Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus).

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Characterization and Expression Analysis of Genes from Megalobrama amblycephala Encoding Hemoglobins with Extracellular Microbicidal Activity.

Hemoglobin (Hb) usually comprises two α and two ß subunits, forming a tetramer responsible for oxygen transportation and storage. Few studies have elucidated fish hemoglobin immune functions. Megalobrama amblycephala is a freshwater-cultured fish prevalent in China. We identified two M. amblycephala hemoglobin subunits and analyzed their expression patterns and antibacterial activities. The respective full-length cDNA sequences of the M. amblycephala Hb α (MaHbα) and ß (MaHbß) subunits were 588 and 603 bp, encoding 143 and 148 amino acids. MaHbα and MaHbß were highly homologous to hemoglobins from other fish, displaying typical globin-like domains, most heme-binding sites, and tetramer interface regions highly conserved in teleosts. In phylogenetic analyses, the hemoglobin genes from M. amblycephala and other cypriniformes clustered into one branch, and those from other fishes and mammals clustered into other branches, revealing fish hemoglobin conservation. These M. amblycephala Hb subunits exhibit different expression patterns in various tissues and during development. MaHbα is mainly expressed in the blood and brain, while MaHbß gene expression is highest in the muscle. MaHbα expression was detectable and abundant post-fertilization, with levels fluctuating during the developmental stages. MaHbß expression began at 3 dph and gradually increased. Expression of both M. amblycephala Hb subunits was down-regulated in most examined tissues and time points post-Aeromonas hydrophila infection, which might be due to red blood cell (RBC) and hematopoietic organ damage. Synthetic MaHbα and MaHbß peptides showed excellent antimicrobial activities, which could inhibit survival and growth in five aquatic pathogens. Two M. amblycephala hemoglobin subunits were identified, and their expression patterns and antibacterial activities were analyzed, thereby providing a basis for the understanding of evolution and functions of fish hemoglobins.

Saliva proteomic profile of early childhood caries and caries-free children.

OBJECTIVE: Saliva plays an important antimicrobial role and it is related to the pathogenesis of early childhood caries (ECC). The aim of this study was to compare the proteomic profile of unstimulated saliva of children aged 3-5 years who had ECC and caries-free (CF) children. MATERIALS AND METHODS: After the saliva collection from 20 children (ECC: n = 10; CF: n = 10), the samples were processed for proteomic analysis on a mass spectrometer. RESULTS: 1638 proteins were identified, of which 355 were present in both groups. A total of 579 proteins were exclusively identified in the CF group and included Leucine-rich alpha-2-glycoprotein, Protein S100-A5, Protein S100-A8 and Mucin-2. Moreover, 704 proteins were exclusively identified in the ECC group, including Enamelin. The differential expression analysis revealed that 112 proteins were up-regulated in the CF group. Among these proteins, we highlighted Hemoglobin subunit gamma-1 (343-fold increase), gamma-2 (336-fold increase) and alpha (40-fold increase). CONCLUSIONS: The proteomic profile of the saliva varied substantially between the groups. Hemoglobin subunit gamma-1, gamma-2 and alpha may play a protective role in children with ECC. These proteins should be evaluated in future studies, because they may be possible good candidates to be included in anti-caries dental products.

Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.

TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.

Advances in mass spectrometric methods for detection of hemoglobin disorders.

Hemoglobin disorders are caused due to alterations in the hemoglobin molecules. These disorders are categorized in two broad classes - hemoglobin variants and thalassemias. The hemoglobin variants arise due to point mutations in the alpha (α), beta (ß), gamma (γ), delta (δ), or epsilon (ε) globin chains of these proteins, while thalassemias are caused due to the under-production of α or ß globin chain. Hemoglobin disorders account for 7 % of the major health issues globally. Mass Spectrometry is an extensively used analytical tool in the field of protein identification, protein-protein interaction, biomarker discovery and diagnosis of several impairments including hemoglobin related disorders. The remarkable advancements in the technology and method development have enormously augmented the clinical significance of mass spectrometry in these fields. The present review describes hemoglobin disorders and the recent advancements in mass spectrometry in the detection of such disorders, including its advantages, lacunae, and future directions. The literature evidence concludes that mass spectrometry can be potentially used as a 'First Line Screening Assay' for the detection of hemoglobin disorders in the near future.

