Search

αβ T cell receptors (αβTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αβTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αβ T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αβ)6γ protein complex. This direct αβTCR–PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αβTCR–peptide–MHC interactions. The crystal structure reveals how six αβTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αβTCR. Here, the αβTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αβTCR can recognise an intact foreign protein in an antibody-like manner. Certain specific antigens have been shown to activate T cells in an MHC independent manner. Here the authors show a phycoerythrin reactive mouse TCR which recognises native protein and characterise the molecular nature of this interaction and that this specific TCR can be selected in the thymus. [ABSTRACT FROM AUTHOR]

Background: Haemoglobinopathy refers to a group of common monogenic inherited conditions associated with variations in the haemoglobin molecule; however, there is relatively limited reporting on abnormal haemoglobinopathy in the Chinese population, especially rare abnormal haemoglobin (Hb). The aim of this study was to explore the clinical characteristics of haemoglobinopathy to supplement data for the epidemiological investigation of Hb variants in Guangdong province of China. Methods: Peripheral blood was collected from five patients (including a family) for Complete blood count, Hb electrophoresis, High‐performance liquid chromatography analysis and degenerative globin body testing. Hb variants were further analysed by PCR and DNA sequencing. Results: The research subjects were diagnosed with different types of abnormal Hb. The blood routine of the Hb Fukuyama (HBB:c.232C>T) diagnosed individual showed microcytic hypochromic anaemia, with a lower Hb level (64 g/L), mean corpuscular volume (MCV) of 71.5 fL and mean corpuscular haemoglobin (MCH) of 21.5 pg. Individuals diagnosed with Hb Port Phillip (HBA2:c.275T>C) exhibit a MCH level that is slightly below average, at 26.4 pg. The Hb Saint Etienne (HBB:c.279C>G) diagnosed individual showed macrocytic hypochromic anaemia, and the proband had a low Hb level (116 g/L), MCV of 102.2 fL and MCH of 29.4 pg. Conclusion: We confirmed the presence of Hb Fukuyama (HBB:c.232C>T) in China for the first time. Three rare patients with the Hb Saint Etienne (HBB:c.279C>G) phenotype and one patient with Hb Port Phillip (HBA2:c.275T>C) phenotype were included. Our research enriches the gene mutation map of haemoglobinopathy in Guangdong province and improves the detection system of haemoglobinopathy for population prevention and eugenics. [ABSTRACT FROM AUTHOR]

Previously, we reported the development of a human Aγ-globin gene lentivirus (LV), GbG, which expresses high levels of HbF to correct the sickle cell anemia (SCA) phenotype in the Berkeley SCA mouse model, and then modified the γ-globin gene by substituting glycine at codon 16 with aspartic acid in the Aγ-globin gene to generate GbGM LV. In the present study, we evaluated the long-term safety of human Aγ-globin gene carrying GbGM LV in wild-type mice after primary and secondary transplants of GbGM-modified hematopoietic stem cells (HSC) over 18 months. The safety of the GbGM bone marrow transplant was assessed by monitoring the effects on body weight, hematology, histopathology, malignancy formation, and survival. Mice transplanted with Mock-transduced and spleen focus forming virus (SFFV) γ-retroviral vector (RV)-transduced HSC served as negative and positive controls, respectively. The mean donor-cell engraftment was comparable across Mock, GbGM LV, and SFFV RV groups. There were no significant differences in body weight, clinical signs, immunophenotype, or histopathology in the GbGM-treated mice compared to controls. Four SFFV RV-treated mice, but none of the GbGM-treated mice, developed donor-derived, vector-positive lymphomas as demonstrated by flow cytometry analysis and in situ hybridization. These results highlight the safety of the administration of GbGM LV-modified HSC with long-term follow-up after primary and secondary transplants in mice. This data supported the initiation of phase 1/2 first-in-human SCA clinical trial in the United States. [ABSTRACT FROM AUTHOR]

Hell's Gate globin-I (HGb-I) is a thermally stable globin from the aerobic methanotroph Methylacidiphilium infernorum. Here we report that HGb-I interacts with lipids stoichiometrically to induce structural changes in the heme pocket, changing the heme iron distal ligation coordination from hexacoordinate to pentacoordinate. Such changes in heme geometry have only been previously reported for cytochrome c and cytoglobin, linked to apoptosis regulation and enhanced lipid peroxidation activity, respectively. However, unlike cytoglobin and cytochrome c, the heme iron of HGb-I is altered by lipids in ferrous as well as ferric oxidation states. The apparent affinity for lipids in this thermally stable globin is highly pH-dependent but essentially temperature-independent within the range of 20–60 °C. We propose a mechanism to explain these observations, in which lipid binding and stability of the distal endogenous ligand are juxtaposed as a function of temperature. Additionally, we propose that these coupled equilibria may constitute a mechanism through which this acidophilic thermophile senses the pH of its environment. [ABSTRACT FROM AUTHOR]

Haemoglobin disorders represent a heterogeneous group of inherited conditions that involve at least one genetic abnormality in one or more of the globin chains, resulting in changes in the structure, function, and/or amount of haemoglobin molecules, which are very important for their related clinical aspects. Detecting and characterizing these disorders depends primarily on laboratory methods that employ traditional approaches and, when necessary, newer methodologies essential for solving a number of diagnostic challenges. This review provides an overview of key laboratory techniques in the diagnosis of haemoglobinopathies, focusing on the challenges, advancements, and future directions in this field. Moreover, many haemoglobinopathies are benign and clinically silent, but it is not uncommon to find unexpected variants during routine laboratory tests. The present work reported a rare and clinically interesting case of identification of haemoglobin fractions in an adult man by the determination of glycated haemoglobin (HbA1c) during a routine laboratory assessment, highlighting how the correct use of laboratory data can modify and improve the patient's clinical management. [ABSTRACT FROM AUTHOR]

