Trienone analogs of curcuminoids induce fetal hemoglobin synthesis via demethylation at Gγ-globin gene promoter.

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for ß-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from ß-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in ß-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Transcription factor competition at the γ-globin promoters controls hemoglobin switching.

BCL11A, the major regulator of fetal hemoglobin (HbF, α2γ2) level, represses γ-globin expression through direct promoter binding in adult erythroid cells in a switch to adult hemoglobin (HbA, α2ß2). To uncover how BCL11A initiates repression, we used CRISPR-Cas9, dCas9, dCas9-KRAB and dCas9-VP64 screens to dissect the γ-globin promoters and identified an activator element near the BCL11A-binding site. Using CUT&RUN and base editing, we demonstrate that a proximal CCAAT box is occupied by the activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate repression. Occupancy of NF-Y is rapidly established following BCL11A depletion, and precedes γ-globin derepression and locus control region (LCR)-globin loop formation. Our findings reveal that the switch from fetal to adult globin gene expression within the >50-kb ß-globin gene cluster is initiated by competition between a stage-selective repressor and a ubiquitous activating factor within a remarkably discrete region of the γ-globin promoters.

Quadrupole-Time-of-Flight Mass Spectrometric Identification of Hemoglobin Subunits α, ß, γ and δ in Unknown Peaks of High Performance Liquid Chromatography of Hemoglobin in ß-Thalassemias.

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, ß, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with ß-thalassemia (ß-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, ß-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with ß-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.

Protein Mixture Segregation at Coffee-Ring: Real-Time Imaging of Protein Ring Precipitation by FTIR Spectromicroscopy.

During natural drying process, all solutions and suspensions tend to form the so-called "coffee-ring" deposits. This phenomenon, by far, has been interpreted by the hydrodynamics of evaporating fluids. However, in this study, by applying Fourier transform infrared imaging (FTIRI), it is possible to observe the segregation and separation of a protein mixture at the "ring", hence we suggest a new way to interpret "coffee-ring effect" of solutions. The results explore the dynamic process that leads to the ring formation in case of model plasma proteins, such as BGG (bovine γ globulin), BSA (bovine serum albumin), and Hfib (human fibrinogen), and also report fascinating discovery of the segregation at the ring deposits of two model proteins BGG and BSA, which can be explained by an energy kinetic model, only. The investigation suggests that the coffee-ring effect of solute in an evaporating solution drop is driven by an energy gradient created from change of particle-water-air interfacial energy configuration.

Exploring the interaction of silver nanoparticles with lysozyme: Binding behaviors and kinetics.

The role of nanoparticle interaction with biomolecules to form a biocorona is the key to nanoparticle behavior and its consequences in the physiological environment. Since the adsorbed biocorona decides the fate of a nanomaterials in vivo, and thus a comprehensive understanding of the dynamic interactions of the proteins with the nanoparticle is imperative. Herein we investigate the interaction of a model protein, lysozyme with silver nanoparticles (AgNPs) using fluorescence, synchronous fluorescence, UV-vis absorption spectrum and circular dichroism (CD) techniques under the physiological conditions. The results indicated that the binding of AgNPs to lysozyme may be a static quenching mechanism. With the analysis of the fluorescence spectral data, the binding constants and the thermodynamic parameters were determined, which suggests that the binding of AgNPs to lysozyme is a spontaneous process. Moreover, it was demonstrated that the main acting forces between AgNPs and lysozyme may be hydrophobic interactions. At the same time, the conformational change of lysozyme induced by AgNPs was investigated with synchronous fluorescence spectroscopy and CD techniques. The results of kinetic studies reveal that the adsorption of lysozyme on AgNPs surface tends to follow pseudo-second-order kinetic characteristic with obvious hysteresis effect.

Effect of halloysite nanotubes on the structure and function of important multiple blood components.

