% vs. 1.5%, respectively, both P < 0.05)., Conclusion: Lead exposure more significantly inhibits Hb synthesis in children who live in e-waste dismantling areas than in those who live in non-e-waste dismantling areas. Other toxins released from e-waste may also contribute to the inhibition of Hb synthesis and may lead to anaemia in local children. Further investigations are needed to provide evidence for the development of relevant protective measures.

Background: Anemia is a common complication of chronic kidney disease (CKD). Treating renal anemia with erythropoiesis-stimulating agents (ESAs) or erythropoietin analogs is effective but has side effects. Therefore, we performed a meta-analysis to assess the efficacy and safety of roxadustat in treating CKD-induced anemia., Methods: We searched publications online and conducted a meta-analysis and calculated relative risks with 95% confidence intervals (CIs) for dichotomous data and mean differences (MD) with 95% CIs for continuous data., Results: Of 110 articles, nine were included that contained 12 data sets and 11 randomized control trials on roxadustat. In the non-dialysis-dependent (NDD) high-dose/low-dose subgroups, the change in hemoglobin (Hb) levels was significantly higher in the roxadustat group than in the placebo group ( P <0.0001, P =0.001, respectively). The Hb response rate of the roxadustat is higher in the NDD subgroup than in the placebo group ( P <0.00001, MD=6.92, 95% CI: 4.03, 11.89). However, in the dialysis-dependent subgroup, there was no significant difference in the change in Hb levels or the Hb response rate between the roxadustat and ESA groups. There was no change in the mortality in the roxadustat group compared to that in the placebo/ESA group. Hyperkalemia may be a side effect of roxadustat., Conclusions: Roxadustat elevated the serum Hb levels in a manner similar to that observed for ESAs. Roxadustat raised the Hb levels more significantly than the placebo and showed a higher Hb response rate than the placebo group in NDD patients. Roxadustat is a safe and effective drug for anemia in CKD patients.

Hemoglobin is a cofactor-containing protein with heme that plays important roles in transporting and storing oxygen. Hemoglobins have been widely applied as acellular oxygen carriers, bioavailable iron-supplying agents, and food-grade coloring and flavoring agents. To meet increasing demands and overcome the drawbacks of chemical extraction, the biosynthesis of hemoglobin has become an attractive alternative. Several hemoglobins have recently been synthesized by various microorganisms through metabolic engineering and synthetic biology. In this review, we summarize the novel strategies that have been used to biosynthesize hemoglobin. These strategies can also serve as references for producing other heme-binding proteins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Studies in adults showed a relationship between low hemoglobin (Hb) and acute kidney injury (AKI). We performed this study to evaluate this association in newborns., Methods: We evaluated 1891 newborns from the Assessment of Worldwide AKI Epidemiology in Neonates (AWAKEN) database. We evaluated the associations for the entire cohort and 3 gestational age (GA) groups: <29, 29-<36, and ≥36 weeks' GA., Results: Minimum Hb in the first postnatal week was significantly lower in neonates with AKI after the first postnatal week (late AKI). After controlling for multiple potential confounders, compared to neonates with a minimum Hb ≥17.0 g/dL, both those with minimum Hb ≤12.6 and 12.7-14.8 g/dL had an adjusted increased odds of late AKI (aOR 3.16, 95% CI 1.44-6.96, p = 0.04) and (aOR 2.03, 95% CI 1.05-3.93; p = 0.04), respectively. This association was no longer evident after controlling for fluid balance. The ability of minimum Hb to predict late AKI was moderate (c-statistic 0.68, 95% CI 0.64-0.72) with a sensitivity of 65.9%, a specificity of 69.7%, and a PPV of 20.8%., Conclusions: Lower Hb in the first postnatal week was associated with late AKI, though the association no longer remained after fluid balance was included., Impact: The current study suggests a possible novel association between low serum hemoglobin (Hb) and neonatal acute kidney injury (AKI). The study shows that low serum Hb levels in the first postnatal week are associated with increased risk of AKI after the first postnatal week. This study is the first to show this relationship in neonates. Because this study is retrospective, our observations cannot be considered proof of a causative role but do raise important questions and deserve further investigation. Whether the correction of low Hb levels might confer short- and/or long-term renal benefits in neonates was beyond the scope of this study.

The development of new technologies for the efficient expression of recombinant hemoglobin (rHb) is of interest for experimental studies of protein biochemistry and the development of cell-free blood substitutes in transfusion medicine. Expression of rHb in Escherichia coli host cells has numerous advantages, but one disadvantage of using prokaryotic systems to express eukaryotic proteins is that they are incapable of performing post-translational modifications such as NH 2 -terminal acetylation. One possible solution is to coexpress additional enzymes that can perform the necessary modifications in the host cells. Here, we report a new method for synthesizing human rHb with proper NH 2 -terminal acetylation. Mass spectrometry experiments involving native and recombinant human Hb confirmed the efficacy of the new technique in producing correctly acetylated globin chains. Finally, functional experiments provided insights into the effects of NH 2 -terminal acetylation on O 2 binding properties. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Gene synthesis and cloning the cassette to the expression plasmid Basic Protocol 2: Selection of E. coli expression strains for coexpression Basic Protocol 3: Large-scale recombinant hemoglobin expression and purification Support Protocol 1: Measuring O 2 equilibration curves Support Protocol 2: Mass spectrometry to confirm NH 2 -terminal acetylation., (© 2020 Wiley Periodicals LLC.)