Hemoglobin subunit beta interacts with the capsid, RdRp and VPg proteins, and antagonizes the replication of rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease virus (RHDV) causes a highly contagious disease in rabbits that is associated with high mortality. Because of the lack of a suitable cell culture system for RHDV, its pathogenic mechanism and replication remain unclear. This study found that the expression level of host protein rabbit hemoglobin subunit beta (HBB) was significantly downregulated in RHDV-infected cells. To investigate the role of HBB in RHDV replication, small interfering RNAs for HBB and HBB eukaryotic expression plasmids were used to change the expression level of HBB in RK-13 cells and the results showed that the RHDV replication level was negatively correlated with the expression level of HBB. It was also verified that HBB inhibited RHDV replication using constructed HBB stable overexpression cell lines and HBB knockout cell lines. The interaction of HBB with viral capsid protein VP60, replicase RdRp, and VPg protein was confirmed, as was the activation of the expression of interferon γ by HBB. The results of this study indicated that HBB may be an important host protein in host resistance to RHDV infection.

Genetic signature related to heme-hemoglobin metabolism pathway in sepsis secondary to pneumonia.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated inflammatory response to pathogens. Bioinformatics and transcriptomics studies contribute to get a better understanding of the pathogenesis of sepsis. These studies revealed differentially expressed genes (DEGs) in sepsis involved in several pathways. Here we investigated the gene expression profiles of blood leukocytes using three microarray datasets of sepsis secondary to pneumonia, focusing on the heme/hemoglobin metabolism pathway. We demonstrate that the heme/hemoglobin metabolism pathway was found to be enriched in these three cohorts with four common genes (ALAS2, AHSP, HBD, and CA1). Several studies show that these four genes are involved in the cytoprotection of non-erythrocyte cells in response to different stress conditions. The upregulation of heme/hemoglobin metabolism in sepsis might be a protective response of white cells to the hostile environment present in septic patients (follow-up samples).

Heterogeneity between Two α Subunits of α2ß2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the ß subunit of α2ß2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)ß(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the ß subunit: Hb M Hyde Park (ß92His → Tyr), Hb M Saskatoon (ß63His → Tyr), and Hb M Milwaukee (ß67Val → Glu). In contrast with the ß subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2ß2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)ß(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)ß(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)ß(Fe3+)ß(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2ß2 tetramer changes the other α subunit to the R structure.

Fetal hemoglobin regulation in ß-thalassemia: heterogeneity, modifiers and therapeutic approaches.

INTRODUCTION: Stress erythropoiesis induces fetal hemoglobin (HbF) expression in ß-thalassemias, however the level of expression is highly variable. The last decade has seen dramatic advances in our understanding of the molecular regulators of HbF production and the genetic factors associated with HbF levels, leading to the promise of new methods of the clinical induction of HbF. Areas covered: This article will review the heterogeneity and genetic modifiers of HbF and HbF induction therapy in ß-thalassemia. Expert commentary: One promising curative ß-thalassemia therapy is to induce HbF synthesis in ß-thalassemic erythrocytes to therapeutic levels before clinical symptom occurs. Further understanding of HbF level variation and regulation is needed in order to predict the response from HbF-inducing approaches.

Identification and Expression Analysis of Upregulated Genes in the Resting Egg-Producing Water Flea (Daphnia pulex).

