HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis.

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg-1 ·d-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

Human Hemoglobin Subunit Beta Functions as a Pleiotropic Regulator of RIG-I/MDA5-Mediated Antiviral Innate Immune Responses.

Hemoglobin is an important oxygen-carrying protein and plays crucial roles in establishing host resistance against pathogens and in regulating innate immune responses. The hemoglobin subunit beta (HB) is an essential component of hemoglobin, and we have previously demonstrated that the antiviral role of the porcine HB (pHB) is mediated by promoting type I interferon pathways. Thus, considering the high homology between human HB (hHB) and pHB, we hypothesized that hHB also plays an important role in the antiviral innate immunity. In this study, we characterized hHB as a regulatory factor for the replication of RNA viruses by differentially regulating the RIG-I- and MDA5-mediated antiviral signaling pathways. Furthermore, we showed that hHB directly inhibited MDA5-mediated signaling by reducing the MDA5-double-stranded RNA (dsRNA) interaction. Additionally, hHB required hHB-induced reactive oxygen species (ROS) to promote RIG-I-mediated signaling through enhancement of K63-linked RIG-I ubiquitination. Taken together, our findings suggest that hHB is a pleiotropic regulator of RIG-I/MDA5-mediated antiviral responses and further highlight the importance of the intercellular microenvironment, including the redox state, in regulating antiviral innate immune responses.IMPORTANCE Hemoglobin, the most important oxygen-carrying protein, is involved in the regulation of innate immune responses. We have previously reported that the porcine hemoglobin subunit beta (HB) exerts antiviral activity through regulation of type I interferon production. However, the antiviral activities and the underlying mechanisms of HBs originating from other animals have been poorly understood. Here, we identified human HB (hHB) as a pleiotropic regulator of the replication of RNA viruses through regulation of RIG-I/MDA5-mediated signaling pathways. hHB enhances RIG-I-mediated antiviral responses by promoting RIG-I ubiquitination depending on the hHB-induced reactive oxygen species (ROS), while it blocks MDA5-mediated antiviral signaling by suppressing the MDA5-dsRNA interaction. Our results contribute to an understanding of the crucial roles of hHB in the regulation of the RIG-I/MDA5-mediated signaling pathways. We also provide novel insight into the correlation of the intercellular redox state with the regulation of antiviral innate immunity.

A Unique Case of a Compound Heterozygosity of Hemoglobin Korle-Bu and Sickle Cell Trait in a Military Trainee.

Hemoglobin Korle-Bu (Hb KB) is a rare and likely under-reported hemoglobin (Hb) variant resulting from an unusual point mutation on the beta-globin chain. Hb KB is typically clinically silent, and there are limited reports of Hb KB heterozygosity compounded with other hemoglobinopathies that can present with varying clinical phenotypes. Here, we report a case of compound Hb KB heterozygosity with Hb S in an asymptomatic military trainee with a positive sickle cell screening test. Hb capillary and gel electrophoresis predicted a compound Hb S/D-Punjab overlap, which foretells a severe clinical phenotype. Sequencing of the Hb beta gene HBB demonstrated Hb KB, allowing for a diagnosis that fit his asymptomatic clinical phenotype and allowed for retention in the military.

Hemoglobin subunit beta protein as a novel marker for time since deposition of bloodstains at crime scenes.