In adults with vitamin B12 deficiency, an inverse correlation between the severity of megaloblastic anemia and the degree of neurological dysfunction has been reported. We aimed to evaluate the association between hematological findings and the results of neurodevelopmental assessment in infants. Denver-II developmental screening test (DDST II) was performed in vitamin B12-deficient infants (n = 122), and its relationship with hematological findings was evaluated. DDST II was abnormal in 15 (12.3%), suspect in 20 (16.4%) and normal in 87 (71.3%) cases. Among the infants aged ≥ 4 months (n = 89), cases with an abnormal DDST II had lower levels of hemoglobin (7.49 ± 3.13 vs. 9.87 ± 1.77 g/dL; P = 0.015), whereas they had higher levels of mean corpuscular volume (MCV) (90.05 ± 19.31 vs. 69.90 ± 10.51 fL; P = 0.002), mean corpuscular hemoglobin (MCH) (28.96 ± 7.50 vs. 22.03 ± 4.58 pg; P = 0.001), homocysteine (44.31 ± 11.51 vs. 21.05 ± 9.23 µmol/L; P < 0.001), transferrin saturation index (25.84 ± 17.72 vs. 9.55 ± 6.38%; P = 0.004) and ferritin (87.28 ± 82.21 vs. 26.59 ± 31.67 ng/mL; P = 0.040) than those with a normal DDST II. The receiver operator characteristic analysis could distinguish infants with an abnormal DDST II from those with a normal DDST II by using a hemoglobin level < 8.75 g/dL [sensitivity: 71.4%, specificity: 76.4%; area under curve (AUC): 0.744], an MCV > 88.4 fL (sensitivity: 76.9%, specificity: 98.2%; AUC 0.813), an MCH > 28.5 pg (sensitivity: 76.9%, specificity: 96.4%; AUC: 0.822), and a homocysteine level > 27.35 µmol/L (sensitivity: 92.9%, specificity: 85.5%; AUC: 0.907). Even mild abnormalities of some commonly evaluated laboratory variables (such as MCV and MCH) in an infant should alert the physicians for the possibility of an underlying vitamin B12 deficiency with some degree of neurological impairment.

The oxygen transport function of hemoglobin (HB) is thought to have arisen ∼500 million years ago, roughly coinciding with the divergence between jawless (Agnatha) and jawed (Gnathostomata) vertebrates. Intriguingly, extant HBs of jawless and jawed vertebrates were shown to have evolved twice, and independently, from different ancestral globin proteins. This raises the question of whether erythroid-specific expression of HB also evolved twice independently. In all jawed vertebrates studied to date, one of the HB gene clusters is linked to the widely expressed NPRL3 gene. Here we show that the nprl3-linked hb locus of a jawless vertebrate, the river lamprey (Lampetra fluviatilis), shares a range of structural and functional properties with the equivalent jawed vertebrate HB locus. Functional analysis demonstrates that an erythroid-specific enhancer is located in intron 7 of lamprey nprl3, which corresponds to the NPRL3 intron 7 MCS-R1 enhancer of jawed vertebrates. Collectively, our findings signify the presence of an nprl3-linked multiglobin gene locus, which contains a remote enhancer that drives globin expression in erythroid cells, before the divergence of jawless and jawed vertebrates. Different globin genes from this ancestral cluster evolved in the current NPRL3-linked HB genes in jawless and jawed vertebrates. This provides an explanation of the enigma of how, in different species, globin genes linked to the same adjacent gene could undergo convergent evolution., (© 2020 by The American Society of Hematology.)

Background: Thalassemia is a group of inherited hemoglobic disorders resulting from defects in the synthesis of one or more of the hemoglobin chains, which is one of the most prevalent inherited disorders in southern China. Only few studies reported the molecular characterization of α- and β-Thalassemia in Hubei Province in the central of China., Methods: A total of 4889 clinically suspected cases of thalassemia were analyzed by Gap-PCR, PCR-based reverse dot blot (RDB)., Results: 1706 (33.8%) subjects harbored thalassemia mutations, including 539 (11.0%) subjects with α-thalassemia, 1140 (23.3%) subjects with β-thalassemia mutations, and 25 (0.51%) subjects with both α- and β-thalassemia mutations. Seven genotypes of α-thalassemia mutations and 29 genotypes of β-thalassemia mutations were characterized. -- SEA /αα (66.05%), -α 3.7 /αα (24.12%), and -α 4.2 /αα (3.71%) accounted for 93.88% of the α-thalassemia mutations. βIVS-II-654/βN, βCD41-42/βN, βCD17/βN, βCD27-28/βN, βCD71-72/βN, β - 28/βN, β - 29/βN, βCD43/βN, βE/βN, accounting for 96.40% of all β-thalassemia genotypes. Furthermore, mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) were sensitive markers for both β-thalassemia and α-thalassemia with -- SEA /αα, but not -α 3.7 /αα and -α 4.2 /αα., Conclusions: Our data indicated great heterogeneity and extensive spectrum of thalassemias in Hubei province of China.

Anaemia is a severe public health problem amongst all vulnerable age groups in India. The National Nutritional Anaemia Prophylaxis Programme initiated in 1970, was revised and expanded to include beneficiaries from all age groups namely children aged 6-59 months, 5-10 yr, adolescents aged 10-19 yr, pregnant and lactating women and women in reproductive age group under the National Iron Plus Initiative (NIPI) programme in 2011. The dose of iron, frequency and duration of iron supplementation and roles and responsibilities of the functionaries were described. At present, the coverage of beneficiaries with iron and folic acid has been poor at the national level. The prevalence of anaemia has continued to remain high during the last 60 years, and there has been no significant change in the scenario due to various reasons. The constraints in implementation and measures to improve the NIPI programme are discussed in the current article., Competing Interests: None

