Red blood cells (RBCs) are the primary mediators of oxygen transport in the human body, and their function is mainly achieved through conformational changes of hemoglobin (Hb). Hb is a tetramer composed of four subunits, with HbA being the predominant Hb in healthy adults, existing in two forms: tense state (T state) and relaxed state (R state). Endogenous regulators of Hb conformation include 2,3-diphosphoglyceric acid, carbon dioxide, protons, and chloride ions, while exogenous regulators include inositol hexaphosphate, inositol tripyrophosphate, benzabate, urea derivative L35, and vanillin, each with different mechanisms of action. The application of Hb conformational regulators provides new insights into the study of hypoxia oxygen supply issues and the treatment of sickle cell disease.
红细胞是人体内血液运输氧气最主要的媒介，其功能主要是由血红蛋白(hemoglobin，Hb)的构象变化实现的。Hb是由4个亚单位组成的四聚体，HbA是健康成人中主要的Hb，以紧张态(T态)和松弛态(R态)2种形式存在。Hb构象的内源性调节器包括2,3-双磷酸甘油酸、二氧化碳、质子和氯化物等，外源性调节器包括肌醇六磷酸、肌醇三焦磷酸、苯扎贝特、尿素衍生物L35、香兰素等，其作用机制各不相同。Hb构象调节剂的应用为研究缺氧供氧问题和治疗镰状细胞病提供了新的思路。.

For the last two decades, the aromatic aldehyde 5-hydroxymethyl-furfural (5-HMF) has been the subject of several investigations for its pharmacologic potential. In 2004, the Safo group reported that 5-HMF has potent antisickling activity by targeting and ameliorating the primary pathophysiology of hypoxia-induced sickling of erythrocytes (red blood cells [RBC]). Following the encouraging outcome of the preclinical and phase I/II clinical studies of 5-HMF for the treatment of sickle cell disease (SCD), there have been multiple studies suggesting 5-HMF has several other biological or pharmacologic activities, including anti-allergic, antioxidant, anti-hypoxic, anti-ischemic, cognitive improvement, anti-tyrosinase, anti-proliferation, cytoprotective, and anti-inflammatory activities. The wide range of its effects makes 5-HMF a potential candidate for treating a variety of diseases including cognitive disorders, gout, allergic disorders, anemia, hypoxia, cancers, ischemia, hemorrhagic shock, liver fibrosis, and oxidative injury. Several of these therapeutic claims are currently under investigation and, while promising, vary in terms of the strength of their evidence. This review presents the research regarding the therapeutic potential of 5-HMF in addition to its sources, physicochemical properties, safety, absorption, distribution, metabolism, and excretion (ADME) profiles.

Aerobic methanotrophs establish a symbiotic association with denitrifiers to facilitate the process of aerobic methane oxidation coupled with denitrification (AME-D). However, the symbiosis has been frequently observed in hypoxic conditions continuing to pose an enigma. The present study has firstly characterized an electrically induced symbiosis primarily governed by Methylosarcina and Hyphomicrobium for the AME-D process in a hypoxic niche caused by Comammox Nitrospira. The kinetic analysis revealed that Comammox Nitrospira exhibited a higher apparent oxygen affinity compared to Methylosarcina. While the coexistence of comammox and AME-D resulted in an increase in methane oxidation and nitrogen loss rates, from 0.82 ± 0.10 to 1.72 ± 0.09 mmol CH4 d-1 and from 0.59 ± 0.04 to 1.30 ± 0.15 mmol N2 d-1, respectively. Furthermore, the constructed microbial fuel cells demonstrated a pronounced dependence of the biocurrents on AME-D due to oxygen competition, suggesting the involvement of direct interspecies electron transfer in the AME-D process under hypoxic conditions. Metagenomic and metatranscriptomic analysis revealed that Methylosarcina efficiently oxidized methane to formaldehyde, subsequently generating abundant NAD(P)H for nitrate reduction by Hyphomicrobium through the dissimilatory RuMP pathway, leading to CO2 production. This study challenges the conventional understanding of survival mechanism employed by AME-D symbionts, thereby contributing to the characterization responsible for limiting methane emissions and promoting nitrogen removal in hypoxic regions.

Non-aerated bacteria-algae system gaining O2 through photosynthesis presents an alternative for costly mechanical aeration. This study investigated oxygen supply and performance of nutrients removal at low and high light intensity (LL and HL). The results showed that P removal was high and robust (LL 97 ± 1.8 %, HL 95 % ± 2.9 %), while NH4+-N removal fluctuated dramatically (LL 66 ± 14.7 %, HL 84 ± 8.6 %). Oxygen generated at illumination of 200 μmol m-2 s-1, 6 h was sufficient to sustain aerobic phase for 2.25 g/L MLSS. However, O2 produced by algae was preferentially captured in the order of heterotrophic bacteria (HB), ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB). Oxygen affinity coupled with light intensity led to NOB suppression with stable nitrite accumulation ratio of 57 %. Free nitrous acid (FNA) and light stimulated the abundance of denitrifying polyphosphate accumulating organism (DPAO) of Flavobacterium, but with declined P-accumulating metabolism (PAM) of P release, P/C, K/P and Mg/P ratios. Flavobacterium and cyanobacteria Leptolyngbya, along with biologically induced CaP in extracellular polymeric substances was the key to robust P removal. AOB of Ellin6067 and DPAO of Flavobacteria offer a promising scenario for partial nitrification-denitrifying phosphorus removal.