It is now widely recognised that the environment in space activates a diverse set of genes involved in regulating fundamental cellular pathways. This includes the activation of genes associated with blood homoeostasis and erythropoiesis, with a particular emphasis on those involved in globin chain production. Haemoglobin biology provides an intriguing model for studying space omics, as it has been extensively explored at multiple -omic levels, spanning DNA, RNA, and protein analyses, in both experimental and clinical contexts. In this study, we examined the developmental expression of haemoglobin over time and space using a unique suite of multi-omic datasets available on NASA GeneLab, from the NASA Twins Study, the JAXA CFE study, and the Inspiration4 mission. Our findings reveal significant variations in globin gene expression corresponding to the distinct spatiotemporal characteristics of the collected samples. This study sheds light on the dynamic nature of globin gene regulation in response to the space environment and provides valuable insights into the broader implications of space omics research. Here the authors analyse the impact of space on haemoglobin gene regulation using data from NASA, JAXA and SpaceX i4 missions. They find that globin gene down-regulation leads to space anaemia with post-flight recovery, and reveal an adult-to-foetal globin switch activation. [ABSTRACT FROM AUTHOR]

In order to become bioactive, proteins must be translated and protected from aggregation during biosynthesis. The ribosome and molecular chaperones play a key role in this process. Ribosome-bound nascent chains (RNCs) of intrinsically disordered proteins and RNCs bearing a signal/arrest sequence are known to interact with ribosomal proteins. However, in the case of RNCs bearing foldable protein sequences, not much information is available on these interactions. Here, via a combination of chemical crosslinking and time-resolved fluorescence-anisotropy, we find that nascent chains of the foldable globin apoHmp1–140 interact with ribosomal protein L23 and have a freely-tumbling non-interacting N-terminal compact region comprising 63–94 residues. Longer RNCs (apoHmp1–189) also interact with an additional yet unidentified ribosomal protein, as well as with chaperones. Surprisingly, the apparent strength of RNC/r-protein interactions does not depend on nascent-chain sequence. Overall, foldable nascent chains establish and expand interactions with selected ribosomal proteins and chaperones, as they get longer. These data are significant because they reveal the interplay between independent conformational sampling and nascent-protein interactions with the ribosomal surface. [ABSTRACT FROM AUTHOR]

β-Thalassemia is an inherited genetic disorder associated with β-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of β-thalassemia. This study conducted a virtual screening of certain compounds similar to 5′-deoxy-5′methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of β-thalassemia. [ABSTRACT FROM AUTHOR]

Background ß-thalassemia major is a genetic issue depicted by a diminished speed of hemoglobin creation, provoking insufficient association of no less than one globin chain. The reality of shortcomings could change depending upon the specific sickness. The model for this study involved not being set in stone to have ß-thalassemia, while an additional fifty individuals without the condition were picked as the benchmark bunch. The Ibn Albaladi Thalassemia Center assembled blood tests During the period from 1st June to 31 October 31, 2022. The place of this study was to assess the levels of serum immunological markers, expressly IL-6 and IL-8, in patients from Iraq who are assailed with ß-thalassemia. The survey plan for postgraduate assessments at the School of Baghdad has been embraced by the Panel of the Association of Innate Planning and Biotechnology. The protein-associated immunosorbent look (ELISA) was utilized to assess the unions of human interleukin-6 (IL-6) and interleukin-8 (IL-8). The discoveries of the review showed that people determined to have ß-thalassemia displayed strikingly raised serum groupings of IL-6 and IL-8, with factual importance. It was found that individuals with ß-thalassemia displayed IL-6 focuses estimating 30.90±3.39 pg/ml, though the benchmark group exhibited degrees of 20.21±2.88 pg/ml. Likewise, it was seen that the IL-8 level in the benchmark group was estimated to be 74.86±21.01 pg/ml, though the patient gathering showed a higher IL-8 degree of 219.10±47.26 pg/ml. The discoveries of this study show that people with thalassemia display an insusceptible dysregulation portrayed by simultaneous immunosuppression and irritation. [ABSTRACT FROM AUTHOR]

[Display omitted] Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) in the two species of Eremias lizards dwelling on the Qinghai-Tibet Plateau. By measuring O2 equilibrium curves of purified Hbs at different pH and temperature in the absence and presence of ATP and/or Cl−, the study found that the high-altitude populations of the two species of Eremias lizards exhibit convergent high Hb-O2 affinity compared to the respective lowland counterparts while demonstrating distinct ATP-mediated allosteric regulation. Hbs of the highland E. argus showed high ATP sensitivity and ATP-dependent strong Bohr effect compared to E. multiocellata. The underlying mechanisms of these functional variations may be attributed to the varying β2/β1 globin ratios, combined with substitutions on the β2-type globin, as suggested by Hb isoform identification and structural analysis of tetrameric Hbs. In addition, Hbs of these Eremias lizards have similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could minimize the impact of temperature fluctuations on Hb-O2 affinity and facilitate the release of O2 in the cold extremities at low temperatures. The functional adaptation and underlying molecular mechanisms of hemoglobins (Hbs) have primarily concentrated on mammals and birds, with few reports on reptiles. This study aimed to investigate the convergent and species-specific high-altitude adaptation mechanisms of Hbs in two Eremias lizards from the Qinghai-Tibet Plateau. The Hbs of high-altitude E. argus and E. multiocellata were characterized by significantly high overall and intrinsic Hb-O2 affinity compared to their low-altitude populations. Despite the similarly low Cl− sensitivities, the Hbs of high-altitude E. argus exhibited higher ATP sensitivity and ATP-dependent Bohr effects than that of E. multiocellata, which could facilitate O2 unloading in respiring tissues. Eremias lizards Hbs exhibited similarly low temperature sensitivities and relatively high Bohr effects at lower temperatures, which could help to stably deliver and release O2 to cold extremities at low temperatures. The oxygenation properties of Hbs in high-altitude populations might be attributed to varying ratios of β2/β1 globin and substitutions on the β2-type globin. Notably, the Asn12Ala in lowland E. argus could cause localized destabilization of the E-helix in the tetrameric Hb by elimination of hydrogen bonds, thereby resulting in its lowest O2 affinity. This study provides a valuable reference for the high-altitude adaptation mechanisms of hemoglobins in reptiles. [ABSTRACT FROM AUTHOR]