Water fleas (Daphnia pulex) normally produce subitaneous eggs that initiate development immediately after oviposition. However, in response to habitat degradation, resting eggs are produced, which are enclosed in a sturdy outer envelope (ephippium) and can survive in harsh environments for an extended time. To understand the molecular mechanism underlying resting egg production in D. pulex, we investigated the genes whose expression patterns played a role in the production and identified the following six candidate genes: Dpfa-1, Dpfa-2, Dpep-1, Dpep-2, Dpep-3, and Dpep-4. These six genes displayed > 40-fold higher expression levels in resting egg-producing animals compared with those in subitaneous egg-producing animals at the period when the ovaries were mature. Dpfa-1 and Dpfa-2 were expressed in the fat cells, and their expression patterns were synchronized with the development of resting egg oocytes in the ovary. In contrast, Dpep-1-4 were expressed in the morphologically altered epidermal cells of the brood chamber with the formation of the ephippium, and their expression patterns were also related to ephippium formation. Our results suggest that the former two genes encode the resting egg-specific components produced by fat cells and that the latter four genes encode the components related to the ephippium formation synthesized by epidermal cells.

Early diagnosis of sepsis using serum hemoglobin subunit Beta.

The development of new sepsis-specific biomarkers is mandatory to improve the detection and monitoring of the disease. Hemoglobin is the main oxygen and carbon dioxide carrier in cells of the erythroid lineage and is responsible for oxygen delivery to the respiring tissues of the body. Hemoglobin subunit beta (HBß) is a component of a larger protein called hemoglobin. The aim of this study was to evaluate blood levels of HBß in septic patients. A prospective study of 82 patients with sepsis was conducted. Furthermore, C57BL/6 mice were subjected to cecal ligation and puncture (CLP) surgery. Alternatively, human umbilical vein endothelial cells (HUVECs) or C57BL/6 mice were exposed to lipopolysaccharide (LPS, 100 ng/ml to HUVECs or 10 mg/kg to mice). The data showed that LPS induced upregulation of the synthesis and secretion of HBß in LPS-treated HUVECs and in LPS-injected and CLP mice. In patients admitted to the intensive care unit with sepsis, circulating levels of HBß were significantly high (sepsis, 64.93-114.76 ng/ml, n = 30; severe sepsis, 157.37-268.69 ng/ml, n = 22; septic shock, 309.98-427.03 ng/ml, n = 30) when compared to the levels of control donors (9.76-12.28 ng/ml, n = 21). Patients with septic shock had higher HBß levels when compared to patients with severe sepsis. Furthermore, the HBß levels in septic patients were higher than those in healthy volunteers. These results suggest that in septic patients, HBß blood level is related to the severity of sepsis and may represent a novel endothelial cell dysfunction marker. Moreover, HBß can be used as a biomarker to determine the severity of sepsis.

Multicolor staining of globin subtypes reveals impaired globin switching during erythropoiesis in human pluripotent stem cells.

Adult hemoglobin composed of α- and ß-globin reflects a change from expression of embryonic ε- and fetal γ-globin to adult ß-globin in human erythroid cells, so-called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ-globin with little ß-globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of ß-globin and the corresponding silencing of γ-globin at the single-cell level during cord blood CD34(+) cell-derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous ß-globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ-globin. These hPSC-derived erythroid cells also displayed reduced expression of BCL11A-L. However, doxycycline-induced overexpression of BCL11A-L in selected hPSCs promoted γ-globin silencing. These results strongly suggest that impaired γ-globin silencing is associated with downregulated BCL11A-L in hPSC-derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.

Hemoglobin Bohr effects: atomic origin of the histidine residue contributions.

The Bohr effect in hemoglobin, which refers to the dependence of the oxygen affinity on the pH, plays an important role in its cooperativity and physiological function. The dominant contribution to the Bohr effect arises from the difference in the pKa values of His residues of the unliganded (deoxy) and liganded (carbonmonoxy) structures. Using recent high resolution structures, the residue pKa values corresponding to the two structures are calculated. The method is based on determining the electrostatic interactions between residues in the protein, relative to those of the residue in solution, by use of the linearized finite difference Poisson-Boltzmann equation and Monte Carlo sampling of protonation states. Given that good agreement is obtained with the available experimental values for the contribution of His residues in HbA to the Bohr effect, the calculated results are used to determine the atomic origin of the pKa shift between deoxy and carbonmonoxy HbA. The contributions to the pKa shift calculated by means of the linear response approximation show that the salt bridge involving His146 plays an important role in the alkaline Bohr effect, as suggested by Perutz but that other interactions are significant as well. A corresponding analysis is made for the contribution of His143 to the acid Bohr effect for which there is no proposed explanation. The method used is summarized and the program by which it is implemented is described in the Appendix .