This study is focused on employing a potential process technology for enhancing hemoglobin peptides production from chicken blood. Effects of surfactants on chicken blood biodegradation and hemoglobin polypeptide accumulation were evaluated and the bioconversion conditions were optimized. Results suggested that surfactants exhibited the positive effect on hemoglobin peptides production during chicken blood bioconversion by Aspergillus niger. Dodecyl glucopyranoside was selected as the optimal surfactant and added at the 48th hour of the fermentation process (64 H) at the concentration of 6.0 g/L. Under the optimized conditions, 104.5 mg·N/mL amino nitrogen, 638.3 mg·N/mL nonprotein nitrogen, and 766.3 mg·N/mL soluble nitrogen were detected, which increased by approximately 0.7-, 3.7-, and 3.8-fold, respectively, compared with the control. Furthermore, the acid protease stability was remarkably intensified and the accumulated peptides were mainly distributed at 500-2,000 Da. Results from this work corroborate the potential of applying dodecyl glucopyranoside in hemoglobin polypeptide production from chicken blood., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Therapeutic induction of fetal hemoglobin (HbF) is one of the most promising approaches to ameliorate the severity of hemoglobinopathies like β-thalassemia and sickle cell anemia. Although several pharmacological agents have been investigated for HbF induction in adults, the majority of these are associated with significant side-effects. While drug repurposing is known to open new doors for the use of approved drugs in unexplored clinical conditions, the primary challenge lies in identifying such candidates. In this study, we aimed to identify repurposing candidates for HbF induction using a novel in silico approach utilizing microRNA-pathway-drug relationships. A computational drug repurposing strategy identified several unique candidates for HbF induction; among which Curcumin, Ginsenoside, Valproate, and Vorinostat were found to be most suitable for future trials. This study identified new drug repurposing candidates for HbF induction and demonstrates an easily adaptable methodology that can be used for other pathophysiological conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Hypertensive cerebral hemorrhage leads to greater mortality and worse functional outcomes at high altitudes. Experimental studies have suggested that hemoglobin can lead to increased perihemorrhagic edema after intracerebral hemorrhage., Methods: Patients were divided into a high-hemoglobin (H-H) group (>180 g/L) and a low-hemoglobin (L-H) group (≤180 g/L). The distance from the cortex to the midline was used to indicate the degree of edema. At 1, 7, 14, and 21 days, the patients' status was scored using the Glasgow coma scale, and survival was plotted using Kaplan-Meier survival curves. Pearson correlation analysis showed that the difference between the postoperative and preoperative Glasgow coma scale score correlated with the hemoglobin concentration. The Glasgow outcome scale was used to assess neurological recovery after 6 months., Results: On days 7, 14, and 21, the edema of the H-H group was significantly greater than that of the L-H group (P < 0.01 and P < 0.001, respectively). The edema of the H-H group peaked at 14 and 21 days, but that of the L-H group peaked at 7 days. The hemoglobin concentration and postoperative neurological recovery had a linear relationship in the H-H group. The L-H group had greater survival compared with the H-H group (P < 0.05). The L-H group had higher Glasgow outcome scale scores compared with the H-H group (P < 0.05)., Conclusion: The hemoglobin concentration affects the mortality and morbidity from hypertensive cerebral hemorrhage in high-altitude regions, and a linear relationship exists between hemoglobin concentration and neurological recovery in the H-H group., (Copyright © 2019 Elsevier Inc. All rights reserved.)

β-Thalassemia (β-thal) is a hemoglobinopathy characterized by reduced or absent β-globin production. Pharmacological reactivation of the γ-globin gene for the production of fetal hemoglobin (Hb F) presents an attractive treatment strategy. In an effort to identify promising therapeutic agents, we evaluated 80 analogues of the histone deacetylase inhibitor MS-275, a known Hb F inducer. The chemical analogues were identified via molecular modeling and targeted chemical modifications. Nine novel agents exhibited significant hemoglobin (Hb)-inducing and erythroid differentiation activities in the human K562 erythroleukemia cell line. Five of them appeared to be stronger inducers than the lead compound, MS-275, demonstrating the effectiveness of our method.

Transcription factor 4 (TCF4) is implicated in lymphoid cell differentiation and its expression predicts outcome in acute myeloid leukemia. Here, we investigated the role of TCF4 in myelopoiesis. Overexpression of TCF4 (TCF4 OE ) in umbilical cord blood (UCB) cells resulted in a twofold increase in erythroid colony forming units (CFU-Es), whereas knock-down (KD) of TCF4 (TCF4 KD ) caused a dramatic decrease in the number of erythroid colonies. In megakaryocyte CFUs (CFU-MKs), both TCF4 KD and TCF4 OE inhibited MK colony formation. TCF4 did not have an impact on granulocyte, macrophage, or granulocyte-macrophage colonies or on the proportion of MK-erythrocyte progenitors (MEPs) in culture. Because TCF4 affects erythroid/MK development and these lineages are affected in myelodysplastic syndrome (MDS), we studied the impact of TCF4 expression in this disease. MDS patients with high (≥median) TCF4 mRNA expression had higher hemoglobin (Hb) levels than MDS patients with low TCF4 expression (mean 9.0 vs. 8.55 g/dL, p = 0.02). Overall, TCF4 mRNA expression was lower in hematopoietic stem cells, common myeloid progenitors, and MEPs from MDS patients, but not in granulocyte-macrophage progenitors, compared with healthy controls. Therefore, in cell fractions with erythroid lineage potential, TCF4 is expressed less in MDS patients than in healthy controls. This correlates with the low overall Hb levels seen in MDS patients compared with healthy individuals and is consistent with the positive impact of TCF4 on erythroid development while not having impact on white colonies. These results indicate a role for TCF4 as a novel factor in erythroid-megakaryocytic differentiation., (Copyright © 2018. Published by Elsevier Inc.)