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis. [ABSTRACT FROM AUTHOR]

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development. [ABSTRACT FROM AUTHOR]

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications. Sickle cell disease (SCD) and β-thalassemia (BT) are globally prevalent inherited blood disorders but, despite extensive research, no ex vivo system exists for SCD and BT. Here, the authors generate pathophysiologically relevant erythroid progenitor models of SCD and BT. [ABSTRACT FROM AUTHOR]

Hypobaric hypoxia is situation that might occur to helicopter pilots in Indonesia who must fly at an altitude of more than 3,048 m such as in Papua. It can be dangerous because hypoxic condition can affect person's performance. So far, the heart is known as an aerobic organ and very sensitive to hypoxic conditions. Hitherto, the effects of hypobaric hypoxia exposure on biomolecular aspects of the heart are still unclear. Therefore, this study assessed cardiac response in rats exposed to intermittent hypobaric hypoxia (IHH) (equivalent to 3,048 meters/10,000 feet). Sprague-Dawley rats were divided into six groups: control; acute hypobaric hypoxia (AHH); and IHH, for 7; 14; 21; and 28 days. We measured super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, reduced glutathione (GSH), malondialdehyde (MDA), cytoglobin, myoglobin, HIF-1α, and Nrf2 level as our parameters. Activities of SOD, CAT, GPx and GSH increased while the levels of MDA, cytoglobin, myoglobin, HIF-1α, and Nrf2 decreased in all IHH groups compared with the AHH group. A biphasic pattern was observed as IHH sessions increased from 14 to 21 or 28. Where the IHH treatment for more than 14 sessions caused a decrease in endogenous antioxidants, but the response to hypoxia and oxidative stress increased. Our findings presented the molecular alterations of cardiac rats exposed to intermittent hypobaric hypoxia. [ABSTRACT FROM AUTHOR]

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia. [ABSTRACT FROM AUTHOR]

Cytoglobin is a heme protein with unresolved physiological function. Genetic deletion of zebrafish cytoglobin (cygb2) causes developmental defects in left-right cardiac determination, which in humans is associated with defects in ciliary function and low airway epithelial nitric oxide production. Here we show that Cygb2 co-localizes with cilia and with the nitric oxide synthase Nos2b in the zebrafish Kupffer's vesicle, and that cilia structure and function are disrupted in cygb2 mutants. Abnormal ciliary function and organ laterality defects are phenocopied by depletion of nos2b and of gucy1a, the soluble guanylate cyclase homolog in fish. The defects are rescued by exposing cygb2 mutant embryos to a nitric oxide donor or a soluble guanylate cyclase stimulator, or with over-expression of nos2b. Cytoglobin knockout mice also show impaired airway epithelial cilia structure and reduced nitric oxide levels. Altogether, our data suggest that cytoglobin is a positive regulator of a signaling axis composed of nitric oxide synthase–soluble guanylate cyclase–cyclic GMP that is necessary for normal cilia motility and left-right patterning. Developmental defects in left-right cardiac determination in humans are associated with ciliary dysfunction and low airway epithelial nitric oxide production. Here, the authors show that cytoglobin is essential for nitric oxide signaling, cilia function, and left-right patterning during zebrafish development. [ABSTRACT FROM AUTHOR]

An increasing number of α-hemoglobin (Hb) variants is causing various clinical symptoms; therefore, accurate identification of these Hb variants is important. This study aimed to describe the molecular and hematological characteristics of novel Hb Doi-Saket that gives rise to a typical α+-thalassemia phenotype in carriers with and without other hemoglobinopathies. Biological samples from a proband and his family members were analyzed. Hematological profiles were analyzed using a standard automated cell counter. Hb was analyzed by capillary electrophoresis and high-performance liquid chromatography. Mutations and globin haplotype were identified by DNA analysis. Novel diagnostic tools based on allele-specific polymerase chain reaction (PCR) and PCR–restriction fragment length polymorphism were developed. Hb analysis showed a major abnormal Hb fraction, moving slower than HbA, and a minor Hb fraction alongside HbA2 in the proband, his father, and son. DNA analysis of the α-globin gene identified the -α3.7 deletion and in cis the C > A mutation on codon 9 of the α2α1 gene, corresponding to Hb Doi-Saket [α9(A7) Asn > Lys]. This mutation could be identified using newly developed allele-specific PCR-based assays. The Hb Doi-Saket al.lele was significantly associated with haplotype [- + M + + 0 -]. Interaction of αDoi-Saket with βE globin chains led to a new Hb variant (HbE Doi-Saket). Phenotypic expression was clinically silent in heterozygotes and might present slight microcytosis. Hb Doi-Saket emphasizes a great diversity present in α-globin gene. The mutation in this family from Thailand was linked to -α3.7 and caused mild microcytosis in the carriers. The combination of this variant with deletions in α genes might cause a severe clinical phenotype. Different methods of separation can provide useful information in diagnosis, and a complete molecular approach is needed for confirmation before considering patient management. The Hb Doi-Saket is a novel α-globin variant mutation occurring in the α2-globin gene in cis to the -α3.7 kb chromosome. The carrier of Hb Doi-Saket may present slight microcytosis and have severe clinical entities when it interacts with deletions in α-globin genes. Hb analysis with the HPLC system could completely separate Hb Doi-Saket and its derivative from other Hbs. [ABSTRACT FROM AUTHOR]

Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high‐active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme‐supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate‐limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450‐BM3, which can be used in the development of artificial meat, high‐cell‐density fermentation, and whole‐cell catalytic synthesis of high‐value‐added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields. [ABSTRACT FROM AUTHOR]

Purpose: Fetal hemoglobin (HbF) upregulation is a mitigating factor in ß-hemoglobinopathies therapy like ß-thalassemia and sickle cell diseases. Finding molecular mechanisms and the key regulators responsible for globin switching could be helpful to develop effective ways to HbF upregulation. In our prior in silico report, we identified a few factors that are likely to be responsible for globin switching. The goal of this study is to experimentally validate the factors. Methods: We established K562 cell line with BCL11A knock down leading to increase in HBG1/2 using CRISPR/Cas9 system. Then, using quantitative polymerase chain reaction (qPCR), we determined the expression level of the factors which were previously identified in our prior in silico study. Results: our analysis showed that BCL11A was substantially knocked down, resulting in the upregulation of HBG1/2 in the BCL11A-ablated K562 cells using CRISPR/Cas9 system. Additionally, the experimental data acquired in this study validated our prior bioinformatics findings about three potentially responsible genes for globin switching, namely HIST1H2Bl, TRIM58, and Al133243.2. Conclusion: BCL11A is a promising candidate for the treatment of ß-hemoglobinopathies, with high HbF reactivation. In addition, HIST1H2BL, TRIM58 and Al133243.2 are likely to be involved in the mechanism of hemoglobin switching. To further validate the selected genes, more experimental in vivo and in vitro studies are required. [ABSTRACT FROM AUTHOR]

We previously reported initial clinical results of post-transcriptional gene silencing of BCL11A expression (NCT 03282656) reversing the fetal to adult hemoglobin switch. A goal of this approach is to increase fetal hemoglobin (HbF) expression while coordinately reducing sickle hemoglobin (HbS) expression. The resulting combinatorial effect should prove effective in inhibiting HbS polymerization at lower physiologic oxygen values thereby mitigating disease complications. Here we report results of exploratory single-cell analysis of patients in which BCL11A is targeted molecularly and compare results with cells of patients treated with hydroxyurea (HU), the current standard of care. We use single-cell assays to assess HbF, HbS, oxygen saturation, and hemoglobin polymer content in RBCs for nine gene therapy trial subjects (BCLshmiR, median HbF% = 27.9) and compare them to 10 HU-treated subjects demonstrating high and comparable levels of HbF (HU High Responders, median HbF% = 27.0). All BCL11A patients achieved the primary endpoint for NCT 03282656, which was defined by an absolute neutrophil count greater than or equal to 0.5 × 109 cells/L for three consecutive days, achieved within 7 weeks following infusion. Flow cytometric assessment of single-RBC HbF and HbS shows fewer RBCs with high HbS% that would be most susceptible to sickling in BCLshmiR vs. HU High Responders: median 42% of RBCs with HbS%>70% in BCLshmiR vs. 61% in HU High Responders (p = 0.004). BCLshmiR subjects also demonstrate more RBCs resistant to HbS polymerization at lower physiologic oxygen tension: median 32% vs. 25% in HU High Responders (p = 0.006). Gene therapy-induced BCL11A down-regulation reverses the fetal-to-adult hemoglobin switch and induces RBCs with higher HbF%, lower HbS%, and greater resistance to deoxygenation-induced polymerization in clinical trial subjects compared with a cohort of highly responsive hydroxyurea-treated subjects. The authors report in full the primary endpoint data of a pilot clinical trial (NCT 03282656) that used post-transcriptional gene silencing of BCL11A expression to reverse the fetal to adult hemoglobin switch in sickle cell disease. They develop new single-cell flow cytometry and microfluidic techniques to predict the efficacy of HbF induction and show that red blood cells from these patients exhibit greater resistance to deoxygenation-induced polymerization than red blood cells from hydroxyurea-responsive patients. [ABSTRACT FROM AUTHOR]

Background: Previous retrospective results are evaluated prospectively and blinded. Methods: A total of 221 eyes previously classified as normal (G1), 279 as moderate risk of glaucoma (G2) and 217 as high risk (G3) according to the Globin Discriminant Function (GDF) Laguna-ONhE index were examined with OCT Spectralis- Results: In G1, the Bruch's Membrane Opening Minimum Rim Width (BMO-MRW) was 332 ± 55 microns; in G2, it was 252 ± 47 (p < 0.0001); and in G3, 231 ± 44 (p < 0.0001). In G1, the 1% and 5% percentiles were 233 and 248, respectively; in G2, they were lower in 28.80% and 42.29% of cases, respectively; and in G3, in 50.23% and 63.59% of cases, respectively. Most of the cases were normal-tension glaucomas. Laguna-ONhE indices showed a curvilinear correlation with BMO-MRW results. The Retinal Nerve Fibre Layer (RNFL) showed a poor relationship with BMO. Assuming G1 to be truly normal, BMO-MRW would have a Receiver operating characteristic (ROC) curve area of 0.901 for G2 and G3 and 0.651 for RNFL. A significant reduction in pixels corresponding to vessels was found in G2 and G3 vs. G1 (p < 0.0001). Conclusions: In some cases, these defects appear to be mainly glaucomatous, and in others, they are associated with diabetic microangiopathy. In normal tension glaucoma, RNFL defects may be less severe than those inside the nerve. [ABSTRACT FROM AUTHOR]

