Structure Of Hb Research Articles

A Rare Case of Homozygous Hemoglobin G-Philadelphia & Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

Hemoglobin-PEG Interactions Probed by Small-Angle X-ray Scattering: Insights for Crystallization and Diagnostics Applications.

Protein-protein interactions, controlling protein aggregation in the solution phase, are crucial for the formulation of protein therapeutics and the use of proteins in diagnostic applications. Additives in the solution phase are factors that may enhance the protein's conformational stability or induce crystallization. Protein-PEG interactions do not always stabilize the native protein structure. Structural information is needed to validate excipients for protein stabilization in the development of protein therapeutics or use proteins in diagnostic assays. The present study investigates the impact of polyethylene glycol (PEG) molecular weight and concentration on the spatial structure of human hemoglobin (Hb) at neutral pH. Small-angle X-ray scattering (SAXS) in combination with size-exclusion chromatography is employed to characterize the Hb structure in solution without and with the addition of PEG. Our results evidence that human hemoglobin maintains a tetrameric conformation at neutral pH. The dummy atom model, reconstructed from the SAXS data, aligns closely with the known crystallographic structure of methemoglobin (metHb) from the Protein Data Bank. We established that the addition of short-chain PEG600, at concentrations of up to 10% (w/v), acts as a stabilizer for hemoglobin, preserving its spatial structure without significant alterations. By contrast, 5% (w/v) PEG with higher molecular weights of 2000 and 4000 leads to a slight reduction in the maximum particle dimension (Dmax), while the radius of gyration (Rg) remains essentially unchanged. This implies a reduced hydration shell around the protein due to the dehydrating effect of longer PEG chains. At a concentration of 10% (w/v), PEG2000 interacts with Hb to form a complex that does not distort the protein's spatial configuration. The obtained results provide a deeper understanding of PEG's influence on the Hb structure in solution and broader knowledge regarding protein-PEG interactions.

Nitric Oxide Ameliorates the Effects of Hypoxia in Mice by Regulating Oxygen Transport by Hemoglobin.

Xiaoying Zhou, Wenting Su, Quanwei Bao, Yu Cui, Xiaoxu Li, Yidong Yang, Chengzhong Yang, Chengyuan Wang, Li Jiao, Dewei Chen, and Jian Huang. Nitric oxide ameliorates the effects of hypoxia in mice by regulating oxygen transport by hemoglobin. High Alt Med Biol. 25:174-185, 2024.-Hypoxia is a common pathological and physiological phenomenon in ischemia, cancer, and strenuous exercise. Nitric oxide (NO) acts as an endothelium-derived relaxing factor in hypoxic vasodilation and serves as an allosteric regulator of hemoglobin (Hb). However, the ultimate effects of NO on the hematological system invivo remain unknown, especially in extreme environmental hypoxia. Whether NO regulation of the structure of Hb improves oxygen transport remains unclear. Hence, we examined whether NO altered the oxygen affinity of Hb (Hb-O2 affinity) to protect extremely hypoxic mice. Mice were exposed to severe hypoxia with various concentrations of NO, and the survival time, exercise capacity, and other physical indexes were recorded. The survival time was prolonged in the 5 ppm NO (6.09 ± 1.29 minutes) and 10 ppm NO (6.39 ± 1.58 minutes) groups compared with the 0 ppm group (4.98 ± 1.23 minutes). Hypoxia of the brain was relieved, and the exercise exhaustion time was prolonged when mice inhaled 20 ppm NO (24.70 ± 6.87 minutes vs. 20.23 ± 6.51 minutes). In addition, the differences in arterial oxygen saturation (SO2%) (49.64 ± 7.29% vs. 42.90 ± 4.30%) and arteriovenous SO2% difference (25.14 ± 8.95% vs. 18.10 ± 6.90%) obviously increased. In ex vivo experiments, the oxygen equilibrium curve (OEC) left shifted as P50 decreased from 43.77 ± 2.49 mmHg (0 ppm NO) to 40.97 ± 1.40 mmHg (100 ppm NO) and 38.36 ± 2.78 mmHg (200 ppm NO). Furthermore, the Bohr effect of Hb was enhanced by the introduction of 200 ppm NO (-0.72 ± 0.062 vs.-0.65 ± 0.051), possibly allowing Hb to more easily offload oxygen in tissue at lower pH. The crystal structure reveals a greater distance between Asp94β-His146β in nitrosyl -Hb(NO-Hb), NO-HbβCSO93, and S-NitrosoHb(SNO-Hb) compared to tense Hb(T-Hb, 3.7 Å, 4.3 Å, and 5.8 Å respectively, versus 3.5 Å for T-Hb). Moreover, hydrogen bonds were less likely to form, representing a key limitation of relaxed Hb (R-Hb). Upon NO interaction with Hb, hydrogen bonds and salt bridges were less favored, facilitating relaxation. We speculated that NO ameliorated the effects of hypoxia in mice by promoting erythrocyte oxygen loading in the lung and offloading in tissues.