Dried bloodstains at crime scenes provide abundant information for analyzing criminal identity of victims or suspects, morphological characteristics, and biological and chemical compounds. Therefore, they are considered important evidence by investigators at crime scenes. Moreover, the age of bloodstains can be used to determine the timeline of incidents at crime scenes; Inappropriately handled bloodstains may cause degradation of blood components. In this study, we identified a novel marker, hemoglobin subunit beta protein, as an internal standard to determine the age of bloodstains at crime scenes. We found that the target spot between 20 and 30 kDa in two-dimensional electrophoresis gradually increased in size. The hemoglobin subunit beta protein was identified from this spot using liquid chromatography-tandem mass spectrometry and verified using western blotting. Sample bloodstains were exposed to various environmental conditions (humidity: 30%, 60%, 90% at room temperature [RT]). Furthermore, the hemoglobin subunit protein extracted from the sample bloodstains at various time points (0 h to 30 d) was dissolved in our newly developed buffer solution and in deionized or distilled water. We also analyzed the expression levels of the protein in the sample bloodstains, dried at RT and under various humidity over time, using western blotting. In addition, we evaluated the protein extraction capacity of deionized or distilled water and the newly developed buffer from the sample bloodstains over time. At RT and 60% humidity, using the newly developed buffer, the hemoglobin subunit beta protein levels showed a gradually increasing pattern. Finally, we quantitated human hemoglobin subunit beta protein using western blotting and enzyme-linked immunosorbent assay, which revealed significant differences among the samples. In particular, the time points from 36 h to 30 days were considered for analysis. Thus, the hemoglobin subunit beta protein dried at RT and 60% humidity and further dissolved in the newly developed buffer solution can be used to determine the age of bloodstains at crime scenes.

Hematological and molecular analyses of the HbS allele among the Sudanese population.

OBJECTIVE: The purpose of this study was to perform hematological and molecular analyses of the HbS allele of the hemoglobin subunit beta gene in the Sudanese population. METHODS: This was a descriptive cross-sectional study. Hematological parameters and fetal hemoglobin (HbF) levels were assessed in all participants. Data were gathered through the use of questionnaires and laboratory investigations. The ßS-globin haplotypes, S allele distributions, and hematological parameters with HbF levels were investigated using PCR-restriction fragment length polymorphism, gel electrophoresis, and a Sysmex hematology analyzer, respectively. RESULTS: According to our findings, the Bantu (BA) haplotype was found in 10.8% of participants with homozygous uncontested haplotypes, followed by Benin (BA) and Sudan (SU), each in 9.8% of participants. This Sudanese group from Northern Kordofan lacked the Arab-Indian haplotype. Two heterozygous versions of undisputed haplotypes were found in 17.3% of participants: SU/BA in 10.8% and CA/BE in 6.5%. CONCLUSION: As a result of sickle cell anemia, this investigation found changes in hematological parameters. In the Sudanese population, a new haplotype of the S gene was discovered.

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

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

Phosphoproteomic analysis of the striatum from pleiotrophin knockout and midkine knockout mice treated with cocaine reveals regulation of oxidative stress-related proteins potentially underlying cocaine-induced neurotoxicity and neurodegeneration.

The neurotrophic factors pleiotrophin (PTN) and midkine (MK) are highly upregulated in different brain areas relevant to drug addiction after administrations of different drugs of abuse, including psychostimulants. We have previously demonstrated that PTN and MK modulate amphetamine-induced neurotoxicity and that PTN prevents cocaine-induced cytotoxicity in NG108-15 and PC12 cells. In an effort to dissect the different mechanisms of action triggered by PTN and MK to exert their protective roles against psychostimulant neurotoxicity, we have now used a proteomic approach to study protein phosphorylation, in which we combined phosphoprotein enrichment, by immobilized metal affinity chromatography (IMAC), with two-dimensional gel electrophoresis and mass spectrometry, in order to identify the phosphoproteins regulated in the striatum of PTN knockout, MK knockout and wild type mice treated with a single dose of cocaine (15mg/kg, i.p.). We identified 7 differentially expressed phosphoproteins: 5'(3')-deoxyribonucleotidase, endoplasmic reticulum resident protein 60 (ERP60), peroxiredoxin-6 (PRDX6), glutamate dehydrogenase 1 (GLUD1), aconitase and two subunits of hemoglobin. Most of these proteins are related to neurodegeneration processes and oxidative stress and their variations specially affect the PTN knockout mice, suggesting a protective role of endogenous PTN against cocaine-induced neural alterations. Further studies are needed to validate these proteins as possible targets against neural alterations induced by cocaine.