The most prevalent hereditary monogenic disorders that claim millions of lives globally are thalassemic syndromes. A thalassemia is an inherited condition, at least one parent must carry the disease's gene. Perhaps a genetic mutation/ defective globin chain or the loss of specific important gene segments is the main cause. Thalassemic illnesses started to strain the healthcare systems of several nations worldwide. Management of thalassemia is now seen as a lifelong treatment that requires continuous monitoring. In this review, we seek to compile and analyze recent research on thalassemia diagnosis and treatment, including papers, studies, and clinical trials. We also intend to present a concise yet comprehensive study. A thalassemia is an inherited condition, at least one parent must carry the disease's gene. Perhaps a genetic mutation/ defective globin chain or the loss of specific important gene segments is the main cause. [ABSTRACT FROM AUTHOR]

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias. [ABSTRACT FROM AUTHOR]

This article discusses a case study of a fetus with homozygous alpha-thalassemia, a type of inherited anemia prevalent in Southeast Asians. The condition leads to inadequate production of alpha-globin chains, resulting in the formation of abnormal hemoglobin and causing severe anemia and tissue hypoxia. The fetus in the case study exhibited limb defects, an enlarged heart, and cortical malformations. The study suggests that homozygous alpha-thalassemia should be considered in fetuses with cortical malformations and limb defects, particularly in Southeast Asian couples with signs of anemia during pregnancy. [Extracted from the article]

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Neuroglobin, which is a heme protein from the globin family that is predominantly expressed in nervous tissue, can promote a neuronal survivor. However, the molecular mechanisms underlying the neuroprotective function of Ngb remain poorly understood to this day. The interactions between neuroglobin and mitochondrial cytochrome c may serve as at least one of the mechanisms of neuroglobin-mediated neuroprotection. Interestingly, neuroglobin and cytochrome c possibly can interact with or without electron transfer both in the cytoplasm and within the mitochondria. This review provides a general picture of molecular interactions between neuroglobin and cytochrome c based on the recent experimental and computational work on neuroglobin and cytochrome c interactions. [ABSTRACT FROM AUTHOR]

Pectobacterium carotovorum is an important plant pathogen responsible for the destruction of crops through bacterial soft rot, which is modulated by oxygen (O2) concentration. A soluble globin coupled sensor protein, Pcc DgcO (also referred to as PccGCS) is one way through which P. carotovorum senses oxygen. DgcO contains a diguanylate cyclase output domain producing c-di-GMP. Synthesis of the bacterial second messenger c-di-GMP is increased upon oxygen binding to the sensory globin domain. This work seeks to understand regulation of function by DgcO at the transcript level. RNA sequencing and differential expression analysis revealed that the deletion of DgcO only affects transcript levels in cells grown under aerobic conditions. Differential expression analysis showed that DgcO deletion alters transcript levels for metal transporters. These results, followed by inductively coupled plasma--mass spectrometry showing decreased concentrations of six biologically relevant metals upon DgcO deletion, provide evidence that a globin coupled sensor can affect cellular metal content. These findings improve the understanding of the transcript level control of O2- dependent phenotypes in an important phytopathogen and establish a basis for further studies on c-di-GMP-dependent functions in P. carotovorum. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a debilitating neurodegenerative disease characterised by the accumulation of amyloid-beta and tau in the brain, leading to the progressive loss of memory and cognition. The causes of its pathogenesis are still not fully understood, but some risk factors, such as age, genetics, and hormones, may play a crucial role. Studies show that postmenopausal women have a higher risk of developing AD, possibly due to the decrease in hormone levels, especially oestrogen, which may be directly related to a reduction in the activity of oestrogen receptors, especially beta (ERβ), which favours a more hostile cellular environment, leading to mitochondrial dysfunction, mainly affecting key processes related to transport, metabolism, and oxidative phosphorylation. Given the influence of hormones on biological processes at the mitochondrial level, hormone therapies are of clinical interest to reduce the risk or delay the onset of symptoms associated with AD. One drug with such potential is tibolone, which is used in clinics to treat menopause-related symptoms. It can reduce amyloid burden and have benefits on mitochondrial integrity and dynamics. Many of its protective effects are mediated through steroid receptors and may also be related to neuroglobin, whose elevated levels have been shown to protect against neurological diseases. Its importance has increased exponentially due to its implication in the pathogenesis of AD. In this review, we discuss recent advances in tibolone, focusing on its mitochondrial-protective effects, and highlight how valuable this compound could be as a therapeutic alternative to mitigate the molecular pathways characteristic of AD. [ABSTRACT FROM AUTHOR]

Plants continuously interact with fungi, some of which, such as Fusarium oxysporum, are lethal, leading to reduced crop yields. Recently, nitric oxide (NO) has been found to play a regulatory role in plant responses to F. oxysporum, although the underlying mechanisms involved are poorly understood. In this study, we show that Arabidopsis mutants with altered levels of phytoglobin 1 (Glb1) have a higher survival rate than wild type (WT) after infection with F. oxysporum, although all the genotypes analyzed exhibited a similar fungal burden. None of the defense responses that were analyzed in Glb1 lines, such as phenols, iron metabolism, peroxidase activity, or reactive oxygen species (ROS) production, appear to explain their higher survival rates. However, the early induction of the PR genes may be one of the reasons for the observed survival rate of Glb1 lines infected with F. oxysporum. Furthermore, while PR1 expression was induced in Glb1 lines very early on the response to F. oxysporum, this induction was not observed in WT plants. [ABSTRACT FROM AUTHOR]