Molecular Insights of an Avian Species with Low Oxygen Affinity, the Crystal Structure of Duck T-State Methemoglobin.

Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C2221 with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and Rfree: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1β1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from Anas platyrhynchos.

Structural and resting-state connection abnormalities of habenula in obsessive-compulsive disorder.

Previous studies have suggested that the habenula (Hb) may be involved in the mechanism of obsessive-compulsive disorder (OCD). However, the specific role of Hb in OCD remains unclear. This study aimed to explore the structural and functional abnormalities of Hb in OCD and their relationship with the clinical symptoms. Eighty patients with OCD and 85 healthy controls (HCs) were recruited as the primary dataset. The grey matter volume, resting-state functional connectivity (FC), and effective connectivity (EC) of the Hb were calculated and compared between OCD group and HCs. An independent replication dataset was used to verify the stability and robustness of the results. Patients with OCD exhibited smaller Hb volume and increased FC of right Hb-left hippocampus than HCs. Dynamic causal model revealed an increased EC from left hippocampus to right Hb and a less inhibitory causal influence from the right Hb to left hippocampus in the OCD group compared to HCs. Similar results were found in the replication dataset. This study suggested that abnormal structure of Hb and hippocampus-Hb connectivity may contribute to the pathological basis of OCD.

Development of novel polymer haemoglobin based particles as an antioxidant, antibacterial and an oxygen carrier agents

This innovative work aims to develop highly biocompatible and degradable nanoparticles by encapsulating haemoglobin (Hb) within poly-ε-caprolactone for novel biomedical applications. We used a modified double emulsion solvent evaporation method to fabricate the particles. A Scanning electron microscope (SEM) characterized them for surface morphology. Fourier Transform Infrared Spectroscopy (FTIR) and Ultraviolet–visible spectroscopies (UV–visible) elucidated preserved chemical and biological structure of encapsulated haemoglobin. The airproof equilibrium apparatus obtained the oxygen-carrying capacity and P50 values. The DPPH assay assessed free radical scavenging potential. The antibacterial properties were observed using four different bacterial strains by disk diffusion method. The MTT assay investigates the cytotoxic effects on mouse fibroblast cultured cell lines (L-929). The MTT assay showed that nanoparticles have no toxicity over large concentrations. The well-preserved structure of Hb within particles, no toxicity, high oxygen affinity, P50 value, and IC50 values open the area of new research, which may be used as artificial oxygen carriers, antioxidant, and antibacterial agents, potential therapeutic agents as well as drug carrier particles to treat the cancerous cells. The novelty of this work is the antioxidant and antibacterial properties of developed nanoparticles are not been reported yet. Results showed that the prepared particles have strong antioxidant and antibacterial potential.

High-Oxygen-Affinity Hemoglobins-Case Series and Review of the Literature.

