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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.

Induction of fetal haemoglobin (HbF) is a promising therapeutic approach for the treatment of β-thalassaemia and sickle cell disease (SCD). Several pharmacological agents, such as hydroxycarbamide (HC) and butyrates, have been shown to induce the γ-globin genes (HBG1, HBG2). However, their therapeutic use is limited due to weak efficacy and an inhibitory effect on erythroid differentiation. Thus, more effective agents are needed. The histone deacetylase (HDAC) inhibitors are potential therapeutic haemoglobin (Hb) inducers able to modulate gene expression through pleiotropic mechanisms. We investigated the effects of a HDAC inhibitor, Givinostat (GVS), on erythropoiesis and haemoglobin synthesis and compared it with sodium butyrate and HC. We used an in vitro erythropoiesis model derived from peripheral CD34⁺ cells of healthy volunteers and SCD donors. GVS effects on erythroid proliferation and differentiation and on Hb synthesis were investigated. We found that GVS at high concentrations delayed erythroid differentiation with no specific effect on HBG1/2 transcription. At a low concentration (1 nmol/l), GVS induced Hb production with no effects on cells proliferation and differentiation. The efficacy of GVS 1 mol/l in Hb induction in vitro was comparable to that of HC and butyrate. Our results support the evaluation of GVS as a new candidate molecule for the treatment of the haemoglobinophathies due to its positive effects on haemoglobin production at low and non-toxic concentrations., (© 2014 John Wiley & Sons Ltd.)

Sotatercept (ACE-011), a recombinant human fusion protein containing the extracellular domain of the human Activin receptor IIA, binds to and inhibits activin and other members of the transforming growth factor -β (TGF-β) superfamily. Administration of sotatercept led to a rapid and sustained increase in red blood cell (RBC) count and haemoglobin (Hb) in healthy volunteers (phase I clinical trials), but the mechanism is not fully understood. Mice treated with RAP-011 (murine ortholog of ACE-011) respond with a rapid (within 24 h) increase in haematocrit, Hb, and RBC count. These effects are accompanied by an equally rapid stimulation of late-stage erythroid precursors in the bone marrow (BM). RAP-011 also induces a significant increase in erythroid burst-forming units and erythropoietin, which could contribute to additional, sustained effects on RBC production. Further in vitro co-culture studies demonstrate that BM accessory cells are required for RAP-011 effects. To better understand which TGF-β family ligand(s) mediate RAP-011 effects, we evaluated the impact of several of these ligands on erythroid differentiation. Our data suggest that RAP-011 may act to rescue growth differentiation factor 11/Activin A-induced inhibition of late-stage erythropoiesis. These data define the mechanism of action of a novel agent that regulates RBC differentiation and provide the rationale to develop sotatercept for the treatment of anaemia and ineffective erythropoiesis., (© 2014 Celgene Corporation. British Journal of Haematology published by John Wiley & Sons Ltd.)

Background: Our objective was to determine if a biofortified variety of black bean can provide more bioavailable-iron (Fe) than a standard variety. Two lines of black beans (Phaseolus-vulgaris L.), a standard (DOR500; 59μg Fe/g) and biofortified (MIB465; 88μg Fe/g) were used. The DOR500 is a common commercial variety, and the MIB465 is a line developed for higher-Fe content. Given the high prevalence of Fe-deficiency anemia worldwide, it is important to determine if Fe-biofortified black beans can provide more absorbable-Fe., Methods: Black bean based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (dietary Fe-concentrations were 39.4±0.2 and 52.9±0.9 mg/kg diet, standard vs. biofortified, respectively). Birds (n=14) were fed the diets for 6-weeks. Hemoglobin-(Hb), liver-ferritin and Fe-related transporter/enzyme gene-expression were measured. Hemoglobin-maintenance-efficiency and total-body-Hb-Fe values were used to estimate Fe-bioavailability., Results: Hemoglobin-maintenance-efficiency values were higher (P<0.05) in the group consuming the standard-Fe beans on days 14, 21 and 28; indicating a compensatory response to lower dietary-Fe. Final total-Hb-Fe body content was higher in the biofortified vs. the standard group (26.6±0.9 and 24.4±0.8 mg, respectively; P<0.05). There were no differences in liver-ferritin or in expression of DMT-1, Dcyt-B, and ferroportin. In-vitro Fe-bioavailability assessment indicated very low Fe-bioavailability from both diets and between the two bean varieties (P>0.05). Such extremely-low in-vitro Fe-bioavailability measurement is indicative of the presence of high levels of polyphenolic-compounds that may inhibit Fe-absorption. High levels of these compounds would be expected in the black bean seed-coats., Conclusions: The parameters of Fe-status measured in this study indicate that only a minor increase in absorbable-Fe was achieved with the higher-Fe beans. The results also raise the possibility that breeding for increased Fe-concentration elevated the levels of polyphenolic-compounds that can reduce bean Fe-bioavailability, although the higher levels of polyphenolics in the higher-Fe beans may simply be coincidental or an environmental effect. Regardless, Fe-biofortified beans remain a promising vehicle for increasing intakes of bioavailable-Fe in human populations that consume high levels of these beans as a dietary staple, and the bean polyphenol profile must be further evaluated and modified if possible in order to improve the nutritional quality of higher-Fe beans.

The author presents a novel algorithm for anaemia based on the erythrocyte haemoglobin content. The scheme is based on the aberrations of erythropoiesis and not on the pathophysiology of anaemia. The hemoglobin content of one erytrocyte is between 28-35 picogram. Any disturbance in hemoglobin synthesis can lead to a lower than 28 picogram hemoglobin content of the erythrocyte which will lead to hypochromic anaemia. In contrary, disturbances of nucleic acid metabolism will result in a hemoglobin content greater than 36 picogram, and this will result in hyperchromic anaemia. Normochromic anemia, characterised by hemoglobin content of erythrocytes between 28 and 35 picogram, is the result of alteration in the proliferation of erythropoeisis. Based on these three categories of anaemia, a unique system can be constructed, which can be used as a model for basic laboratory investigations and work-up of anaemic patients.