The protozoan disease is a major global health concern. Amoebiasis, leishmaniasis, Chagas disease, and African sleeping sickness affect several million people worldwide, leading to millions of deaths annually and immense social and economic problems. Iron is an essential nutrient for nearly all microbes, including invading pathogens. The majority of iron in mammalian hosts is stored intracellularly in proteins, such as ferritin and hemoglobin (Hb). Hb, present in blood erythrocytes, is a very important source of iron and amino acids for pathogenic microorganisms ranging from bacteria to eukaryotic pathogens, such as worms, protozoa, yeast, and fungi. These organisms have developed adequate mechanisms to obtain Hb or its byproducts (heme and globin) from the host. One of the major virulence factors identified in parasites is parasite-derived proteases, essential for host tissue degradation, immune evasion, and nutrient acquisition. The production of Hb-degrading proteases is a Hb uptake mechanism that degrades globin in amino acids and facilitates heme release. This review aims to provide an overview of the Hb and heme-uptake mechanisms utilized by human pathogenic protozoa to survive inside the host. [ABSTRACT FROM AUTHOR]

During the storage and processing of mung beans, proteins and polyphenols are highly susceptible to interactions with each other. Using globulin extracted from mung beans as the raw material, the study combined it with ferulic acid (FA; phenolic acid) and vitexin (flavonoid). Physical and chemical indicators were combined with spectroscopy and kinetic methods, relying on SPSS and peak fit data to statistically analyze the conformational and antioxidant activity changes of mung bean globulin and two polyphenol complexes before and after heat treatment and clarify the differences and the interaction mechanism between globulin and the two polyphenols. The results showed that, with the increase in polyphenol concentration, the antioxidant activity of the two compounds increased significantly. In addition, the antioxidant activity of the mung bean globulin–FA complex was stronger. However, after heat treatment, the antioxidant activity of the two compounds decreased significantly. The interaction mechanism of the mung bean globulin–FA/vitexin complex was static quenching, and heat treatment accelerated the occurrence of the quenching phenomenon. Mung bean globulin and two polyphenols were combined through a hydrophobic interaction. However, after heat treatment, the binding mode with vitexin changed to an electrostatic interaction. The infrared characteristic absorption peaks of the two compounds shifted to different degrees, and new peaks appeared in the areas of 827 cm−1, 1332 cm−1, and 812 cm−1. Following the interaction between mung bean globulin and FA/vitexin, the particle size decreased, the absolute value of zeta potential increased, and the surface hydrophobicity decreased. After heat treatment, the particle size and zeta potential of the two composites decreased significantly, and the surface hydrophobicity and stability increased significantly. The antioxidation and thermal stability of the mung bean globulin–FA were better than those of the mung bean globulin–vitexin complex. This study aimed to provide a theoretical reference for the protein–polyphenol interaction mechanism and a theoretical basis for the research and development of mung bean functional foods. [ABSTRACT FROM AUTHOR]

Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed. [ABSTRACT FROM AUTHOR]

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme–albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins. [ABSTRACT FROM AUTHOR]

Nitrobindins (Nbs) are all-β-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3−. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2− to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M−1 s−1, 9.3 M−1 s−1, 1.4 × 101 M−1 s−1, and 5.8 M−1 s−1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2−-based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-β-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-β-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged. [ABSTRACT FROM AUTHOR]

The globin protein superfamily has diverse functions. Haemoglobin has been found in non-erythroid locations, including within the mitochondria. Using co-immunoprecipitation and in silico methods, we investigated the interaction of mitochondrial haemoglobin with ATP synthase and its associated proteins, including inhibitory factor 1 (IF1). We measured the expression of mitochondrial haemoglobin in response to hypoxia. In vitro and in silico evidence of interactions between mitochondrial haemoglobin and ATP synthase were found, and we report upregulated mitochondrial haemoglobin expression in response to hypoxia within skeletal muscle tissue. Our observations indicate that mitochondrial pH and ATP synthase activity are implicated in the mitochondrial haemoglobin response to hypoxia. [ABSTRACT FROM AUTHOR]

Imbalanced globin chain output contributes to thalassemia pathophysiology. Hence, induction of fetal hemoglobin in β-thalassemia and other β-hemoglobinopathies are of continuing interest for therapeutic approaches. Genome-wide association studies have identified three common genetic loci: namely β-globin (HBB), an intergenic region between MYB and HBS1L, and BCL11A underlying quantitative fetal hemoglobin production. Here, we report that knockdown of HBS1L (all known variants) using shRNA in early erythroblast obtained from β0-thalassemia/HbE patients triggers an upregulation of γ-globin mRNA 1.69 folds. There is modest perturbation of red cell differentiation assessed by flow cytometry and morphology studies. The levels of α- and β-globin mRNAs are relatively unaltered. Knockdown of HBS1L also increases the percentage of fetal hemoglobin around 16.7 folds when compared to non-targeting shRNA. Targeting HBS1L is attractive because of the potent induction of fetal hemoglobin and the modest effect on cell differentiation. [ABSTRACT FROM AUTHOR]

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), –SEA/αCSα (1.5%), –SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations. [ABSTRACT FROM AUTHOR]