Modifications of the hemoglobin (Hb) structure in regions involving the regulation of oxygen transport may lead to an increased oxygen affinity for the hemoglobin molecule and impaired oxygen delivery to the tissues. Herein, we present six patients with high-oxygen-affinity Hb variants, either in heterozygous form or in compound heterozygosity (such as heterozygosity for Hb Hiroshima, Köln, Crete, and compound heterozygosity Hb Crete with β or δβ thalassemia), in order to demonstrate the need for prompt and accurate diagnosis and enrich the limited literature due to the rarity of such cases. Hb Crete, Hb Hiroshima, and Hb Köln have distinct pathophysiologies and may result in different clinical phenotypes. In conclusion, high-oxygen-affinity hemoglobins are rare and inherited within a dominant autosomal manner, have various clinical presentations, and should always be suspected in patients with erythrocytosis. Their management (as phlebotomy or low-dose aspirin) should be based on an individualized assessment of the risk of complications, the medical history, concomitant symptoms, and quality of life.

Green Synthesis of Silver Nanoparticles Using Drymaria cordata and Their Biocompatibility with Hemoglobin: A Therapeutic Potential Approach.

In this study, we present the synthesis and characterization of AgNPs using Drymaria cordata along with an assessment of their antioxidant, antibacterial, and antidiabetic activities. Antibacterial activities using four bacterial strains, free radical scavenging assays (DPPH and ABTS), and carbohydrate hydrolyzing enzyme inhibition assays were done to examine the therapeutic efficacy of AgNPs. Additionally, herein, we also evaluated the biocompatibility of the AgNPs using hemoglobin (Hb) as a model protein. A comprehensive analysis of Hb and AgNP interactions was carried out by using various spectroscopic, imaging, and size determination studies. Spectroscopic results showed that the secondary structure of Hb was not altered after its interaction with AgNPs. Furthermore, the thermal stability was also well maintained at different concentrations of nanoparticles. This study demonstrated a low-cost, quick, and eco-friendly method for developing AgNPs using D. cordata, and the biocompatible nature of AgNPs was also established. D. cordata-mediated AgNPs have potential applications against bacteria and diabetes and may be utilized for targeted drug delivery.

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the β subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep β-globin locus would recapitulate SCD’s complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to β2-Glu6 and β1-Asp73 (β1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep β-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Atomistic simulations of RNA duplex thermal denaturation: Sequence- and forcefield-dependence

Double-stranded RNA is the end-product of template-based replication, and is also the functional state of some biological RNAs. Similarly to proteins and DNA, they can be denatured by temperature, with important physiological and technological implications. Here, we use an in silico strategy to probe the thermal denaturation of RNA duplexes. Following previous results that were obtained on a few different duplexes, and which nuanced the canonical 2-state picture of nucleic acid denaturation, we here specifically address three different aspects that greatly improve our description of the temperature-induced dsRNA separation. First, we investigate the effect of the spatial distribution of weak and strong base-pairs among the duplex sequence. We show that the deviations from the two-state dehybridization mechanism are more pronounced when a strong core is flanked with weak extremities, while duplexes with a weak core but strong extremities exhibit a two-state behavior, which can be explained by the key role played by base fraying. This was later verified by generating artificial hairpin or circular states containing one or two locked duplex extremities, which results in an important reinforcement of the entire HB structure of the duplex and higher melting temperatures. Finally, we demonstrate that our results are little sensitive to the employed combination of RNA and water forcefields. The trends in thermal stability among the different sequences as well as the observed unfolding mechanisms (and the deviations from a two-state scenario) remain the same regardless of the employed atomistic models. However, our study points to possible limitations of recent reparametrizations of the Amber RNA forcefield, which sometimes results in duplexes that readily denature under ambient conditions, in contradiction with available experimental results.