Hemoglobin is produced mainly in erythroid cells. However, it has been reported in non-erythroid cells of human and rodents. We have shown previously that neuroglobin, cytoglobin and hemoglobin are expressed in human glioblastoma multiforme (GBM) cells. We sought to determine whether hemoglobin expression is upregulated by hypoxia, and whether its expression is restricted to the cancer stem cell populations in different GBM cell lines or GBM brain tumor initiating cells (BTICs). Flow cytometry, magnetic cell sorting and qRT-PCR were used to examine the hypoxic upregulation of hemoglobins as well as erythropoietin (EPO) and erythropoietin receptor (EPOR) in GBM cell lines (M006x, M059J, M059K, U87R and U87T) and GBM-BTICs. The data showed significantly increased expression in globins (α, β, γ, δ, ζ and ε), EPO and EPOR mRNA levels under hypoxia. Globin expression is not limited to the stem cell populations or GBM-BTICs but is a property of the entire GBM population. We assumed that the total expression of mRNA of different normalized globins (α, β, γ, δ, ζ and ε) at different time‑points for the same cell line is 100%. Under aerobic conditions, ε globin was predominantly expressed, and then decreased gradually with increasing time in hypoxia. This was coupled to a concomitant increase in α and γ globins. Our findings suggest that hypoxic upregulation of hemoglobin expression in GBM cells may be a part of a repertoire of active defence and adaptation mechanisms enabling these cells to acquire resistance to aggressive multimodality treatments of chemotherapy and radiotherapy. New therapeutic strategies to interfere with hemoglobin expression or function in GBM cells are required.

Astrocytes and microglia become reactive in many neurological disorders resulting in phenotypic and functional alterations. Both cell types might also display functional changes during normal aging. To identify gene signatures and changes in basal cellular functions of astrocytes and microglia in relation to aging, we isolated viable astrocytes and microglia from young adult and aged mouse cortices and determined their gene expression profile. Aged astrocytes, compared with young astrocytes, showed an increased inflammatory phenotype and increased 'zinc ion binding.' Young astrocytes showed higher expression of genes involved in 'neuronal differentiation' and hemoglobin synthesis. Astrocyte expression of genes involved in neuronal signaling remains high throughout age. Aged microglia had higher expression of genes involved in 'vesicle release,' 'zinc ion binding,' and genes within the tumor necrosis factor-ligand family and young microglia had increased transcript levels of C-C motif chemokines. These data provide a transcriptome database of cell-type enriched genes of astrocytes and microglia from adult mice and give insight into the differential gene signature of astrocytes and microglia in relation to normal aging., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (α2β2). In this work we evaluated the expression of different combinations of α and β peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group., (© 2013 Published by International Metabolic Engineering Society on behalf of International Metabolic Engineering Society.)

Anemia is a common complication in several types of cancer including hepatocellular carcinoma (HCC). The prognostic potential of hemoglobin (Hb) levels has not yet been investigated in HCC patients. One hundred and ninety-nine patients were prospectively recruited and Hb levels were determined. Hb levels were compared to the stages of liver cirrhosis and HCC stages. The association of the Hb levels and overall survival (OS) was assessed by univariate and multivariate Cox regression models. The relation of Hb levels and OS was further validated in an independent cohort of 87 HCC patients. Hb levels negatively correlated with the stage of liver cirrhosis (model of end stage liver disease score and Child-Pugh stage) and differed between stages of HCC. Low Hb levels (≤ 13 g/dl) were associated with higher mortality in the test [hazard ratio (HR) 2.422, 95 % confidence interval (CI) 1.357-4.322, P = 0.003] as well in the validation cohort (HR 2.486, 95 % CI 1.097-5.632, P = 0.029) in univariate Cox regression model. Low Hb levels were associated with mortality independently from the tumor stage, age, gender and the C-reactive protein levels in a multivariate Cox regression model. Anemia should be considered as a risk factor for mortality in HCC patients.

Antiretroviral (ARV) prophylaxis for prevention of mother to child transmission (MTCT) of HIV could affect hemoglobin (Hb) development of infants. A cross-sectional descriptive study was conducted in 24 HIV-infected and 21 HIV-uninfected pregnancies. ARV drugs were administered to HIV-infected pregnancies at 21 weeks of gestational age and at labor. Their infants received zidovudine (ZDV) until 4 weeks of age. Blood samples of ARV-exposed and - unexposed infants were collected at delivery, 1, 2 and 4 months of age. Molecular analyses for α-thalassemia-1 Southeast Asian (SEA) type deletion, β-thalassemia mutations and Hb E were performed for excluding the thalassemia carrier infants. Hemoglobinopathy and Hb A, Hb F and Hb A2 were analyzed by using capillary electrophoresis (CE) while hematological parameters were measured using an automated blood counter. At delivery, 1 and 2 months of age, ARVexposed infants had significantly lower levels of RBC counts than ARV-unexposed infants (3.56 vs 4.90, 2.66 vs 4.62 and 3.01 vs 4.05 x10(12)/L; P <0.001, <0.001 and 0.001, respectively). At delivery, there was a trend for low hemoglobin level in the group of ARV-exposed infants as compared to the group of ARV-unexposed infants (149 vs 154 g/L; P = 0.09) and the significantly different levels were observed among the two groups at 1 and 2 months of age (89 vs 136 and 87 vs 110 g/L; P < 0.001 and 0.001, respectively). The development of Hb A, Hb F and Hb A2 levels from delivery to 4 months of age among the two groups was not significantly different. Therefore, ARV treatments for prevention of MTCT of HIV decreased RBC counts and hemoglobin but did not alter the development of Hb A, Hb F and Hb A2 of non-thalassemia carrier infants.