Proteomic analysis of the effect of triterpenes from Patrinia heterophylla on leukemia K562 cells.

For centuries, Patrinia heterophylla had been used in China to treat many diseases including tumor. Triterpenes has been identified as the major active constituents in Patrinia heterophylla. To elucidate the antitumor mechanism of triterpenes from Patrinia heterophylla1 (TPH), a proteomic analysis is carried out with TPH treatment in K562 cells. The total proteins extracted from TPH treated K562 cells are analyzed by two dimensional gel electrophoresis (2-DE) and compared with those untreated K562 cells. Mass spectrometry is applied to identify the differentially expressed proteins. Twenty-three differentially expressed significant proteins are discovered. Eight proteins are later identified by mass spectrometry (MALDI-TOF-MS) and Mascot software. Among them, four proteins are up-regulated (Aldolase A, Glyceraldehyde-3-phosphate dehydrogenase, Flavin reductase and Hemoglobin subunit) and four proteins were down-regulated (Heat-shock protein 90 〈Alpha〉 (HSP90-〈Alpha〉), Eukaryotic translation initiation factor 5A, Moesin, tublin) by TPH treatment in K562 cells. The identified proteins are associated with energy metabolism, oxidative stress, apoptosis, signal transduction, differential induction, and protein biosynthesis. These findings might provide valuable insights into the antitumor mechanism of TPH in K562 cells.

Swim bladder nematodes (Anguillicoloides crassus) disturb silvering in European eels (Anguilla anguilla).

The introduced parasite Anguillicoloides crassus is thought to play an important role in the decline of freshwater eel (Anguilla spp.) populations. These nematodes are known to negatively affect many fitness-related traits in eels. We used experimental infections to study the effect of A. crassus on the relative size or mass of organs, and the expression of functionally relevant genes (total of 12 parameters) that are involved in the silvering process of Anguilla anguilla. Our results showed that the liver mass, the hemoglobin α-chain, and androgen receptors α expression levels were significantly higher in infected eels, whereas the freshwater rod opsin expression level and the gut mass were significantly lower in infected eels. Our results suggested that infected eels were at a more advanced stage in the silvering process than uninfected counterparts of similar size. These results may be explained by 2 hypotheses. First, A. crassus could trigger physiological mechanisms involved in the silvering process as a side-effect of infection. Second, eels may adjust their life history traits in response to infection. The implications for eel migration and reproductive success may be either negative or positive, depending on whether the response to A. crassus infection results in an additional cost of the parasite or is due to the phenotypic plasticity of the host.

Relationship between maternal hemoglobin concentration and neonatal birth weight.

OBJECTIVE: Pregnancy considerably increases iron needs in a mother and her fetus. The purpose of this study was to assess the relationship between maternal hemoglobin concentration in labor with neonatal birth weight. METHODS: A population-based study in Shahid Sadughi Hospital in Yazd, Iran, was performed by comparing 1842 singleton term pregnancies of patients with and without anemia and their newborns. Maternal characteristics, including hemoglobin values, were recorded at the labor visit. Maternal anemia was defined as hemoglobin concentration lower than 10 g/dl during pregnancy. Main outcome measures included birth weight and Apgar score. Linear and logistic regression models were used to analyze data. RESULTS: Anemia (Hb < 10 g/dl) was associated with a significantly increased risk of low birth weight (< 2500 g). High hemoglobin (> 13 g/dl) increased the risk of low birth weight but it was not significant. The risk of a low Apgar score was significantly increased in women with anemia. The minimum incidence of low birth weight occurs in association with a hemoglobin concentration of 10-13 g/dl. CONCLUSIONS: Maternal anemia was significantly associated with effect on birth weight. Also Hb > 13 g/dl was also associated with an increased risk of low birth weight.