Doxorubicin bioavailability to human hemoglobin and cancer cells via MOF-A520

Cancer is one of the most serious and challenging issues in the life sciences, with complicated and multifactorial mechanisms making it difficult to manage. With this complexity comes various strategies for attacking tumors, including using nanomaterials to target tumor cells, bioavailability and deliver anticancer drugs directly. Doxorubicin (DOX) is a well-known and powerful anticancer drug, but its high toxicity limits its application. Intending to reduce the cytotoxic effects of DOX on normal cell line and deliver it to cancerous cell line, the present study attempts to design and synthesize a modified metal–organic framework (MOF) named A520 and a composite containing DOX (A520@DOX). The synthesized MOFs were characterized using X-ray diffraction (XRD), electron microscopy (TEM and FESEM), and FTIR. Subsequently, the toxicity of DOX was assessed at the cellular and molecular levels using MTT assays (for the cell lines MCF −10A and MCF-7) and various biophysical assays targeting human hemoglobin (Hb). Following expectations, free DOX was highly cytotoxic and strongly affected Hb structure (converting it into methemoglobin species), while the toxicity was significantly reduced in the A520@DOX composite form. It is important to mention that DOX released from the A520@DOX was significantly pH-sensitive, so it was more pronounced in acidic pH 5 (resembling a cancerous pH environment). Furthermore, the effect of DOX released from A520@DOX resulted in more oxyHb and deoxyHb species as functional Hb species. The present study used an effective DOX delivery and bioavailability nanosystem to control cytotoxicity and ensure slow drug release.

Hemoglobin Monza: A New Variant of Unstable Hemoglobin

Introduction: Unstable hemoglobins are variants with structural abnormalities; while not hindering protein formation, they lead to protein destabilization and can cause hemolytic crises. Most unstable hemoglobins are caused by point mutations that result in the premature dissociation of heme from the globin chain and usually involve His residues (Weatherall et al. 2001 Nat.Rev.Genetics; Scheps et al. 2018 EJH). Here a new unstable hemoglobin, named Hb-Monza is presented, exhibiting a long duplication in the HBB gene without apparent alterations in α-β subunit interaction or involvement of specific His residues. The variant was identified in a family, with four out of six members involved and is associated with mild chronic hemolysis and acute severe hemolytic crises during febrile infections. Methods: The presence of erythrocyte membrane abnormalities was tested by glycerol lysis, Pink, osmotic resistance and by EMA binding test. G6PD and PK activity levels were tested both during the hemolytic crisis and at resolution. Abnormal variants were screened by HPLC and electrophoresis. Stability of the Hb variant was assessed by the Isopropanol Precipitation Test. DNA was extracted from peripheral blood leukocytes, followed by PCR amplification of HBB, HBA1 and HBA2 genes. Sequencing was performed using Genetic Analyzer 3500. A 3D model of the variants was produced by SWISS-MODEL using the crystal structure of the deoxy human hemoglobin (PDB 1A3N) as template and refined by Maestro Suite. Results: The index patient (pt) was a 13-year-old girl of Chinese origin; she was the firstborn of 4 children (2 sisters and 1 brother) and came to ER with fever and hyperchromic urine. Blood tests revealed severe anemia (Hb 5.2 g/dL) and signs of hemolysis. Direct Coombs test was negative both for warm and cold antibodies. She tested positive for Mycoplasma Pneumoniae, Epstein Barr Virus (EBV) and Parvovirus B19 (IgG and IgM) antibodies. The child's infection cleared during hospitalization, and hemoglobin levels increased without transfusion. Her hemoglobin further rose to 11 g/dL in outpatient follow-up although signs of low-level chronic hemolysis persisted. All affected family members exhibit mild chronic hemolytic anemia, with all, except the mother, experiencing at least one acute severe hemolytic state triggered by infection. HPLC showed the presence of an abnormal fraction of hemoglobin with slow migration speed in alkaline electrophoresis and extremely slow elution in HPLC (4.8 min). The abnormal Hb fraction in the proband, mother, brother and sister was 20.8%, 17.7%, 28.1%, and 25.6% respectively. The father and the youngest sibling did not show this band. Sequencing of HBB demonstrated an in-frame duplication of 69 bases corresponding to 23 amino acids located in exon 2 (c.176_244dup; p.Pro59_Asn81dup); this was present in all pts exhibiting the abnormal fraction at HPLC. A functional validation of Hb Monza utilizing the isopropanol test confirmed the unstable nature of the variant (Figure 1). The 3D-model analysis (Figure 2) showed that the duplication is situated amidst helix E and F of HBB resulting in a disordered conformation protruding from the Hb structure The binding of the heme group remains unaffected and the coordination of the heme group with His residues of both the E and F helix is preserved. The tertiary structure of the Hb beta chain is scarcely affected. Conclusions: This report introduces a novel HBB gene variant, Hb Monza (HBB: c.176_244dup), associated with subclinical chronic hemolysis and acute hemolytic crises during fever-inducing infections. Only a few HBB duplications exceeding 20 bp have been documented, all leading to Beta-Thalassemia clinical features through production of a truncated beta globin chain or failure of HBB to bind the alpha chain (e.g. Frischknecht et al. 2007 Haematologica). However, 3D-modeling of Hb Monza indicates minimal tertiary structure alteration. The inclusion of 23 additional amino acids results in a disordered conformation protruding from the Hb structure possibly facilitates direct bonding between the iron molecule in the heme group and internal amino acid side chains due to increased structural flexibility. This could lead during thermal or metabolic stress, to Hb precipitation. Further data from molecular dynamic experiments will be presented. Dr. Civettini and Dr. Corti contributed equally to this article