Hemoglobin (Hb) synthesis is a complex, well-coordinated process that requires molecular chaperones. These intervene in different steps: regulating epigenetic mechanisms necessary for the adequate expression of the α- and β-globin clusters, binding the nascent peptides and helping them acquire their native structure, preventing oxidative damage by free globin chains and preventing the cleavage of essential erythroid transcription factors. This study analyzed the distribution of the single nucleotide polymorphism (SNP) rs4296276 in intron 1 of the α-globin chaperone α Hb-stabilizing protein (AHSP) in the Argentinean population. The risk allele was found in thalassemia patients who exhibited more severe phenotypes than expected. Future studies may help establish the role of these chaperones as modifiers in pathological states with globin chain imbalance, such as thalassemia.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Ahaptoglobinaemia have been indicated in blacks from West Africa. Owing to the clinical and biologicimportance of haptoglobin (hpt), this work explores the situation in a Nigerian cohort since there are no published values ofhaptoglobin levels of individuals in this locality. The study was aimed at determining the amount of haptoglobin in the bloodof normal healthy Nigerians. Haptoglobin was quantitatively estimated in one hundred and fifty-two apparently healthyindividuals using highly sensitive immunoassay technology. Blood grouping and haemoglobin genotype were assayed forall subjects to know if they influence haptoglobin levels. The association between haptoglobin and blood group was alsoestablished. Serum levels of haptoglobin among all subjects analyzed revealed a marked decrease in their haptoglobin levelswhen compared to other reference intervals. A further association between haptoglobin and gender did not reveal a statisticalsignificant relationship (p>0.05). However, there was a significant different when haptoglobin levels of different bloodgroups and haemoglobin genotype when compared. Our data suggest that serum levels of haptoglobin are significantly lowerin healthy Nigerians. The lower limit was remarkably lower than the internationally acceptable Caucasian reference rangesuggesting a clear necessity for establishing reference African values.

During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.

Re-expression of fetal hemoglobin (HbF) was proposed as a possible therapeutic strategy for β-haemoglobinopathies. Although several inducers of HbF were tested in clinical trials, only hydroxyurea (HU) received FDA approval. Despite it produced adequate HbF levels only in half of HU-treated SCD patients, and was ineffective at all in β-thalassemia patients, beneficial effects of this approach suggested to continue in this direction identifying further molecules capable of inducing HbF. We tested the potential of essential oil isolated from Ocimum basilicum L. leaves (ObEO) in inducing hemoglobin biosynthesis. Initially, dose-dependent effect and kinetics of hemoglobin accumulation in K562 cells after treatment with ObEO were evaluated. ObEO induced dose-dependent hemoglobin accumulation superior to hydroxyurea and rapamycin and a strongest γ-globin mRNA expression. Terpenes composition of ObEO was studied by GC-MS. Three main constituents, linalool, eugenol and eucalyptol, represented about 75% of total. A blend of these three terpenes fully replicated the ObEO's biological effect, thus indicating that one of them or all together could be the active ingredients. When terpenes were tested individually, eugenol was the only one inducing stable hemoglobin accumulation, while eucalyptol and linalool produced only a small transient response. However, eugenol potential was strongly enhanced in the presence of eucalyptol and linalool, suggesting a synergistic effect on hemoglobin accumulation. By these results, the discovery of a new inducer and the interesting activity of a blend of major terpenes from ObOE on Hb accumulation could have positive fallouts on β-thalassemia and sickle cells anemia., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The iron metabolism of malignant cells, which is altered to ensure higher acquisition and utilization, motivates the investigation of iron chelation strategies in cancer treatment. In a prochelation approach aimed at increasing intracellular specificity, disulfide reduction/activation switches are incorporated on iron-binding scaffolds resulting in intracellularly activated scavengers. Herein, this strategy is applied to several tridentate donor sets including thiosemicarbazones, aroylhydrazones and semicarbazones. The novel prochelator systems are antiproliferative in breast adenocarcinoma cell lines (MCF-7 and metastatic MDA-MB-231) and do not result in the intracellular generation of oxidative stress. Consistent with iron deprivation, the tested prochelators lead to cell-cycle arrest at the G 1 /S interface and induction of apoptosis. Notably, although hemoglobin-synthesizing blood cells have the highest iron need in the human body, no significant impact on hemoglobin production was observed in the MEL (murine erythroleukemia) model of differentiating erythroid cells. This study provides new information on the intracellular effects of disulfide-based prochelators and indicates aroylhydrazone (AH1-S) 2 as a promising prototype of a new class of antiproliferative prochelator systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-β) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-β (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αβ (RAW and A549) or that we transfected to express the globins. We found the following: ( i ) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbβ or Hb-γ) and that hsp90 does not associate with mature Hb. ( ii ) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. ( iii ) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-β, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis., Competing Interests: The authors declare no conflict of interest.

Vertebrate heme synthesis requires three substrates: succinyl-CoA, which regenerates in the tricarboxylic acid cycle, iron and glycine. For each heme molecule synthesized, one atom of iron and eight molecules of glycine are needed. Inadequate delivery of iron to immature erythroid cells leads to a decreased production of heme, but virtually nothing is known about the consequence of an insufficient supply of extracellular glycine on the process of hemoglobinization. To address this issue, we exploited mice in which the gene encoding glycine transporter 1 (GlyT1) was disrupted. Primary erythroid cells isolated from fetal livers of GlyT1 knockout (GlyT1 -/- ) and GlyT1-haplodeficient (GlyT1 +/- ) embryos had decreased cellular uptake of [2 -14 C]glycine and heme synthesis as revealed by a considerable decrease in [2- 14 C]glycine and 59 Fe incorporation into heme. Since GlyT1 -/- mice die during the first postnatal day, we analyzed blood parameters of newborn pups and found that GlyT1 -/- animals develop hypochromic microcytic anemia. Our finding that Glyt1-deficiency causes decreased heme synthesis in erythroblasts is unexpected, since glycine is a non-essential amino acid. It also suggests that GlyT1 represents a limiting step in heme and, consequently, hemoglobin production., (Copyright© 2017 Ferrata Storti Foundation.)