Striped mullet (Mugil cephalus) hemoglobin system: multiplicity and functional properties.

The most frequent (90%) phenotype of the hemoglobin system of M. cephalus presented two major hemoglobins, the more anodal HbI accounting for approximately 70% of the total. The two hemoglobin components separated by ion-exchange chromatography were analyzed by reverse-phase HPLC and electrospray ionization-mass spectrometry which revealed a more complex pattern: HbI consists in four different globins, two ß (named ß1 and ß3) and two co-eluting α chains (α1 and α2); HbII consists in three globins, one ß chain (named ß2) and the same α1 and α2 present in HbI. The oxygen-binding properties of both hemoglobin components purified by DEAE cellulose were almost identical to those of the hemolysate: stripped hemoglobin showed a large Bohr effect which was enhanced by chloride ions and, at a larger extent, by organic phosphates which, at acidic pH values gave rise to the Root effect. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of physiological amounts of chloride. The results indicated that hemoglobin do have two sites for GTP binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for both GTP-binding sites. The presence of multiple hemoglobin components with identical properties confirms that hemoglobin heterogeneity that often occurs in fish cannot be only explained as an evolutionary response to the physiological and/or environmental needs of the species.

P-selectin-mediated platelet-neutrophil aggregate formation activates neutrophils in mouse and human sickle cell disease.

OBJECTIVE: To determine the role of platelets in stimulating mouse and human neutrophil activation and pulmonary injury in sickle cell disease (SCD). METHODS AND RESULTS: Both platelet and neutrophil activation occur in SCD, but the interdependence of these events is unknown. Platelet activation and binding to leukocytes were measured in mice and patients with SCD and in controls. Relative to controls, blood obtained from mice or patients with SCD contained significantly elevated platelet-neutrophil aggregates (PNAs). Both platelets and neutrophils found in sickle PNAs were activated. Multispectral imaging (ImageStream) and conventional flow cytometry revealed a subpopulation of activated neutrophils with multiple adhered platelets that expressed significantly more CD11b and exhibited greater oxidative activity than single neutrophils. On average, wild-type and sickle PNAs contained 1.1 and 2.6 platelets per neutrophil, respectively. Hypoxia/reoxygenation induced a further increase in PNAs in mice with SCD and additional activation of both platelets and neutrophils. The pretreatment of mice with SCD with clopidogrel or P-selectin antibody reduced the formation of PNAs and neutrophil activation and decreased lung vascular permeability. CONCLUSIONS: Our findings suggest that platelet binding activates neutrophils and contributes to a chronic inflammatory state and pulmonary dysfunction in SCD. The inhibition of platelet activation may be useful to decrease tissue injury in SCD, particularly during the early stages of vaso-occlusive crises.

Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

Differences in response to fetal hemoglobin induction therapy in beta-thalassemia and sickle cell disease.

Inducers of fetal hemoglobin (HbF) have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in beta-thalassemia (beta-Thal). To understand the basis of these differences in clinical effectiveness, we compared the effects of butyrate and hemin on the expression of the different globin genes in progenitors-derived erythroid cells from patients with beta-Thal intermedia and SCD. Exposure to butyrate resulted in an augmentation of gamma-globin mRNA levels in both SCD and beta-Thal. Interestingly, butyrate exposure increased alpha-globin expression in beta-Thal, while alpha-globin mRNA levels decreased in SCD in response to butyrate. As a result, the favorable effects of the butyrate-induced increase in gamma-globin expression on alpha:beta-like globin mRNA imbalance in beta-Thal were reduced as a result of the associated increase in alpha-globin expression. Hemin had similar but less profound effects on all three globin genes in both categories of patients. Although the majority of patients with beta-Thal did not correct their globin imbalance in response to butyrate or hemin induction of HbF in a minority of patients resulted in marked reduction in globin imbalance. Thus, we believe that the poor clinical response in a majority of patients with beta-Thal to inducers of gamma-globin expression may be a reflection of unfavorable effects of these agents on the other globin genes.