The Spectrum of α-Thalassemia Mutations in Syrian Patients

α-Thalassemia (α-thal) is a globally prevalent genetic disorder of hemoglobin (Hb) structure where the rate of α-globin chain synthesis is reduced or absent due to the presence of α-globin mutation(s). The aim of this study is to define the spectrum of α-globin gene mutations and evaluate their allele frequency in a group of α-thal carriers. A total of 55 individuals with possible α-thal patients were referred from the thalassemia centers in Syria. They have unexplained hypochromia and microcytosis. All patients were genetically tested for 21 common α-globin gene mutations using reverse hybridization kit. Seven different α-globin gene mutations and 13 different genotypes were detected in 55 patients. The two most frequently encountered mutations were -α3.7 deletion (47.1%) and --MED mutation (21.4%). The most commonly observed genotype was -α3.7/αα (40%), followed by --MED/αα genotype (21.8%). We determined the most common α thalassemia mutations in the Syrian patients. α-Thalassemia mutations with deletions were mostly observed in our study.

Rapid Assessment of Hemoglobin-Oxygen Dissociation

Introduction: Oxygen (O 2) delivery to tissues depends on the hemoglobin (Hb)-O 2 affinity and dissociation of O 2 from Hb in red blood cells (RBCs). Mutations that impact α and β-globin subunits of Hb often alter the Hb-O2 affinity of Hb and Hb-O 2 dissociation. A point mutation in the β-globin gene yields sickle hemoglobin (HbSS) which results in sickle cell disease (SCD). Recently, there has been an upsurge in improving SCD pathophysiology through modifying HbSS O 2 affinity. The ability to alter cellular phosphate such as 2,3-DPG that influences Hb-O 2 affinity is also under investigation. In this context, measuring Hb-O 2 affinity has gained additional significance as a clinical and laboratory parameter. Hb-O 2 affinity is determined by measuring Hb-O 2 saturation (SO 2, %) vs. partial pressure of oxygen (pO 2, mmHg). The Hb-O 2 affinity is commonly represented by the p50 value, which is the pO 2 value at 50% SO 2. p50 measurement is underutilized in clinical practice mainly due to cost, complexity, and limited throughput of existing instruments. Herein, we present a rapid assay that can detect Hb-O 2 affinity disorders, determine cellular oxygen availability, and examine the effectiveness of existing and novel therapies for SCD patients. Methods: In this assay, we optically measure the Hb-O 2 dissociation by induction of physiologic acidic pH (6.9). We exploited the acidic pH environment to expedite chemically induced partial deoxygenation of Hb (deoxy-Hb) during which the conformational changes in the tertiary structure of Hb are manifested as significant peak shifts in the optical absorption spectra. Hb absorption spectra in the 350-700 nm wavelength range primarily reflect the conformation of heme-heme interactions, which are sensitive indicators of changes in the Hb-O 2 affinity. We validated the assay by comparing it with the reference standard Hemox analyzer using blood from healthy individuals (HbAA) and from patients with SCD (HbSS) i.e., low Hb-O 2 affinity. Venous blood samples were collected in EDTA-coated tubes from subjects with HbSS (n=44), HbAS (n=14), and HbAA (n=17) following an IRB-approved study protocol. 