Iron is required for hemoglobin production, and it plays a key role during erythropoiesis. Systemic iron homeostasis is mainly negatively regulated by the peptide hormone hepcidin, coded by the gene HAMP. Hepcidin excess may cause iron deficiency, iron-restricted erythropoiesis, and anemia. Conversely, hepcidin insufficiency leads to iron overload and oxidative damage in multiple tissues. During regulation of hepcidin synthesis, multiple promoter elements in the HAMP gene respond to variable signaling pathways corresponding to different extracellular situations. It has been reported that hepcidin expression can be suppressed by secreted erythroid factors, including GDF15, TWSG1, GDF11, and ERFE, thereby increasing iron availability for hemoglobin synthesis. These potential erythroid factors act via intricate mechanisms that remain controversial. However, it is clear that hepcidin affects erythropoiesis, and promising therapies targeting hepcidin have been developed to treat erythroid disorders. These therapeutic strategies include suppressing or activating HAMP gene expression, mimicking or activating hepcidin activity, and blocking the ability of hepcidin to bind to its target ferroportin., (Copyright © 2017 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

Hemoglobin (Hb) is the major protein in erythrocytes and carries oxygen (O 2 ) throughout the body. Recently, Hb has been found synthesized in atypical sites, including the brain. Hb is highly expressed in A9 dopaminergic (DA) neurons of the substantia nigra (SN), whose selective degeneration leads to Parkinson's disease (PD). Here we show that Hb confers DA cells' susceptibility to 1-methyl-4-phenylpyridinium (MPP + ) and rotenone, neurochemical cellular models of PD. The toxic property of Hb does not depend on O 2 binding and is associated with insoluble aggregate formation in the nucleolus. Neurochemical stress induces epigenetic modifications, nucleolar alterations and autophagy inhibition that depend on Hb expression. When adeno-associated viruses carrying α- and β-chains of Hb are stereotaxically injected into mouse SN, Hb forms aggregates and causes motor learning impairment. These results position Hb as a potential player in DA cells' homeostasis and dysfunction in PD.

The uterus is a highly dynamic organ, undergoing dramatic physiological changes during normal cyclicity and pregnancy. Many of these changes involve remodeling of the uterine vasculature in order to provide oxygen and nutrients to the developing embryo/fetus. Vasculogenesis, angiogenesis, vasodilation/vasoconstriction, and vascular permeability are coordinated by a vast network of autocrine, paracrine, and endocrine-signaling factors that derive from a number of cellular sources at the maternal:fetal interface, as well as from tissue outside the uterus. In this chapter, the dynamic changes that occur in uterine vasculature during pregnancy are described, and some of the hemodynamic regulatory factors are reviewed. These include uterine natural killer cells, sex steroid hormones, the calcitonin gene-related peptide family, angiopoietins, sphingolipids, and the renin-angiotensin system. Aberrancies in these factors are associated with disorders of uterine vascular remodeling, leading to conditions such as early pregnancy loss, preeclampsia, uterine hemorrhage, and intrauterine growth restriction. In addition, we introduce the role of the mas-related gene family in angiotensin signaling and endothelial function during pregnancy. Finally, this chapter introduces the novel concept that in addition to remodeling the vasculature to bring oxygenated maternal blood to the embryo, the gravid uterus synthesizes its own hemoglobin. Overall, this chapter provides an overview of the regulators of uterine vascular remodeling and hemodynamics during pregnancy and pregnancy-associated pathologies., (© 2017 Elsevier Inc. All rights reserved.)

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro-Hb, and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA. In contrast, bovine Hb (bHb) has no quantity constraints for its ample resource. Thus, bHb is of potential to function as an alternative substrate to obtain a PEGylated bHb (bHb-PEG). bHb-PEG was prepared under the same reaction condition as HbA-PEG, using maleimide chemistry. The structural, functional, solution and physiological properties of bHb-PEG were determined and compared with those of HbA-PEG. bHb-PEG showed higher hydrodynamic volume, colloidal osmotic pressure, viscosity and P 50 than HbA-PEG. The high P 50 of bHb can partially compensate the PEGylation-induced perturbation in the R to T state transition of HbA. bHb-PEG was non-vasoactive and could efficiently recover the mean arterial pressure of mice suffering from hemorrhagic shock. Thus, bHb-PEG is expected to function as a potent HBOC for its high oxygen delivery and strong plasma expanding ability. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:252-260, 2017., (© 2016 American Institute of Chemical Engineers.)

Protein synthesis continues in platelets and maturing reticulocytes, although these blood cells lack nuclei and do not make new mRNA or ribosomes. Here, we analyze translation in primary human cells from anucleate lineages by ribosome profiling and uncover a dramatic accumulation of post-termination unrecycled ribosomes in the 3' UTRs of mRNAs. We demonstrate that these ribosomes accumulate as a result of the natural loss of the ribosome recycling factor ABCE1 during terminal differentiation. Induction of the ribosome rescue factors PELO and HBS1L is required to support protein synthesis when ABCE1 levels fall and for hemoglobin production during blood cell development. Our observations suggest that this distinctive loss of ABCE1 in anucleate blood lineages could sensitize them to defects in ribosome homeostasis, perhaps explaining in part why genetic defects in the fundamental process of ribosome production ("ribosomopathies") often affect hematopoiesis specifically., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC-MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV-Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein-ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.

High-throughput single-cell technologies have great potential to discover new cell types; however, it remains challenging to detect rare cell types that are distinct from a large population. We present a novel computational method, called GiniClust, to overcome this challenge. Validation against a benchmark dataset indicates that GiniClust achieves high sensitivity and specificity. Application of GiniClust to public single-cell RNA-seq datasets uncovers previously unrecognized rare cell types, including Zscan4-expressing cells within mouse embryonic stem cells and hemoglobin-expressing cells in the mouse cortex and hippocampus. GiniClust also correctly detects a small number of normal cells that are mixed in a cancer cell population.

Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34(+) human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis.