Background: Diamond-Blackfan anemia is a rare congenital red blood cell dysplasia that develops soon after birth. RPL11 mutations account for approximately 4.8% of human DBA cases with defective hematopoietic phenotypes. However, the mechanisms by which RPL11 regulates hematopoiesis in DBA remain elusive. In this study, we analyzed the transcriptome using deep sequencing data from an Rpl11-deficient zebrafish model to identify Rpl11-mediated hematopoietic failure and investigate the underlying mechanisms., Results: We characterized hematological defects in Rpl11-deficient zebrafish embryos by identifying affected hematological genes, hematopoiesis-associated pathways, and regulatory networks. We found that hemoglobin biosynthetic and hematological defects in Rpl11-deficient zebrafish were related to dysregulation of iron metabolism-related genes, including tfa, tfr1b, alas2 and slc25a37, which are involved in heme and hemoglobin biosynthesis. In addition, we found reduced expression of the hematopoietic stem cells (HSC) marker cmyb and HSC transcription factors tal1 and hoxb4a in Rpl11-deficient zebrafish embryos, indicating that the hematopoietic defects may be related to impaired HSC formation, differentiation, and proliferation. However, Rpl11 deficiency did not affect the development of other blood cell lineages such as granulocytes and myelocytes., Conclusion: We identified hematopoietic failure of Rpl11-deficient zebrafish embryos using transcriptome deep sequencing and elucidated potential underlying mechanisms. The present analyses demonstrate that Rpl11-deficient zebrafish may serve as a model of DBA and may provide insights into the pathogenesis of mutant RPL11-mediated human DBA disease.

The lack of knowledge about the mechanism of erythrocyte biogenesis through self-replication makes the in vitro generation of large quantities of cells difficult. We show that transduction of c-MYC and BCL-XL into multipotent hematopoietic progenitor cells derived from pluripotent stem cells and gene overexpression enable sustained exponential self-replication of glycophorin A(+) erythroblasts, which we term immortalized erythrocyte progenitor cells (imERYPCs). In an inducible expression system, turning off the overexpression of c-MYC and BCL-XL enabled imERYPCs to mature with chromatin condensation and reduced cell size, hemoglobin synthesis, downregulation of GCN5, upregulation of GATA1, and endogenous BCL-XL and RAF1, all of which appeared to recapitulate normal erythropoiesis. imERYPCs mostly displayed fetal-type hemoglobin and normal oxygen dissociation in vitro and circulation in immunodeficient mice following transfusion. Using critical factors to induce imERYPCs provides a model of erythrocyte biogenesis that could potentially contribute to a stable supply of erythrocytes for donor-independent transfusion.

Background: Although low preoperative haemoglobin (Hb) concentration is a well known risk factor for adverse outcome, little is known about decreases in Hb and postoperative Hb concentrations., Objectives: The aim of this study was to evaluate the prognostic impact of both pre- and postoperative Hb concentrations (divided into low, intermediate and high tertiles) as well as Hb decrease, defined as preoperative minus postoperative Hb (g dl(-1)), on postoperative cardiovascular events in vascular surgery patients., Design: A retrospective observational cohort study., Setting: Erasmus University Medical Centre, Rotterdam, the Netherlands, from 1 January 2002 to 31 December 2011., Patients: One thousand four hundred and eighty-four patients underwent elective open or endovascular abdominal aortic repair (aneurysm or stenosis), lower extremity arterial repair or carotid surgery. Patients for whom pre or postoperative Hb concentrations were not available were excluded., Main Outcome Measures: The study endpoint was 30-day postoperative cardiovascular events, including myocardial infarction, heart failure, arrhythmias, stroke, asymptomatic troponin-T release and cardiovascular death., Results: In 1041 patients, both pre and postoperative Hb concentrations were available. Thirty-day cardiovascular events occurred in 221 (21%) patients. Multivariable logistic regression analyses, adjusting for age, sex, Revised Cardiac Risk Index (high-risk surgery, coronary heart disease, heart failure, cerebrovascular disease, diabetes mellitus, renal insufficiency), hypertension and hypercholesterolaemia, demonstrated that low preoperative Hb (8.7 to 12.9 g dl(-1)) was associated with 30-day events [odds ratio (OR) 1.7; 95% confidence interval (CI) 1.1 to 2.5]. Intermediate (10.6 to 12.1 g dl(-1)) and low (7.4 to 10.5 g dl(-1)) postoperative Hb and Hb decrease were also associated with an independently increased risk of 30-day events (intermediate Hb: OR 1.7; 95% CI 1.1 to 2.7; low Hb: OR 3.1; 95% CI 2.0 to 4.8; and Hb decrease: OR 1.2; 95% CI 1.1 to 1.3, respectively). Sensitivity analyses excluding patients with transfusions (n=314) demonstrated that only postoperative Hb concentrations remained associated with a high risk of 30-day cardiovascular events (intermediate Hb: OR 1.8; 95% CI 1.0 to 3.3 and low Hb: OR 2.0; 95% CI 1.0 to 4.0)., Conclusion: Pre and postoperative Hb concentrations and Hb decrease are all related to 30-day cardiovascular events in elective vascular surgery patients. Postoperative Hb concentrations are the strongest predictor of 30-day cardiovascular events.

In this issue of Blood, Cooke et al demonstrate the potential of a fully human anti-hepcidin antibody as a novel therapeutic for iron-restricted anemias such as anemia of inflammation, cancer, or chronic kidney disease (formerly known as “anemia of chronic diseases”).

Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.

The physiological switch in expression of the embryonic, fetal, and adult β-like globin genes has garnered enormous attention from investigators interested in transcriptional mechanisms and the molecular basis of hemoglobinopathies. These efforts have led to the discovery of cell type-specific transcription factors, unprecedented mechanisms of transcriptional coregulator function, genome biology principles, unique contributions of nuclear organization to transcription and cell function, and promising therapeutic targets. Given the vast literature accrued on this topic, this article will focus on the master regulator of erythroid cell development and function GATA-1, its associated proteins, and its frontline role in controlling hemoglobin synthesis. GATA-1 is a crucial regulator of genes encoding hemoglobin subunits and heme biosynthetic enzymes. GATA-1-dependent mechanisms constitute an essential regulatory core that nucleates additional mechanisms to achieve the physiological control of hemoglobin synthesis.