75 µl of whole blood diluted 100-fold with pH 6.9 phosphate buffer was incubated for 2 minutes at room air to attain full oxygenation. The mixture of blood and the buffer was then deoxygenated with 0.053M of sodium metabisulphite (Na 2S 2O 5) for 3 minutes to achieve partial deoxy-Hb. We then obtained 350-450 nm spectra of deoxy-Hb using a spectroscopy microplate reader with a 2 nm resolution. We recorded each sample's Soret band shift in 4 minutes using the 414 nm Hb fully oxygenated absorption peak as the reference. Data were reported as SEM. Results: Hb exhibits three characteristic light absorption peaks. The Soret peak is located at 414 nm under full oxygenation at pO 2 of 162 mmHg, and upon full deoxy-Hb at pO 2 of 5 mmHg, the peak shifts to 430 nm ( Figure 1A). We initially tested different concentrations of Na 2S 2O 5 and the time of deoxygenation to achieve partial deoxy-Hb. At 75 mmHg we analyzed the Soret peak which was used as an optical marker of oxygen affinity, an equivalent to the p50 value. The peak shift significantly correlated to the p50 value measured by the Hemox device (PCC=0.81, p=0.004, n=28) ( Figure 1A). We also observed significant differences between HbAA (n=17), HbAS(n=14), SCD samples on various treatments (transfusion (Tx) (n=8), and Hydroxyurea (n=15) and SCD treatment-naïve patients' samples (n=20). HbAA vs. HbAS; p=0.01, HbAS vs. HbSS+Tx; p=0.02, HbSS+Tx vs. HbSS+Hu; p=0.04, HbAA vs. HbSS treatment naïve; p=0.000, HbSS treatment Naive vs. HbAS; p=0.001 ( Figure 1B). Additionally, the peak shift for HbSS correlated with the percentage of HbSS (PCC = 0.87, p = 0.000, n=20, data not shown). Discussion: We demonstrate fast, effective differentiation of abnormal Hb-O 2 affinity and characterize the changes in the HbSS-O 2 affinity in response to treatments by using chemically induced partial deoxygenation of Hb at a physiologic acidic pH.The ubiquity of optical absorption spectrophotometers positions this assay as an accessible, equitable, and accurate Hb-O 2 dissociation measurement resource for research and clinical use. The assay will facilitate the diagnosis and prognosis of red blood cells and Hb disorders requiring minimal technical expertise and laboratory infrastructure and assist with testing the effect of new targeted and genome-editing-based therapies for SCD.

Habenula Volume and Functional Connectivity Changes Following Laparoscopic Sleeve Gastrectomy for Obesity Treatment

BackgroundNeuroimaging studies have revealed alterations in habenular (Hb) structure and functional connectivity (FC) in psychiatric conditions. The Hb plays a particularly critical role in regulating negative emotions, which trigger excessive food intake and obesity. However, obesity and weight loss intervention (i.e., laparoscopic sleeve gastrectomy [LSG])–associated changes in Hb structure and FC have not been studied. MethodsWe used voxel-based morphometry analysis to measure changes in gray matter volume (GMV) in the Hb in 56 patients with obesity at pre-LSG and 12 months post-LSG and in 78 normal-weight (NW) control participants. Then, we conducted Hb seed-based resting-state FC (RSFC) to examine obesity-related and LSG-induced alterations in RSFC. Finally, we used mediation analysis to characterize the interrelationships among Hb GMV, RSFC, and behaviors. ResultsCompared with NW participants, Hb GMV was smaller in patients at pre-LSG and increased at 12 months post-LSG to levels equivalent to that of NW; in addition, increases in Hb GMV were correlated with reduced body mass index (BMI). Compared with NW participants, pre-LSG patients showed greater RSFCs of the Hb–insula, Hb–precentral gyrus, and Hb–rolandic operculum and weaker RSFCs of the Hb–thalamus, Hb–hypothalamus, and Hb–caudate; LSG normalized these RSFCs. Decreased RSFC of the Hb-insula was correlated with reduced BMI, Yale Food Addiction Scale rating, and emotional eating; reduced hunger levels were correlated with increased RSFCs of the Hb–thalamus and Hb–hypothalamus; and reduced BMI and Yale Food Addiction Scale ratings were correlated with increased RSFCs of the Hb–thalamus and Hb–hypothalamus, respectively. The bidirectional relationships between Hb GMV and RSFC of the Hb–insula contributed to reduced BMI. ConclusionsThese findings indicate that LSG increased Hb GMV and that its related improvement in RSFC of the Hb–insula may mediate long-term benefits of LSG for eating behaviors and weight loss.