Almost all insects are equipped with a tracheal system, which appears to be sufficient for O2 supply even in phases of high metabolic activity. Therefore, with the exception of a few species dwelling in hypoxic habitats, specialized respiratory proteins had been considered unnecessary in insects. The recent discovery and apparently universal presence of intracellular hemoglobins in insects has remained functionally unexplained. The fruitfly Drosophila melanogaster harbors three different globin genes (referred to as glob1-3). Glob1 is the most highly expressed globin and essentially occurs in the tracheal system and the fat body. To better understand the functions of insect globins, the levels of glob1 were modulated in Drosophila larvae and adults by RNAi-mediated knockdown and transgenic over-expression. No effects on the development were observed in flies with manipulated glob1 levels. However, the knockdown of glob1 led to a significantly reduced survival rate of adult flies under hypoxia (5% and 1.5% O2). Surprisingly, the glob1 knockdown flies also displayed increased resistance towards the reactive oxygen species-forming agent paraquat, which may be explained by a restricted availability of O2 resulting in decreased formation of harmful O2(-). In summary, our results suggest an important functional role of glob1 in O2 homeostasis, possibly by enhancing O2 supply., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: During Streptococcus zooepidemicus fermentation, most carbon sources are used to synthesize lactic acid, which can inhibit strain growth and hyaluronic acid production. Here, we expressed bacterial haemoglobin (Vhb) in Strep. zooepidemicus. Due to highly efficient oxygen use, only 15·26 g l(-1) lactic acid was produced, which is 0·73 times the quantity produced by the control strain. Compared with the control strain (1·61 g l(-1) ), hyaluronic acid (HA) production in this strain did not substantially increase, only to 2·16 g l(-1) . Next, we used a series of N-methyl-N'-nitro-N-nitroso-guanidine (NTG) treatments and selection programmes. Finally, we generated a hyaluronidase-negative and rifampin-resistant mutant strain that produces high levels of HA. The optimum carbon concentration for maximum hyaluronic acid production is only 30 g l(-1) of sucrose, which is lower than the control strain (60 g l(-1) ). The oxygen transfer rate coefficient KL a increased significantly to 372 ± 53 h(-1) from 18 ± 4 h(-1) of the control. The optimum carbon source for this strain is 21 g l(-1) of sucrose, 9 g l(-1) of maltose and 5 g l(-1) of glutamic acid. Hyaluronic acid accumulated at 6·7 g l(-1) in the culture broth. However, the molecular weight of HA decreased from 1835 KDa (Control) to 429 kDa. The prepared low-molecular weight HA could function as potential antiangiogenic substances, antiviral and antitumour agents to possibly be used as functional food ingredients., Significance and Impact of the Study: Hyaluronic acid (HA) has been used for a wide range of applications in health, cosmetic and clinical fields. During fermentation of Streptococcus to produce HA, 80-85% of the carbon source is used to produce lactic acid and acetic acid, and only approx. 5 and 10% of the carbon source is used to produce HA and biomass respectively. Here, we expressed bacteria haemoglobin (Vhb) in Streptococcus zooepidemicus, which can dramatically inhibit lactic acid production. After NTG treatments and selection programmes, we identified a mutant strain with highly efficient hyaluronic acid production (6·7 g l(-1) ) under economic fermentation conditions., (© 2016 The Society for Applied Microbiology.)

Anemia management, based on erythropoiesis stimulating agents (ESA) and iron supplementation, has become an increasingly challenging problem in hemodialysis patients. Maintaining hemodialysis patients within narrow hemoglobin targets, preventing cycling outside target, and reducing ESA dosing to prevent adverse outcomes requires considerable attention from caregivers. Anticipation of the long-term response (i.e. at 3 months) to the ESA/iron therapy would be of fundamental importance for planning a successful treatment strategy. To this end, we developed a predictive model designed to support decision-making regarding anemia management in hemodialysis (HD) patients treated in center. An Artificial Neural Network (ANN) algorithm for predicting hemoglobin concentrations three months into the future was developed and evaluated in a retrospective study on a sample population of 1558 HD patients treated with intravenous (IV) darbepoetin alfa, and IV iron (sucrose or gluconate). Model inputs were the last 90 days of patients' medical history and the subsequent 90 days of darbepoetin/iron prescription. Our model was able to predict individual variation of hemoglobin concentration 3 months in the future with a Mean Absolute Error (MAE) of 0.75 g/dL. Error analysis showed a narrow Gaussian distribution centered in 0 g/dL; a root cause analysis identified intercurrent and/or unpredictable events associated with hospitalization, blood transfusion, and laboratory error or misreported hemoglobin values as the main reasons for large discrepancy between predicted versus observed hemoglobin values. Our ANN predictive model offers a simple and reliable tool applicable in daily clinical practice for predicting the long-term response to ESA/iron therapy of HD patients.

Subarachnoid hemorrhage (SAH) usually results from ruptured aneurysm, but how leaked hemoglobin regulates the microcirculation in the pathophysiology of early brain injury after SAH is still unclear. In the present study, we sought to investigate the role and possible mechanism of hemoglobin induced pericyte phenotype transformation in the regulation of microcirculation after SAH. Endovascular perforation SAH rat model, brain slices and cultured pericytes were used, and intervened with endothelial nitric oxide synthase (eNOS) antagonist L-NNA and its agonist scutellarin, hemoglobin, DETA/NO (nitric oxide(NO) donor), PITO (NO scavenger), 8-Br-cGMP (cGMP analog). We found modulating eNOS regulated pericyte α-SMA phenotype transformation, microcirculation, and neurological function in SAH rats. Modulating eNOS also affected eNOS expression, eNOS activity and NO availability after SAH. In addition, we showed hemoglobins penetrated into brain parenchyma after SAH. And hemoglobins significantly reduced the microvessel diameters at pericyte sites, due to the effects of hemoglobin inducing α-SMA expressions in cultured pericytes and brain slices via inhibiting NO/cGMP pathway. In conclusion, pericyte α-SMA phenotype mediates acute microvessel constriction after SAH possibly by hemoglobin suppressing NO/cGMP signaling pathway. Therefore, by targeting the eNOS and pericyte α-SMA phenotype, our present data may shed new light on the management of SAH patients.