Bone marrow is generally considered the main source of erythroid cells. Here we report that a single hypoxia-mimic chemical, CoCl2, can increase the size of fibroblasts and cancer cells and lead to formation of polyploidy giant cells (PGCs) or polyploidy giant cancer cells (PGCCs), activation of stem cell marker expression, increased growth of normal and cancer spheroid, and lead to differentiation of the fibroblasts and epithelial cells toward erythroid lineage expressing hemoglobins both in vitro and in vivo. Immunohistochemical examination demonstrated that these cells are predominantly made of embryonic hemoglobins, with various levels of fetal and adult hemoglobins. Ectopic expression of c-Myc induced the generation of nucleated erythoid cells expressing variable levels of embryonic and fetal hemoglobins. Generation of these erythroid cells can be also observed via histological examination of other cancer cell lines and human tumor samples. These data suggest that normal and solid cancer cells can directly generate erythroid cells to obtain oxygen in response to hypoxia and may explain the ineffectiveness of conventional anti-angiogenic therapies for cancer, which are directed at endothelium-dependent vessels, and offer new targets for intervention., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Erythropoiesis results from a complex combination of the expression of several transcription factor genes and cytokine signaling. However, the overall view of erythroid differentiation remains unclear. First, we screened for erythroid differentiation-related genes by comparing the expression profiles of high differentiation-inducible and low differentiation-inducible murine erythroleukemia cells. We identified that overexpression of α-1,6-fucosyltransferase (Fut8) inhibits hemoglobin production. FUT8 catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α-1,6 linkage, leading to core fucosylation in mammals. Expression of Fut8 was down-regulated during chemically induced differentiation of murine erythroleukemia cells. Additionally, expression of Fut8 was positively regulated by c-Myc and c-Myb, which are known as suppressors of erythroid differentiation. Second, we found that FUT8 is the only fucosyltransferase family member that inhibits hemoglobin production. Functional analysis of FUT8 revealed that the donor substrate-binding domain and a flexible loop play essential roles in inhibition of hemoglobin production. This result clearly demonstrates that core fucosylation inhibits hemoglobin production. Third, FUT8 also inhibited hemoglobin production of human erythroleukemia K562 cells. Finally, a short hairpin RNA study showed that FUT8 down-regulation induced hemoglobin production and increase of transferrin receptor/glycophorin A-positive cells in human erythroleukemia K562 cells. Our findings define FUT8 as a novel factor for hemoglobin production and demonstrate that core fucosylation plays an important role in erythroid differentiation.

As the use of single donor apheresis platelets and plateletpheresis procedures done steadily increases in India, the plateletpheresis donors are at an increased risk of postpheresis anemia and thrombocytopenia. This study was planned in order to evaluate the effect of plateletpheresis on the hematological parameters in the local donor population and to evaluate the possibility of following the 2day deferral for repeat plateletpheresis. A total of 60 plateletpheresis procedures performed over a period of 1year with CS 3000 Plus Baxter Fenwal were evaluated. All the post-donation hematological parameters showed a significant decrease from the pre-donation values (p<0.001). Post-donation hemoglobin <12g, which is defined by WHO as anemia irrespective of the gender occurred in 25% (n=15) of the donors. The post-donation platelet count was less than 100×10(9)/L in 16.6% (n=10) of the procedure. The ability of these donors to serve as repeat plateletpheresis donor after 2days as recommended by AABB needs to be evaluated by further studies done post donation. Keeping in view the hematological profile of our donor population we recommend that donors who already have a low or borderline pre-donation platelet count and hemoglobin should be assessed and monitored post-donation for decrements in these parameters. While serving as repeat donors, their pre donation hemoglobin and platelet counts should be tested again., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objective: Hemoglobin (Hb) is a prognostic factor in cervical cancer, but the underlying mechanisms remain unknown. In this study, we hypothesized that low Hb level, either before or during radiotherapy (RT), is a surrogate for a more infiltrative and therefore aggressive disease, with uterine corpus invasion and nodal metastases., Methods and Materials: Prospectively collected data of patients with locally advanced cervical cancer treated with curative intent using chemoradiation at a tertiary academic center was reviewed. All eligible patients had a positron emission tomographic scan and pelvic magnetic resonance imaging. Hemoglobin levels before RT and Hb nadir during RT were collected from the medical record., Results: The median follow-up for 263 eligible patients was 38.7 months. Ninety-six patients (36.5%) had both uterine corpus invasion and positron emission tomography-positive nodal disease (C+N+). Patients with pretreatment Hb level of less than 120 g/L were more likely to have C+N+ disease (47%) compared with patients with a high pretreatment Hb level (32%; P = 0.034). The 3-year disease-free survival and overall survival (OS) were significantly lower in the C+N+ group compared with the remaining patients (40.1% vs 76.1%, P < 0.001, and 59.7% vs 83.1%, P < 0.001, respectively). Patients with low Hb nadir were more likely to have a C+N+ disease (P < 0.001), and low Hb nadir during RT was significantly an indicator of a higher recurrence rate (P = 0.002) and lower OS (P < 0.001). In multifactor analysis, statistically significant prognostic factors for OS included histology, high-echelon nodal involvement, tumor volume on magnetic resonance imaging, C+N+ status, and Hb nadir during treatment. Pretreatment Hb level was not an independent prognostic factor., Conclusions: The combination of corpus invasion and nodal metastases is associated with lower Hb level and inferior prognosis. Because C+N+ state is related to tumor growth from early invasion to the time of presentation, it is unlikely that the correction of Hb level during treatment will have a major impact on outcome.

Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferrin saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound (58) Fe, the amount of (58) Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to bone morphogenetic protein (BMP)-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. In conclusion, testosterone inhibits hepcidin transcription through its interaction with BMP/Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells., (© 2013 The Authors Aging Cell © 2013 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Changes in the endocannabinoid system are implicated in numerous diseases, making it an attractive target for pharmaceutical development. The endocannabinoid receptors have traditionally been thought to act through the effects of lipophilic messengers called cannabinoids. The exciting finding of endocannabinoid system modulation by the nonapeptide hemopressin and its N-terminal extensions has highlighted the complexity of cannabinoid biology and pharmacology and sparked interest for therapeutic purposes. However, many questions surrounding the generation and regulation of the hemopressin peptides, the self-assembly of hemopressin and the potential for drug development based on hemopressin remain and are discussed in this review., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Diseases affecting hemoglobin synthesis and function are extremely common worldwide. More than 1000 naturally occurring human hemoglobin variants with single amino acid substitutions throughout the molecule have been discovered, mainly through their clinical and/or laboratory manifestations. These variants alter hemoglobin structure and biochemical properties with physiological effects ranging from insignificant to severe. Studies of these mutations in patients and in the laboratory have produced a wealth of information on hemoglobin biochemistry and biology with significant implications for hematology practice. More generally, landmark studies of hemoglobin performed over the past 60 years have established important paradigms for the disciplines of structural biology, genetics, biochemistry, and medicine. Here we review the major classes of hemoglobin variants, emphasizing general concepts and illustrative examples.

During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50-60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p < 0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.

Since the approval of recombinant insulin from Escherichia coli for its clinical use in the early 1980s, the amount of recombinant pharmaceutical proteins obtained by microbial fermentations has significantly increased. The recent advances in genomics together with high throughput analysis techniques (the so-called-omics approaches) and integrative approaches (systems biology) allow the development of novel microbial cell factories as valuable platforms for large scale production of therapeutic proteins. This review summarizes the main achievements and the current situation in the field of recombinant therapeutics using yeast Saccharomyces cerevisiae as a model platform, and discusses the future potential of this platform for production of blood proteins and substitutes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Menstruation is the expulsion of the endometrial lining of the uterus following a nearly month long preparation for embryo implantation and pregnancy. Increasingly, the health of the endometrium is being recognized as a critical factor in female fertility, and proteomes and transcriptomes from endometrial biopsies at different stages of the menstrual cycle have been studied for both diagnostic and therapeutic purposes (1 Kao, L. C., et al. 2003 Endocrinology 144, 2870-2881; Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617-630; DeSouza, L., et al. 2005 Proteomics 5, 270-281). Disorders of the uterus ranging from benign to malignant tumors, as well as endometriosis, can cause abnormal menstrual bleeding and are frequently diagnosed through endometrial biopsy (Strowitzki, Tet al. 2006 Hum. Reprod. Update 12, 617-630; Ferenczy, A. 2003 Maturitas 45, 1-14). Yet the proteome of menstrual blood, an easily available noninvasive source of endometrial tissue, has yet to be examined for possible causes or diagnoses of infertility or endometrial pathology. This study employed five different methods to define the menstrual blood proteome. A total of 1061 proteins were identified, 361 were found by at least two methods and 678 were identified by at least two peptides. When the menstrual blood proteome was compared with those of circulating blood (1774 proteins) and vaginal fluid (823 proteins), 385 proteins were found unique to menstrual blood. Gene ontology analysis and evaluation of these specific menstrual blood proteins identified pathways consistent with the processes of the normal endometrial cycle. Several of the proteins unique to menstrual blood suggest that extramedullary uterine hematopoiesis or parenchymal hemoglobin synthesis may be occurring in late endometrial tissue. The establishment of a normal menstrual blood proteome is necessary for the evaluation of its usefulness as a diagnostic tool for infertility and uterine pathologies. Identification of unique menstrual blood proteins should aid the forensic community in distinguishing menstrual blood from circulating blood.

Anemia is a major clinical symptom of a wide variety of pathological conditions a common related to reduced erythropoiesis. Whereas erythropoietin treatment showed an improvement in the patients' condition, it revealed increased risks of thromboembolic and cardiovascular events. Herein we describe stimulation of erythropoiesis by the multifunctional 1-(butyryloxy)ethyl-5-amino-4-oxopentanoate, (AlaAcBu), a 5-aminolevulinic-acid (ALA) derivative, which undergoes metabolic hydrolysis yielding two erythroid differentiation inducers, ALA and butyric acid (BA), each acting through a different mechanism. ALA, the first precursor in the heme biosynthesis, accelerates heme synthesis and BA, a histone deacetylase inhibitor (HDACI) that activates the transcription of globin mRNA. Our results show that the AlaAcBu mutual prodrug is a potent chemical differentiation inducer of K562 human erythroleukemia cells manifested by augmentation of heme and globin synthesis and assembly of hemoglobin. Exposure of K-562 cells to AlaAcBu resulted in an increase in heme synthesis and globin expression. Stimulation of the heme pathway was evident by the over-expression of porphobilinogen deaminase (PBGD) and ferrochelatase. AlaAcBu promoted cellular erythroid differentiation depicted by the expression of the marker glycophorin A and cellular maturation characterized by cytoplasm hemoglobinization, polar arrangement of mitochondria and a developed central vacuolar system preceding nuclear extrusion. The ability of AlaAcBu to promote differentiation along the erythroid lineage and to dramatically induce hemoglobin synthesis presented in this report., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: Kawasaki disease (KD) is a systemic febrile vasculitis complicated by coronary artery lesions (CAL). Anemia is common in patients with KD and is associated with a prolonged duration of active inflammation. Hepcidin is a central modulator of inflammation-associated anemia, acting via control of iron absorption and a direct inhibitory effect on erythropoiesis. The aims of this study were to investigate the role of inflammation-induced hepcidin in the development of anemia, the occurrence of CAL formation, and IVIG treatment response in patients with KD., Methods: Eighty-six KD patients and 30 febrile controls were enrolled. Levels of interleukin (IL)-6 and serum hepcidin were measured in sera by enzyme-linked immunosorbent assay. Hemoglobin and serum iron levels were also measured., Results: Hemoglobin and iron levels were lower in KD patients than in controls (p < 0.001 and p = 0.009, respectively). Serum hepcidin and IL-6 levels were higher in KD patients than in controls (both p < 0.001) before intravenous immunoglobulin (IVIG) treatment. After IVIG treatment, serum hepcidin, IL-6, and hemoglobin levels decreased significantly (all p < 0.001). In addition, the serum hepcidin levels before IVIG treatment were negatively correlated with hemoglobin levels after IVIG treatment (R = -0.188, p = 0.046) and positively correlated with the changes of hemoglobin levels after IVIG treatment (R = 0.269, p = 0.015). Furthermore, serum hepcidin levels were negatively correlated with serum iron levels (R = -0.412, p = 0.002), which were positively correlated with hemoglobin levels (R = 0.210, p = 0.045). Additionally, the change of hepcidin levels was associated with IVIG treatment response and the occurrence of CAL formation., Conclusions: Inappropriately raised hepcidin levels impair iron metabolism and are associated with decreased hemoglobin levels in KD patients. Inflammation-induced hepcidin is associated with the development of anemia and disease outcomes in patients with KD.