ОЦЕНКА ВОЗДЕЙСТВИЯ ВАКЦИНАЦИИ В РЕСПУБЛИКЕ БЕЛАРУСЬ НА ЭПИДЕМИЧЕСКИЙ ПРОЦЕСС ГЕПАТИТА В

Integration of hepatitis B (HBV) vaccination into national immunization programs has resulted in a significant reduction in hepatitis B virus (HBV) transmission worldwide, and specifically in previously highly endemic countries. A key aspect of HBV elimination strategy is the administration of a birth dose followed by a further hepatitis B vaccination of newborns. Additional preventive measures include screening during pregnancy, administration of antivirals as well as the use of HB immunoglobulin. Despite a significant decrease in the number of HB cases, vaccination of adults, in particular those of high-risk groups, remains an important challenge. Objective. To evaluate the impact of vaccination strategies on HB epidemiology in the Republic of Belarus. Material and methods. HBV incidence in the Republic of Belarus has been estimated according to the data from state statistical reporting forms. The results were statistically processed using Statistica V.10.0 program (Statsoft, USA). Results. Over the period in question there has been noted a marked decrease in the incidence of acute and asymptomatic HBV forms (the rate (ТсН.) equals to -13,25% and -11,99% respectively), while the incidence of chronic hepatitis B (CHB) tends to increase (the rate (Тpr.) equals to +1,09%). Over the period of 2002-2022, CHB prevails in the etiological structure, constituting more than 70% of observations in the total structure of HB since 2018. HBV incidence pattern varies according to the regions. Currently, the leading cohort in the overall incidence is the population aged 30-49. The incidence rate of hepatitis B among individuals of this age cohort (320.57 per 100 thousand people) is 2.35 times higher than that of a 0-29-yearage cohort (136.50 per 100 thousand people) that is accounted for by a low level of preventive vaccination coverage (according to medical records), equal to 39.3% for the cohort aged 30-49. Scheduled vaccine prophylaxis introduced in 2000 has become the basis for the effective HB epidemiology surveillance in the Republic of Belarus. Conclusions. The period in question is characterized by both a marked decrease in the incidence of nosological HBV forms (the rate is 7.5%) and a moderate increase in the incidence of chronic hepatitis B (the rate (Тpr.) equals to +1,09%. Despite the undeniable progress and success of HB vaccination, it should be mentioned that an estimated over 65% of unvaccinated adult population may form a cluster for the potential spread of the virus.

X-ray crystallography and sickle cell disease drug discovery-a tribute to Donald Abraham.

X-ray crystallography and structure-based drug discovery have played a major role in the discovery of antisickling agents that target hemoglobin (Hb) for the treatment of sickle cell disease (SCD). Sickle cell disease, the most common inherited hematologic disorder, occurs as a result of a single point mutation of βGlu6 in normal human adult hemoglobin (HbA) to βVal6 in sickle hemoglobin (HbS). The disease is characterized by polymerization of HbS and sickling of red blood cells (RBCs), leading to several secondary pathophysiologies, including but not limited to vaso-occlusion, hemolytic anemia, oxidative stress, inflammation, stroke, pain crisis, and organ damage. Despite the fact that SCD was the first disease to have its molecular basis established, the development of therapies was for a very long time a challenge and took several decades to find therapeutic agents. The determination of the crystal structure of Hb by Max Perutz in the early 60s, and the pioneering X-ray crystallography research by Donald J. Abraham in the early 80s, which resulted in the first structures of Hb in complex with small molecule allosteric effectors of Hb, gave much hope that structure-based drug discovery (SBDD) could be used to accelerate development of antisickling drugs that target the primary pathophysiology of hypoxia-induced HbS polymerization to treat SCD. This article, which is dedicated to Donald J. Abraham, briefly reviews structural biology, X-ray crystallography and structure-based drug discovery from the perspective of Hb. The review also presents the impact of X-ray crystallography in SCD drug development using Hb as a target, emphasizing the major and important contributions by Don Abraham in this field.