Background: Increased hemoglobin (Hb) accumulation was reported in abdominal aortic aneurysms (AAAs). CD163 is a macrophage receptor involved in tissue Hb clearance, however its role in AAA has not been reported. We investigated the role of Hb on monocyte recruitment and differentiation towards CD163 expressing macrophages ex vivo, in vitro and in human AAA., Methods and Results: CD163 mRNA and protein expression was significantly higher in human AAA (n=7) vs. healthy wall (n=6). CD163 was predominantly found in adventitia of AAA, coinciding with areas rich in hemosiderin and adjacent to neoangiogenic microvessels. Dual CD14/CD163 expression was observed in recently infiltrated monocytes surrounding microvessels. A higher release of soluble CD163 was observed in the conditioned medium from AAA (AAA-CM, n=10), mainly in the adventitial layer. Similar to Hb, AAA-CM induced CD163-dependent monocyte chemotaxis, especially on circulating monocytes from AAA patients. Hb or AAA-CM promoted differentiation towards CD163(high)/HLA-DR(low)-expressing macrophages, with enhanced Hb uptake, increased anti-inflammatory IL-10 secretion and decreased pro-inflammatory IL-12p40 release. All these effects were partially suppressed when Hb was removed from AAA-CM. Separate analysis on circulating monocytes reported increased percentage of pre-infiltrating CD14(++)CD16(+) monocytes in patients with AAA (n=21), as compared to controls (n=14). A significant increase in CD163 expression in CD14(++)CD16(+) monocyte subpopulation was observed in AAA patients., Conclusions: The presence of Hb in the adventitial AAA-wall promotes the migration and differentiation of activated circulating monocytes in AAA patients, explaining the existence of a protective CD163-macrophage phenotype that could take up the Hb present in the AAA-wall, avoiding its injurious effects., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34(+) marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up-regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis-splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation., (© 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

Background: A too short recovery time after blood donation results in a gradual depletion of iron stores and a subsequent decline in hemoglobin (Hb) levels over time. This decline in Hb levels may depend on individual, unobserved characteristics of the donor., Study Design and Methods: We used a data set of 5388 Dutch blood donors from the Donor InSight study. The statistical analysis is based on a Bayesian growth mixture model, which assumes that each donor belongs to one of several groups. Each group implies a different Hb trajectory, and donors with similar longitudinal trajectories belong to the same group. Analyses were performed for male and female donors separately., Results: For both sexes the model identified four groups of donors. Stable Hb trajectories were found among 14% of male donors and 15% of female donors; declining Hb trajectories were observed in the remaining groups of donors. The percentage of donor deferrals differed strongly between groups., Conclusion: The model can be used to predict to which group a donor belongs, and this prediction can be updated after each donation. This is of high practical importance because early identification of donors with declining Hb levels could help to tailor donation intervals and to prevent iron deficiency and donor deferrals., (© 2015 AABB.)

Purpose: Autologous stem-cell transplantation (ASCT) has shown to provide curative benefit in patients with relapsed lymphoma and multiple myeloma (MM), often requiring hematopoietic support until marrow engraftment. Because of Jehovah's Witnesses' (JW) refusal of blood products, treatment challenges arise. This study represents 125 JWs with lymphoma (n = 55), MM (n = 68), or amyloidosis (n = 2), treated with high-dose chemotherapy (HDC) and ASCT without transfusions., Patients and Methods: Priming with intravenous iron and erythropoietin occurred to increase hemoglobin (Hb) pretransplantation. Cytokine mobilization of stem-cells was used. Delay to HDC was done to allow Hb and platelets to approach 11 g/dL and 100 × 10(3)/μL, respectively. Patients with MM received a standard dose of melphalan 200 mg/m(2), with dose reduction for severe kidney dysfunction. Patients with lymphoma received carmustine 300 mg/m(2), cyclophosphamide 1,500 mg/m(2) on days 2 through 5 (total 6 g/m(2)), and etoposide 700 mg/m(2) per day on days 2 through 4 (total 2,100 mg/m(2)). Post-transplantation, a combination of granulocyte colony-stimulating factor, erythropoietin, aminocaproic acid, and phytonadione was administered., Results: There were two major and 15 minor bleeding complications, none occurring at platelets less than 5.0 × 10(3)/μL, with six (4.8%) treatment-related mortalities. The median decrease in Hb was 5.0 g/dL, with median Hb nadir of 7.0 g/dL. The median number of days with platelet count less than 10 × 10(3)/μL was 3, with median platelet nadir of 5.0 × 10(3)/μL. Cardiac complications occurred in 40 patients (32%)., Conclusion: ASCT can safely be performed without transfusion support. A platelet transfusion trigger of ≤ 5 × 10(3)/μL may be appropriate in select patients. Pharmacotherapy and cardiac monitoring are effective in the management of cardiac complications., (© 2015 by American Society of Clinical Oncology.)