Introduction: The number of cases of lead poisoning (LP), a widely known disease with various aetiologies, being reported globally has decreased over the years due to both limited domestic applications of lead and enforcement of stringent safety measures. However, a new presentation of lead poisoning, lead-contaminated opium (LCO), is gradually emerging in our region. This study aimed to determine the prevalence and clinical effects of lead toxicity associated with opium use., Methods: Between November 2006 and December 2007, all patients diagnosed with LP at a central laboratory in Tehran, Iran, were assessed for potential causes of poisoning. Patients with a history of LCO abuse were evaluated and recruited for the study., Results: Overall, there were 240 patients with LP, and poisoning from LCO was diagnosed in 25 patients. The duration of addiction was between three months and 40 years, and the duration of symptoms was 28.1 ± 17.7 days. Mean blood lead levels of the patients were 145 ± 61 (range 61-323) μg/dL. The average creatinine and haemoglobin levels were 77.4 ± 8.1 μmol/L and 105 ± 25 g/L, respectively. The association between the duration of addiction and levels of lead in blood was not statistically significant (r = -0.142, p = 0.54). The most common symptoms were gastrointestinal complaints, followed by musculoskeletal complaints with muscle weakness (92%). Anorexia was also a leading complaint., Conclusion: The results of our study suggest that the possibility of LP should be considered with high suspicion among opium users presenting with acute abdominal symptoms.

1. Hydroxyurea (HU) is a drug used for the treatment of haemoglobinopathies. Hydroxyurea functions by upregulating γ-globin transcription and fetal haemoglobin (HbF) production in erythroid cells. The K562 erythroleukaemia cell line is widely used as a model system in which to study the mechanism of γ-globin induction by HU. However, the transcription factors required for the upregulation of γ-globin expression by HU in K562 cells have not been identified. Similarities between the HU and sodium butyrate (SB) pathways suggest cAMP response element-binding protein (CREB) 1 as a potential candidate. Thus, the aim of the present study was to investigate the possible role of CREB1 in the HU pathway. 2. Experiments were performed using transient and stable RNA interference (RNAi) to show that CREB1 is necessary for HU-mediated induction of γ-globin expression and haemoglobin production in K562 cells. 3. Furthermore, western blot analyses demonstrated that CREB1 becomes phosphorylated in a dose-dependent manner after HU (100-400 µmol/L) treatment of K562 cells for 72 h. 4. We also investigated role of a Gγ promoter CREB1 response element (G-CRE) in this pathway. Quantitative amplification refractory mutation system-polymerase chain reaction experiments were performed to demonstrate that HU induces the expression of both Gγ and Aγ in this cell line. In addition, electrophoretic mobility shift assays were used to show that levels of CREB1 complexes binding to the G-CRE site are increased following HU treatment and are decreased in CREB1-knockdown cells. 5. The results suggest that CREB1 is necessary for γ-globin induction by HU in K562 cells, a role that may be mediated, in part, through the G-CRE element., (© 2012 The Authors Clinical and Experimental Pharmacology and Physiology © 2012 Blackwell Publishing Asia Pty Ltd.)

Resistance exercise increases heme synthesis in the bone marrow and the hemoglobin in iron-deficient rats. Post-exercise early nutrient provision facilitates skeletal muscle protein synthesis compared to late provision. However, the effects of post-exercise nutrition timing on hemoglobin synthesis are unclear. The current study investigated the effect of post-exercise meal timing on the activity of the key enzyme involved in hemoglobin synthesis, δ-aminolevulinic acid dehydratase (ALAD), in the bone marrow and examined the hemoglobin concentration in iron-deficient rats. Male 4-week-old Sprague-Dawley rats were fed an iron-deficient diet containing 12 mg iron/kg and performed climbing exercise (5 min × 6 sets/day, 3 days/week) for 3 weeks. The rats were divided into a group fed a post-exercise meal early after exercise (E) or a group fed the meal 4 h after exercise (L). A single bout of exercise performed after the 3-week training period increased the bone marrow ALAD activity, plasma iron concentration, and transferrin saturation. Although the plasma iron concentration and transferrin saturation were lower in the E group than the L group after a single bout of exercise, the basal hematocrit, hemoglobin, and TIBC after 3 weeks did not differ between the groups. Therefore, resistance exercise increases the bone marrow ALAD activity, while the post-exercise meal timing has no effect on the hemoglobin concentration in iron-deficient rats.