A novel single sensor hemoglobin domain from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 exhibits higher pH but lower thermal stability compared to globins from mesophilic organisms

Thermosynechococcus elongatus-BP1 belongs to the class of photoautotrophic cyanobacterial organisms. The presence of chlorophyll a, carotenoids, and phycocyanobilin are the characteristics that categorize T. elongatus as a photosynthetic organism. Here, we report the structural and spectroscopic characteristics of a novel hemoglobin (Hb) Synel Hb from T.elongatus, synonymous with Thermosynechococcus vestitus BP-1. The X-ray crystal structure (2.15 Å) of Synel Hb suggests the presence of a globin domain with a pre-A helix similar to the sensor domain (S) family of Hbs. The rich hydrophobic core accommodates heme in a penta-coordinated state and readily binds an extraneous ligand (imidazole). The absorption and circular dichroic spectral analysis of Synel Hb reiterated that the heme is in FeIII+ state with a predominantly α-helical structure similar to myoglobin. Synel Hb displays higher resistance to structural perturbations induced via external stresses like pH and guanidium hydrochloride, which is comparable to Synechocystis Hb. However, Synel Hb exhibited lower thermal stability compared to mesophilic hemoglobins. Overall, the data is suggestive of the structural sturdiness of Synel Hb, which probably corroborates its origin in extreme thermophilic conditions. The stable globin provides scope for further investigation and may lead to new insights with possibilities for engineering stability in hemoglobin-based oxygen carriers.

Effects of Temperature, Metal Ions and Biosurfactants on Interaction Mechanism between Caffeic Acid Phenethyl Ester and Hemoglobin.

Caffeic acid phenylethyl ester (CAPE) is a natural polyphenol extracted from propolis, which is reported to have several pharmacological effects such as antibacterial, antitumor, antioxidant and anti-inflammatory activities. Hemoglobin (Hb) is closely related to the transport of drugs, and some drugs, including CAPE, can lead to a change in Hb concentration. Herein, the effects of temperature, metal ions and biosurfactants on the interaction between CAPE and Hb were studied using ultraviolet-visible spectroscopy (UV-Vis), fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering (DLS) and molecular docking analysis. The results showed that the addition of CAPE led to changes in the microenvironment of Hb amino acid residues as well as the secondary structure of Hb. Hydrogen bonding and van der Waals force were found to be the main driving forces for the interaction between CAPE and Hb through fluorescence spectroscopy and thermodynamic parameter data. The results of fluorescence spectroscopy also showed that lowering the temperature, adding biosurfactants (sodium cholate (NaC) and sodium deoxycholate (NaDC)) and the presence of Cu2+ increased the binding force between CAPE and Hb. These results provide useful data for the targeted delivery and absorption of CAPE and other drugs.

Biochemical interaction of human hemoglobin with ionic liquids of noscapinoids: Spectroscopic and computational approach

In this work, we have developed noscapine based ionic liquids i.e., Noscapine (MeNOS) and 9-Bromonoscapine (MeBrNOS) as cation supported with bis(trifluoromethylsulfonyl)amide (NTf2−) as anion. We have reported the mechanism of binding interaction between noscapine based ILs and human hemoglobin (Hb) using various spectroscopic and computational techniques. The corresponding thermodynamics studies showed that the binding is exothermic in nature and major forces responsible for binding are Van der waals and hydrogen bonding interaction. The fluorescence spectra showed that the intensity of Hb decreases in the presence of [MeNOS]NTf2 and [MeBrNOS]NTf2 both shows static quenching. The secondary structural changes in Hb were observed and calculated by using CD spectroscopy. Molecular docking studies revealed that both the ILs show strong binding in β1 fragment of the tetrameric structure of Hb, but the binding of [MeNOS]NTf2 is relatively stronger than [MeBrNOS]NTf2 and the results are supported by MD simulations.