Many researchers have investigated the application of halloysite nanotubes (HNTs) in biomedicine, because of their special nanoscale hollow tubular structure. Although the cytocompatibility of HNTs has been studied, their blood compatibility has not been systematically investigated. In this work, the effect of HNTs on the structure and function of different blood components has been studied, including the morphology and hemolysis of red blood cells (RBCs). Based on scanning electron microscopy (SEM) observations, optical density test and flow cytometry analysis, we found that HNTs can affect the morphology and membrane integrity of RBCs in phosphate buffered saline (PBS) in a content-dependent way. In particular, based on UV-vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra, HNTs can alter the secondary structure and conformation of human fibrinogen and γ-globulins. In addition, the detection of biomarker molecules C3a and C5a in plasma suggests that HNTs can trigger complement activation. In the blood clotting assay, HNTs were found to significantly prolong the activated partial thromboplastin time (APTT), shorten the prothrombin time (PT) of platelet-poor plasma (PPP), and change the thromboelastography (TEG) parameters of whole blood coagulation. Furthermore, confocal laser scanning microscopy and flow cytometry analysis were used to test intracellular uptake by macrophages, and the cellular uptake of HNTs in the RAW 264.7 was found to be content-dependent, but not time-dependent. These findings provide insight for the potential use of HNTs as biofriendly nanocontainers for biomaterials in vivo.

Structural and Functional Insights on an Uncharacterized Aγ-Globin-Gene Polymorphism Present in Four ß0-Thalassemia Families with High Fetal Hemoglobin Levels.

INTRODUCTION: Several DNA polymorphisms have been associated with high production of fetal hemoglobin (HbF), although the molecular basis is not completely understood. In order to identify and characterize novel HbF-associated elements, we focused on five probands and their four families (from Egypt, Iraq and Iran) with thalassemia major (either ß(0)-IVSII-1 or ß(0)-IVSI-1) and unusual HbF elevation (>98 %), congenital or acquired after rejection of bone marrow transplantation, suggesting an anticipated favorable genetic background to high HbF expression. METHODS: Patient recruitment, genomic DNA sequencing, western blotting, electrophoretic mobility shift assays, surface plasmon resonance (SPR) biospecific interaction analysis, bioinformatics analyses based on docking experiments. RESULTS: A polymorphism of the Aγ-globin gene is here studied in four families with ß(0)-thalassemia (ß(0)-IVSII-1 and ß(0)-IVSI-1) and expressing unusual high HbF levels, congenital or acquired after rejection of bone marrow transplantation. This (G→A) polymorphism is present at position +25 of the Aγ-globin genes, corresponding to a 5'-UTR region of the Aγ-globin mRNA and, when present, is physically linked in chromosomes 11 of all the familiar members studied to the XmnI polymorphism and to the ß(0)-thalassemia mutations. The region corresponding to the +25(G→A) polymorphism of the Aγ-globin gene belongs to a sequence recognized by DNA-binding protein complexes, including LYAR (Ly-1 antibody reactive clone), a zinc-finger transcription factor previously proposed to be involved in down-regulation of the expression of γ-globin genes in erythroid cells. CONCLUSION: We found a novel polymorphism of the Aγ-globin gene in four families with ß(0)-thalassemia and high levels of HbF expression. Additionally, we report evidence suggesting that the Aγ-globin gene +25(G→A) polymorphism decreases the efficiency of the interaction between this sequence and specific DNA binding protein complexes.

Why the DNA self-depurination mechanism operates in HB-ß but not in ß-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2.

The human ß-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5'GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5'G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of ß-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and ß-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the ß-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in ß-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human ß-globin paralogs.

Two new γ chain variants: Hb F-Augusta GA [(G)γ59(E3)Lys → Arg; HBG2: c.179A > G] and Hb F-Port Royal-II [(A)γ125(H3)Glu → Ala; HBG1: c.377A > C].

The total number of hemoglobin (Hb) variants so far reported to the HbVar database is 1598 (April 9 2014) and 130 of them are fetal Hb variants. Fetal Hb are categorized as two different subunits, (G)γ- and (A)γ-globin chains, and γ chain variants can be observed in both subunits. There are 72 (G)γ- and 58 (A)γ-globin chain variants. Most of them are clinically silent and detected during newborn screening programs in the USA and outside the USA. In this report, we discuss the molecular characteristics and diagnostic difficulties of two new γ-globin chain variants found in an African American baby with no clinical symptoms. One is a new (G)γ-globin chain variant, Hb F-Augusta GA [(G)γ59(E3)Lys → Arg; HBG2: c.179A > G] and the other one is Hb F-Port Royal-II [(A)γ125(H3)Glu → Ala; HBG1: c.377A > C].