The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.

Significance: Sickle cell disease (SCD) is the most common inherited diathesis affecting mostly underserved populations globally. SCD is characterized by chronic pain and fatigue, severe acute painful crises requiring hospitalization and opioids, strokes, multiorgan damage, and a shortened life span. Symptoms may appear shortly after birth, and, in less developed countries, most children with SCD die before attaining age 5. Hematopoietic stem cell transplant and gene therapy offer a curative therapeutic approach, but, due to many challenges, are limited in their availability and effectiveness for a majority of persons with SCD. A critical unmet need is to develop safe and effective novel targeted therapies. A wide array of drugs currently undergoing clinical investigation hold promise for an expanded pharmacological armamentarium against SCD. Recent Advances: Hydroxyurea, the most widely used intervention for SCD management, has improved the survival in the Western world and more recently, voxelotor (R-state-stabilizer), l-glutamine, and crizanlizumab (anti-P-selectin antibody) have been approved by the Food and Drug Administration (FDA) for use in SCD. The recent FDA approval emphasizes the need to revisit the advances in understanding the core pathophysiology of SCD to accelerate novel evidence-based strategies to treat SCD. The biomechanical breakdown of erythrocytesis, the core pathophysiology of SCD, is associated with intrinsic factors, including the composition of hemoglobin, membrane integrity, cellular volume, hydration, andoxidative stress. Critical Issues and Future Directions: In this context, this review focuses on advances in emerging nongenetic interventions directed toward the therapeutic targets intrinsic to sickle red blood cells (RBCs), which can prevent impaired rheology of RBCs to impede disease progression and reduce the sequelae of comorbidities, including pain, vasculopathy, and organ damage. In addition, given the intricate pathophysiology of the disease, it is unlikely that a single pharmacotherapeutic intervention will comprehensively ameliorate the multifaceted complications associated with SCD. However, the availability of multiple drug options affords the opportunity for individualized therapeutic regimens tailored to specific SCD-related complications. Furthermore, it opens avenues for combination drug therapy, capitalizing on distinct mechanisms of action and profiles of adverse effects.

We present a new hemoglobin variant, Hb Raklev, characterized by the substitution of leucine with glutamine at position 75 in the β-globin chain. This variant was discovered inadvertently during an HbA1c evaluation using high performance liquid chromatography in a symptomless 54-year-old Caucasian woman, with the same variant also identified in her 16-year-old daughter. Purification of the hemoglobin revealed possibly diminished 2,3-bisphosphoglycerate (2,3-BPG) sensitivity, which may result in heightened oxygen affinity. Notably, two variants have been previously documented at this location: the unstable Hb Atlanta and the high-affinity Hb Pasadena.

Cerebral malaria (CM) is a severe manifestation of malaria that commonly occurs in children and is hallmarked by neurologic symptoms and significant Plasmodium falciparum parasitemia. It is currently hypothesized that cerebral hypoperfusion from impaired microvascular oxygen transport secondary to parasitic occlusion of the microvasculature is responsible for cerebral ischemia and thus disease severity. Animal models to study CM, are known as experimental cerebral malaria (ECM), and include the C57BL/6J infected with Plasmodium berghei ANKA (PbA), which is ECM-susceptible, and BALB/c infected with PbA, which is ECM-resistant. Here we sought to investigate whether changes in oxygen (O2) delivery, O2 flux, and O2 utilization are altered in both these models of ECM using phosphorescence quenching microscopy (PQM) and direct measurement of microvascular hemodynamics using the cranial window preparation. Animal groups used for investigation consisted of ECM-susceptible C57BL/6 (Infected, n = 14) and ECM-resistant BALB/c (Infected, n = 9) mice. Uninfected C57BL/6 (n = 6) and BALB/c (n = 6) mice were included as uninfected controls. Control animals were manipulated in the exact same way as the infected mice (except for the infection itself). C57BL/6 ECM animals at day 6 of infection were divided into two cohorts: Early-stage ECM, presenting mild to moderate drops in body temperature (>34 < 36 °C) and Late-stage ECM, showing marked drops in body temperature (<33 °C). Data taken from new experiments conducted with these animal models were analyzed using a general linear mixed model. We constructed three general linear mixed models, one for total O2 content, another for total O2 delivery, and the third for total O2 content as a function of convective flow. We found that in both the ECM-susceptible C57BL/6J model and ECM-resistant BALB/c model of CM, convective and diffusive O2 flux along with pial hemodynamics are impaired. We further show that concomitant changes in p50 (oxygen partial pressure for 50% hemoglobin saturation), only 5 mmHg in the case of late-stage CM C57BL/6J mice, and O2 diffusion result in insufficient O2 transport by the pial microcirculation, and that both these changes are required for late-stage disease. In summary, we found impaired O2 transport and O2 affinity in late-stage ECM, but only the former in either early-stage ECM and ECM-resistant strains.

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.

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd = 23 ± 2 nM) as an example, and also discuss tips and tricks to make the assay work for other oxygen-binding proteins.