Importantly, there exist HO-1 gene promoter polymorphisms that can affect the ability to upregulate expression of the gene in humans, and a relative deficiency of HO-1 gene expression has been associated with a variety of diseases [[37]]. In his paper discussing HO-1 deficiency in nine reported human cases and animal models, Yachie 2021 [[23]] hypothesized a scenario in which the lack of HO-1 results in unregulated activation of macrophages [[24]] and release of inflammatory cytokines as well as overproduction of tissue factor, leading to dysregulation of the clotting system. We then studied several models of HO-1 deficiency and showed that methemalbumin or certain statins (independent of their lipid-lowering properties) could be used as a rescue treatment for conditions caused by a relative deficiency of HO-1 by upregulating the expression of the HO-1 gene [[21], [26]]. Biliverdin and bilirubin have antioxidant, anti-inflammatory, and anti-apoptotic properties [[14]] and bilirubin may even act as a hormone [[15]]. [Extracted from the article]

Pectobacterium carotovorum is an important plant pathogen responsible for the destruction of crops through bacterial soft rot, which is modulated by oxygen (O2) concentration. A soluble globin coupled sensor protein, Pcc DgcO (also referred to as PccGCS) is one way through which P. carotovorum senses oxygen. DgcO contains a diguanylate cyclase output domain producing c-di-GMP. Synthesis of the bacterial second messenger c-di-GMP is increased upon oxygen binding to the sensory globin domain. This work seeks to understand regulation of function by DgcO at the transcript level. RNA sequencing and differential expression analysis revealed that the deletion of DgcO only affects transcript levels in cells grown under aerobic conditions. Differential expression analysis showed that DgcO deletion alters transcript levels for metal transporters. These results, followed by inductively coupled plasma—mass spectrometry showing decreased concentrations of six biologically relevant metals upon DgcO deletion, provide evidence that a globin coupled sensor can affect cellular metal content. These findings improve the understanding of the transcript level control of O2-dependent phenotypes in an important phytopathogen and establish a basis for further studies on c-di-GMP-dependent functions in P. carotovorum.

Intravascular hemolysis occurs in diverse pathological conditions. Extracellular hemoglobin and heme have strong pro-oxidative and pro-inflammatory potentials that can contribute to the pathology of hemolytic diseases. However, many of the effects of extracellular hemoglobin and heme in hemolytic diseases are still not well understood. Here we demonstrate that oxidized hemoglobin (methemoglobin) can modify the antigen-binding characteristics of human immunoglobulins. Thus, incubation of polyclonal or some monoclonal human IgG in the presence of methemoglobin results in an appearance of binding reactivities towards distinct unrelated self-proteins, including the protein constituent of hemoglobin i.e., globin. We demonstrate that a transfer of heme from methemoglobin to IgG is indispensable for this acquisition of antibody polyreactivity. Our data also show that only oxidized form of hemoglobin have the capacity to induce polyreactivity of antibodies. Site-directed mutagenesis of a heme-sensitive human monoclonal IgG1 reveals details about the mechanism of methemoglobin-induced antigen-binding polyreactivity. Further here we assess the kinetics and thermodynamics of interaction of a heme-induced polyreactive human antibody with hemoglobin and myoglobin. Taken together presented data contribute to a better understanding of the functions of extracellular hemoglobin in the context of hemolytic diseases. Oxidized hemoglobin induces polyreactivity and autoreactivity of human IgG through direct transfer and binding of heme to the variable region of IgG, which contributes to a better understanding of the physiopathology of hemolytic diseases. [ABSTRACT FROM AUTHOR]

When profiling blood samples by RNA-sequencing (RNA-seq), RNA from haemoglobin (Hgb) can account for up to 70% of the transcriptome. Due to considerations of sequencing depth and power to detect biological variation, Hgb RNA is typically depleted prior to sequencing by hybridisation-based methods; an alternative approach is to deplete reads arising from Hgb RNA bioinformatically. In the present study, we compared the impact of these two approaches on the outcome of differential gene expression analysis performed using RNA-seq data from 58 human tuberculosis (TB) patient or contact whole blood samples–29 globin kit-depleted and 29 matched non-depleted—a subset of which were taken at TB diagnosis and at six months post-TB treatment from the same patient. Bioinformatic depletion of Hgb genes from the non-depleted samples (bioinformatic-depleted) substantially reduced library sizes (median = 57.24%) and fewer long non-coding, micro, small nuclear and small nucleolar RNAs were captured in these libraries. Profiling published TB gene signatures across all samples revealed inferior correlation between kit-depleted and bioinformatic-depleted pairs when the proportion of reads mapping to Hgb genes was higher in the non-depleted sample, particularly at the TB diagnosis time point. A set of putative "globin-fingerprint" genes were identified by directly comparing kit-depleted and bioinformatic-depleted samples at each timepoint. Two TB treatment response signatures were also shown to have decreased differential performance when comparing samples at TB diagnosis to six months post-TB treatment when profiled on the bioinformatic-depleted samples compared with their kit-depleted counterparts. These results demonstrate that failure to deplete Hgb RNA prior to sequencing has a negative impact on the sensitivity to detect disease-relevant gene expression changes even when bioinformatic removal is performed. [ABSTRACT FROM AUTHOR]