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

Lower hemoglobin levels have been associated with cognitive decline in older persons. The objective of this study was to investigate the relationship between lower hemoglobin levels and common, age-related neuropathologies associated with cognitive decline. Hemoglobin and neuropathology measures were available in 113 deceased, community-dwelling, older adults participating in the Rush Memory and Aging Project, a prospective, observational, clinical pathology study of aging. The mean hemoglobin level was 13.0 g/dL (SD = 1.4) and was measured 3.2 (SD = 1.3) years prior to death. Thirty-five participants had at least one chronic macroscopic infarction and twenty-nine had at least one chronic microscopic infarction. Eleven participants had Lewy Bodies. The mean Alzheimer's disease pathology score based on a summary measure of neuritic plaques, diffuse plaques, and neurofibrillary tangles was 0.56 unit (SD = 0.56; range = 0, 2.34). Using logistic regression models adjusted for age at death, gender, and education, each g/dL lower hemoglobin level increased the odds for having a chronic macroscopic infarction by 37% (95% CI = 1.01, 1.86) but not for having a chronic microscopic infarction (OR = 1.11; 95% CI = 0.82, 1.52) or Lewy Bodies (OR = 1.07; 95% CI = 0.68, 1.68). In an adjusted multiple regression model, hemoglobin level was not associated with the global AD pathology measure (parameter estimate = -0.02, SE = 0.03, p = 0.6). In secondary analyses, lower hemoglobin levels were associated with higher odds of having a chronic macroscopic infarction in a subcortical region but not with higher total subcortical chronic macroscopic infarction volume. In conclusion, lower hemoglobin levels appear to be associated with chronic macroscopic infarctions but not other common age-related neuropathologies.

Introduction: Although genital tuberculosis is a condition that is prevalent worldwide, it is still a diagnostic dilemma. This study aimed to find an effective diagnostic modality for the condition., Methods: A total of 100 infertile women were clinically evaluated with haemoglobin estimation, total and differential count, Mantoux test, tubercle bacilli enzyme-linked immunosorbent assay (TB ELISA), hysterosalpingography, pelvic ultrasonography, laparohysteroscopy, premenstrual endometrial biopsy for histopathology, culture and tubercle bacilli polymerase chain reaction (TBPCR). The womens' Day 2 hormonal profile (luteinising, follicle-stimulating, prolactin and thyroidstimulating hormones) and their husbands' semen analysis were also conducted., Results: A total of 58 women had primary infertility and 42 had secondary infertility. Female factor infertility was present in 63 percent of the cases (mostly tubal; 45.97 percent). 26 women tested positive for endometrial TBPCR. Erythrocyte sedimentation rate, Mantoux test, TB ELISA and hysterosalpingography were found to have high negative predictive value (greater than 80 percent), while the positive predictive value was 35-45 percent. Laparoscopy findings were suggestive of tuberculosis in 13 percent of the women, out of which 83.3 percent were positive for endometrial TBPCR. Hysteroscopy revealed intrauterine adhesions in 34.8 percent of the women, with 68.8 percent being positive for tubercular bacilli., Conclusion: Our study established that in cases of genital tuberculosis, the use of expensive endometrial TBPCR tests may be avoided with a detailed workup, which would also help in the institution of anti-tubercular treatment in early disease, thus enhancing the chance of pregnancy.

Background: Our objective was to compare the capacities of biofortified and standard colored beans to deliver iron (Fe) for hemoglobin synthesis. Two isolines of large-seeded, red mottled Andean beans (Phaseolus vulgaris L.), one standard ("Low Fe") and the other biofortified ("High Fe") in Fe (49 and 71 μg Fe/g, respectively) were used. This commercial class of red mottled beans is the preferred varietal type for most of the Caribbean and Eastern and Southern Africa where almost three quarters of a million hectares are grown. Therefore it is important to know the affect of biofortification of these beans on diets that simulate human feeding studies., Methods: Maize-based diets containing the beans were formulated to meet the nutrient requirements for broiler except for Fe (Fe concentrations in the 2 diets were 42.9 ± 1.2 and 54.6 ± 0.9 mg/kg). One day old chicks (Gallus gallus) were allocated to the experimental diets (n = 12). For 4 wk, hemoglobin, feed-consumption and body-weights were measured., Results: Hemoglobin maintenance efficiencies (HME) (means ± SEM) were different between groups on days 14 and 21 of the experiment (P < 0.05). Final total body hemoglobin Fe contents were different between the standard (12.58 ± 1.0 mg {0.228 ± 0.01 μmol}) and high Fe (15.04 ± 0.65 mg {0.273 ± 0.01 μmol}) bean groups (P < 0.05). At the end of the experiment, tissue samples were collected from the intestinal duodenum and liver for further analyses. Divalent-metal-transporter-1, duodenal-cytochrome-B, and ferroportin expressions were higher and liver ferritin was lower (P < 0.05) in the standard group vs. the biofortified group. In-vitro analysis showed lower iron bioavailability in cells exposed to standard ("Low Fe") bean based diet., Conclusions: We conclude that the in-vivo results support the in-vitro observations; biofortified colored beans contain more bioavailable-iron than standard colored beans. In addition, biofortified beans seems to be a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume these beans as a dietary staple. This justifies further work on the large-seeded Andean beans which are the staple of a large-region of Africa where iron-deficiency anemia is a primary cause of infant death and poor health status.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

We describe a method for production of recombinant human hemoglobin by Escherichia coli grown in a bioreactor. E. coli BL21(DE3) transformed with a plasmid containing hemoglobin genes and Plesiomonas shigelloides heme transport genes reached a cell dry weight of 83.64 g/liter and produced 11.92 g/liter of hemoglobin in clarified lysates.

Atherothrombosis remains one of the main causes of morbidity and mortality in the western countries. Human atherothrombotic disease begins early in life in relation to circulating lipid retention in the inner vascular wall. Risk factors enhance the progression towards clinical expression: dyslipidaemia, diabetes, smoking, hypertension, ageing, etc. The evolution from the initial lipid retention in the arterial wall to clinical events is a continuum of increasingly complex biological processes. Current strategies to fight the consequences of atherothrombosis are orientated either towards the promotion of a healthy life style and preventive treatment of risk factors, or towards late interventional strategies. Despite this therapeutic arsenal, the incidence of clinical events remains dramatically high, dependent, at least in part, on the increasing frequency of type 2 diabetes and ageing. But some medical treatments, focusing only on prevention of the metabolic risk, have failed to reduce cardiovascular mortality, thus illustrating that our understanding of the pathophysiology of human atherothrombosis leading to clinical events remain incomplete. New paradigms are now emerging which may give rise to novel experimental strategies to improve therapeutic efficacy and prediction of disease progression. Recent studies strengthen the concept that the intraplaque neovascularization and bleeding (Figure 1, upper panel) are events that could play a major role in plaque progression and leucocyte infiltration, and may also serve as a measure of risk for the development of future events. The recent advances in our understanding of IntraPlaque Hemorrhage as a critical event in triggering acute clinical events have important implications for clinical research and possibly future clinical practice.