The aim of this study is to explore a cause-oriented therapy for patients with uterine cervical cancer that expresses erythropoietin (Epo) and its receptor (EpoR). Epo, by binding to EpoR, stimulates the proliferation and differentiation of erythroid progenitor cells into hemoglobin-containing red blood cells. In this study, we report that the HeLa cells in the xenografts expressed ε, γ, and α globins as well as myoglobin (Mb) to produce tetrameric α2ε2 and α2γ2 and monomeric Mb, most of which were significantly suppressed with an EpoR antagonist EMP9. Western blotting revealed that the EMP9 treatment inhibited the AKT-pAKT, MAPKs-pMAPKs, and STAT5-pSTAT5 signaling pathways. Moreover, the treatment induced apoptosis and suppression of the growth and inhibited the survival through disruption of the harmonized hemoprotein syntheses in the tumor cells concomitant with destruction of vascular nets in the xenografts. Furthermore, macrophages and natural killer (NK) cells with intense HIF-1α expression recruited significantly more in the degenerating foci of the xenografts. These findings were associated with the enhanced expressions of nNOS in the tumor cells and iNOS in macrophages and NK cells in the tumor sites. The treated tumor cells exhibited a substantial number of perforations on the cell surface, which indicates that the tumors were damaged by both the nNOS-induced nitric oxide (NO) production in the tumor cells as well as the iNOS-induced NO production in the innate immune cells. Taken together, these data suggest that HeLa cells constitutively acquire ε, γ and Mb synthetic capacity for their survival. Therefore, EMP9 treatment might be a cause-oriented and effective therapy for patients with squamous cell carcinoma of the uterine cervix.

Background: Our objective was to compare the capacity of iron (Fe) biofortified and standard pearl millet (Pennisetum glaucum L.) to deliver Fe for hemoglobin (Hb)-synthesis. Pearl millet (PM) is common in West-Africa and India, and is well adapted to growing areas characterized by drought, low-soil fertility, and high-temperature. Because of its tolerance to difficult growing conditions, it can be grown in areas where other cereal crops, such as maize, would not survive. It accounts for approximately 50% of the total world-production of millet. Given the widespread use of PM in areas of the world affected by Fe-deficiency, it is important to establish whether biofortified-PM can improve Fe-nutriture., Methods: Two isolines of PM, a low-Fe-control ("DG-9444", Low-Fe) and biofortified ("ICTP-8203 Fe",High-Fe) in Fe (26 μg and 85 μg-Fe/g, respectively) were used. PM-based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (Fe concentrations were 22.1±0.52 and 78.6±0.51 μg-Fe/g for the Low-Fe and High-Fe diets, respectively). For 6-weeks, Hb, feed-consumption and body-weight were measured (n = 12)., Results: Improved Fe-status was observed in the High-Fe group, as suggested by total-Hb-Fe values (15.5±0.8 and 26.7±1.4 mg, Low-Fe and High-Fe respectively, P<0.05). DMT-1, DcytB, and ferroportin mRNA-expression was higher (P<0.05) and liver-ferritin was lower (P>0.05) in the Low-Fe group versus High-Fe group. In-vitro comparisons indicated that the High-Fe PM should provide more absorbable-Fe; however, the cell-ferritin values of the in-vitro bioassay were very low. Such low in-vitro values, and as previously demonstrated, indicate the presence of high-levels of polyphenolic-compounds or/and phytic-acid that inhibit Fe-absorption. LC/MS-analysis yielded 15 unique parent aglycone polyphenolic-compounds elevated in the High-Fe line, corresponding to m/z = 431.09., Conclusions: The High-Fe diet appeared to deliver more absorbable-Fe as evidenced by the increased Hb and Hb-Fe status. Results suggest that some PM varieties with higher Fe contents also contain elevated polyphenolic concentrations, which inhibit Fe-bioavailability. Our observations are important as these polyphenols-compounds represent potential targets which can perhaps be manipulated during the breeding process to yield improved dietary Fe-bioavailability. Therefore, the polyphenolic and phytate profiles of PM must be carefully evaluated in order to further improve the nutritional benefit of this crop.

In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies.

We have established a method to express soluble heme-bound recombinant crocodile (Crocodylus siamensis) α-globin chain holo-protein in bacteria (Escherichia coli) using an autoinduction system without addition of exogenous heme. This is the first time that heme-bound crocodile α-globin chains have been expressed in bacteria without in vitro heme reconstitution. The observed molecular mass of purified recombinant α-globin is consistent with that calculated from the primary amino acid sequence of native crocodile (C. siamensis) α-globin. Both the monomeric and the dimeric protein configuration formed by intermolecular disulfide bond could be purified as soluble protein. Spectroscopic characterization [UV-visible, circular dichroism (CD), and electron paramagnetic resonance (EPR)] of purified recombinant α-globin demonstrates nearly identical properties as reported for hemoglobin and myoglobin isolated from other organisms. For comparison, cyanide and nitric oxide binding of purified α-globin was also investigated. These results suggested that C. siamensis α-globin expressed in E. coli was folded correctly with proper incorporation of the heme cofactor. The expression method we now describe can facilitate production and isolation of individual globin chains in order to further study the mechanism and assembly of crocodile hemoglobin., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To determine whether the administration of isotonic saline in patients undergoing spinal fusion surgery prevents the development of hyponatremia, thus masking the detection of syndrome of inappropriate antidiuretic hormone secretion (SIADH)., Study Design: Prospective observational cohort study conducted in pediatric patients undergoing spinal fusion surgery. Using established criteria for diagnosing SIADH with the exception of serum sodium as a criterion, we separated patients into those with and without masked SIADH. Random cortisol levels were measured in the perioperative period to test for adrenal insufficiency to exclude it as a cause for natriuresis and hyponatremia., Results: Of the 40 patients included in the study, 13 (32%; 95% CI, 19%-49%) met study criteria for masked SIADH. The serum sodium levels between the 2 groups were not different throughout the postoperative period. The antidiuretic hormone levels were increased at 24-48 hours after surgery (20.4 pg/mL in masked SIADH group vs 6.6 pg/mL in no masked SIADH group, P = .04). Subjects with masked SIADH demonstrated a tendency for weight gain (3.9 kg vs 2.5 kg, P = .058), which was maximal on postoperative day 2. Cortisol levels were similar between the groups., Conclusion: Masked SIADH (SIADH-like state without hyponatremia) commonly occurs in the postoperative period in children and young adults undergoing spinal fusion surgery. Early postoperative evaluation and recognition may result in appropriate management of patient's fluid balance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(•)) in either the ferric or ferrous state and has efficient NO(•) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.