Hydroxyurea (HU) is found to be beneficial in sickle cell anaemia (SCA) patients, due to its ability to increase foetal haemoglobin (HbF), however, patients show a variable response. Differences in HbF levels are attributed to many factors; but, the role of miRNA in HbF regulation is sparsely investigated. In this study, we evaluated the effect of miRNA expression on HbF induction in relation to hydroxyurea therapy in 30 normal controls, 30 SCA patients at baseline, 20 patients after 3 and 6 months of hydroxyurea (HU) therapy. HbF levels were measured by HPLC. Total RNA and miRNA were extracted from CD71+ erythroid cells and the expression was determined using Taqman probes. The mean HbF level increased 7.54 ± 2.44 fold, after 3 months of HU therapy. After the HU therapy 8 miRNAs were significantly up-regulated while 2 were down-regulated. The increase in miR-210, miR16-1, and miR-29a expression and decrease in miR-96 expression were strongly associated with the HU mediated HbF induction. Post HU therapy, decreased miR-96 expression negatively correlate with HbF and γ-globin gene while increased expression of miR-210, miR-16-1 and miR-29a post HU therapy positively corelate with HbF and γ-globin gene. Thus, suggest that miR-210, miR-16-1 and miR-29a are positive regulator of γ-globin gene and miR-96 is negative regulator of γ-globin gene. The study suggests the role of miR-210, miR16-1, miR-29a, and miR-96 in γ-globin gene regulation leading to HbF induction. Identification of the relevant protein targets might be useful for understanding the HU mediated HbF induction. [ABSTRACT FROM AUTHOR]

Biomonitoring of human exposure to reactive electrophilic chemicals such as ethylene oxide (EO) has been commonly based on the determination of adducts with N -terminal valine in blood protein globin, but a systematic search has also been undertaken to find surrogate markers enabling non-invasive sampling. Recently, N -(2-hydroxyethyl)-L-valyl-L-leucine (HEVL) has been identified as an ultimate cleavage product of EO-adducted globin in the urine of occupationally exposed workers. Herein, full validation of the analytical procedure consisting of solid-phase extraction of HEVL from urine samples (2 mL) followed by high-performance liquid chromatography–electrospray ionization–high-resolution mass spectrometry determination using deuterium-labeled HEVL as an internal standard (IS) is described. Method limit of quantitation is 0.25 ng/mL, and its selectivity is excellent as demonstrated by the invariable ratio of the qualifier and quantifier ion intensities across diverse urine samples and synthetic standard. The linear calibration model was applicable over the whole concentration range tested (0.25–10 ng/mL). The method accuracy assessed as a recovery of HEVL using a spiking experiment was 98–100%. Within-day precision of the method ranged from 1.8% to 3.0%, while the results from consecutive analytical runs conducted within 1 week or within 10–150 weeks differed in the range of 2.2–9.7%. The stability study on urine samples (−20°C up to 3 years, freeze-and-thaw up to 10 cycles) as well as on aqueous solutions (5°C up to 4 months) indicated no relevant changes in HEVL concentration (≤4%) over the time tested. Analytical responses of both HEVL and IS correlated with urinary creatinine as an index of matrix composition, but this matrix effect was mostly eliminated using the HEVL/IS peak area ratio, attaining the IS-normalized relative matrix effect <3%. In conclusion, the method complied successfully with the bioanalytical method validation criteria, making it a reliable tool for HEVL determination in human biomonitoring. [ABSTRACT FROM AUTHOR]

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5′-GGTTAT-3′) is located within the 5′-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in β-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify β-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in β-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of β-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment. [ABSTRACT FROM AUTHOR]

Copyright of Brazilian Journal of Biology is the property of Instituto Internacional de Ecologia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Early brain injury is a series of pathophysiological changes and direct damage of brain tissue within 72 h after subarachnoid hemorrhage before cerebral vasospasm occurs. Early brain injury is a key factor affecting the prognosis of subarachnoid hemorrhage, and its possible pathological mechanisms include oxidative stress, cell apoptosis, autophagy, and immune inflammation. Microglia are important immune cells of the central nervous system. Microglia play a dual role in protection and injury. Microglia are involved in the occurrence of brain edema, the processes of neuronal apoptosis, and the blood–brain barrier disruption after subarachnoid hemorrhage (SAH) through the signaling pathways mediated by receptors such as Toll-like receptor 4 (TLR4), calcium-sensing receptor (CaSR), and triggering receptor expressed on myeloid cells-1 (TREM-1), which secrete pro-inflammatory cytokines such as interleukins and tumor necrosis factor α. Conversely, they exert their anti-inflammatory and protective effects by expressing substances such as neuroglobin and heme oxygenase-1. This article reviews the latest developments in single-cell transcriptomics for microglia in early brain injury after subarachnoid hemorrhage and its inflammatory role. [ABSTRACT FROM AUTHOR]

Objective: To determine the frequency of iron deficiency anemia among women in Skardu, Gligit-Baltistan. Study Design: Cross-sectional study. Place and Duration of Study: Combined Military Hospital, Skardu, Pakistan, from Dec 2021 to May 2022. Methodology: A total of 202 women were selected through consecutive sampling from General Medical OPD of Combined Military Hospital, Skardu, Pakistan. Hemoglobin globin levels (Hb gram/deciliter), mean corpuscular volume (MCV fL) and red cell distribution width (RDW%) were determined for all enrolled patients. Results: Mean age of participants in this study was 34.46±10.93 years. Reduced Hb was observed in 62(30.69%) women with a mean hemoglobin level of 9.48±1.59 gl/deciliter and mean corpuscular volume of 79.97±10.7d fL. Among these women, 5(2.47%) were severely anemic (6.56±0.39 gl/deciliter), 28(13.86%) moderately anemic (8.51±0.83 gl/deciliter) while 29(14.35%) were mildly anemic (10.92±0.48 gl/deciliter). Women of younger age group, between18-35 years, were found to suffer more from iron deficiency anemia (39.14%) with mean hemoglobin levels of 11.31±1.80 and mean corpuscular volume of 79.34±10.86 fL. Conclusion: Iron Deficiency Anemia is present in female population of Gilgit-Baltistan in general and woman of childbearing age in particular. Emergency measures for awareness and fortification of basic food items with iron should be undertaken at large scale. [ABSTRACT FROM AUTHOR]