Introduction: A quarter of patients with involuntary weight loss (IWL) have cancer. Inflammation and anemia are associated with cancer, and recent studies showed that red blood cell distribution width (RDW) is a predictor of mortality, including cancer-related death. The aim of this study was to assess the ability of routine hematological and inflammation parameters to diagnose cancer in patients with IWL., Materials and Methods: A total of 253 consecutive patients with IWL admitted in a secondary care university hospital were included. Routine hematological and inflammatory parameters (hemoglobin level, mean corpuscular volume, RDW, serum iron level, erythrocyte sedimentation rate, C-reactive protein level, and ferritin level) were recorded for all patients. The investigative workup was not standardized, but the patients were followed up for 6 months to avoid misclassification concerning the final diagnosis., Results: All parameters, excepting mean cellular volume, were statistically associated with cancer. The areas under the curve were 0.708 (95% confidence interval [CI], 0.627-0.790) for C-reactive protein level, 0.690 (95% CI: 0.620-0.760) for erythrocyte sedimentation rate, 0.651 (95% CI, 0.566-0.735) for serum iron level, 0.607 (95% CI, 0.526-0.687) for hemoglobin level, 0.598 (95% CI, 0.518-0.679) for ferritin level, 0.594 (95% CI, 0.517-0.671) for RDW, and 0.561 (95% CI, 0.474-0.649) for mean cellular volume. In the multivariable analysis, only erythrocyte sedimentation rate remained associated with cancer., Conclusions: In patients with IWL, the hematological and inflammation parameters were statistically different in patients with cancer and in those without cancer. However, in clinical practice, they were modest diagnostic tests for cancer.

Paratuberculosis (Johne's disease) is a chronic granulomatous enteritis affecting ruminants and other species. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF-MS) was used as a platform to identify candidate biomarkers from sheep serum. Multivariate biomarker models which aimed to differentiate sheep with paratuberculosis and vaccinated-exposed sheep from unexposed animals were proposed based on classification and regression tree (CART) and linear discriminant analysis (LDA) algorithms from two array types. The accuracy of classification of sheep into unexposed or exposed groups ranged from 75 to 100% among models. SELDI was used to monitor protein profile changes over time during an experimental infection trial by examining sera collected at 4-, 8- and 13-months post infection. Although three different SELDI instruments were used, nine consistent proteomic features were observed associated with exposure to MAP. Two of the putative serum biomarkers were purified from serum using chromatographic methods and were identified as transthyretin and alpha haemoglobin by tandem mass spectrometry. They belong to highly abundant, acute phase reactants in the serum proteome and have also been discovered as serum biomarkers in human inflammatory conditions and cancer. Their relationship to the pathogenesis of Johne's disease remains to be elucidated., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Benzene is a common occupational hazard and a ubiquitous environmental pollutant. Benzene exposure at the levels even below 1ppm still showed hematotoxicity. It is widely accepted that the metabolites of benzene play important roles in the benzene toxicity to the hematopoietic system, but little is known about the effects of benzene metabolites on erythropoiesis. In present study, erythroid progenitor-like K562 cells were used to determine the effects of phenolic metabolites of benzene, including phenol, hydroquinone and 1,2,4-benzenetriol, on the erythroid differentiation. After the treatment with these benzene metabolites at the concentrations with no obvious cytotoxicity, the hemin-induced hemoglobin synthesis in K562 cells decreased in a concentration- and time-dependent manner, and the expression of CD71 and GPA protein on the surface of K562 cells was also inhibited. The reverse transcription-PCR was used to determine the mRNA level of the erythroid related genes in the K562 cells that were treated with benzene metabolites. The hemin-induced expression of globin genes, including α-, β- and γ-globin genes, and the gene encoding the heme synthesis enzyme porphobilinogen deaminase was inhibited by benzene metabolites. When the K562 cells were pretreated with benzene metabolites, the hemin-induced expression of two transcription factor genes GATA-1 and NF-E2 was distinctly reduced, and the pre-treatment with benzene metabolites promoted the decrease of the mRNA level of transcription factor gene GATA-2 by hemin. These results indicated that benzene metabolites inhibited the hemin-induced erythroid differentiation through affecting the transcription of the erythroid related genes., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Erythropoiesis is a coordinated process by which RBCs are produced. Erythropoietin, a kidney-derived hormone, and iron are critical for the production of oxygen-carrying mature RBCs. To meet the high demands of iron during erythropoiesis, small intestinal iron absorption is increased through an undefined mechanism. In this study, erythropoietic induction of iron absorption was further investigated. Hypoxia-inducible factor-2α (HIF-2α) signaling was activated in the small intestine during erythropoiesis. Genetic disruption of HIF-2α in the intestine abolished the increase in iron absorption genes as assessed by quantitative real-time reverse transcription-PCR and Western blot analyses. Moreover, the increase in serum iron following induction of erythropoiesis was entirely dependent on intestinal HIF-2α expression. Complete blood count analysis demonstrated that disruption of intestinal HIF-2α inhibited efficient erythropoiesis; mice disrupted for HIF-2α demonstrated lower hematocrit, RBCs, and Hb compared with wild-type mice. These data further cement the essential role of HIF-2α in regulating iron absorption and also demonstrate that hypoxia sensing in the intestine, as well as in the kidney, is essential for regulation of erythropoiesis by HIF-2α.