Identification of key overlapping DEGs and molecular pathways under multiple stressors in the liver of Nile tilapia (Oreochromis niloticus).

The identification of key genes and molecular pathways that are involved in the response to stressors is crucial for controlling stress in fish and sustainable aquaculture. Environmental stressors can induce stress responses in aquatic animals, resulting in compromised immune function, inhibited growth, and increased mortality rates. mRNA-seq analysis provides a powerful tool to identify key genes and pathways associated with stress response. In the present study, mRNA-seq analysis was employed to identify key overlapping differentially expressed genes (DEGs) and molecular pathways under salinity, nitrite, copper, and pH stress in the liver of Nile tilapia (Oreochromis niloticus). The pathways associated with the immune response, oxygen transport, homeostasis, and oxidative stress were enriched across all stressors. The top KEGG pathways were complement and coagulation cascades, PPAR signaling pathway, and cardiac muscle contraction. The top GO enrichment terms were oxidoreductase activity, aerobic respiration, endopeptidase inhibitor activity, endopeptidase regulator activity, heme binding, and iron ion binding. The complement genes (C3, C4, C5, factor B, and factor H), alpha-2-macroglobulin (A2M), hemoglobin subunit epsilon (HBE), hemoglobin subunit alpha (HBA), coagulation factor genes (XI and X) and the cytochrome c oxidase (COX) gene family (cox1, cox2, cox3, cytochrome P450) were identified as key shared genes across multiple stressors. The discovery of these genes and molecular pathways provided a better understanding of the molecular mechanism underlying the stress response in Nile tilapia. The results of the present study can facilitate the development of stress management strategies in Nile tilapia.

Spectrum Of HBB Gene Variants And Major Endocrine Complications In Thalassemia Patients Of Pakistan.

OBJECTIVE: To determine the molecular characterisation of beta-thalassemia major patients, pattern of major endocrine complications and its association with haemoglobin subunit beta gene variants. Method: The cross-sectional study was conducted from November 2021 to November 2022 after approval from the ethics review committee of Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan, and comprised of 88 patients with beta thalassemia major aged >8 years and having serum ferritin level >1000 µg/L. The subjects were analysed for haemoglobin subunit beta gene variants and major endocrine complications, like growth retardation, hypogonadism, hypothyroidism, hypoparathyroidism and diabetic abnormalities using an automatic chemistry analyser, fully automatic chemiluminescence immunoassay analyser, enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Data was analysed using SPSS 25. RESULTS: Of the 88 subjects, 40(45.4%) were girls and 48(54.5%) were boys. The overall mean age was 12±2.81 years. Of the total, 55(62.5%) had growth retardation, 41(46.6%) were cases of hypogonadism, 16(18.1%) hypothyroidism, 5(5.7%) hypoparathyroidism, 3(3.4%) diabetes mellitus and 8 (9.1%) had impaired glucose tolerance. Also, 65 (73.9%) patients confronted at least one endocrine complication. Endocrine complications were strongly associated with serum ferritin levels (p=0.000). The most common haemoglobin subunit beta gene variant identified was IVSI-5 (G>C) in 36 (40.9%), and the least identified variant was cluster of differenctiation-CD26(G>A) 1(1.1%). The association between haemoglobin subunit beta gene variants with endocrine complications was statistically non-significant (p>0.05). CONCLUSIONS: IVSI-5 (G>C) was found to be the most frequent haemoglobin subunit beta gene variant among beta- thalassemia major patients.

Characterization and Expression Analysis of Genes from Megalobrama amblycephala Encoding Hemoglobins with Extracellular Microbicidal Activity.

Hemoglobin (Hb) usually comprises two α and two ß subunits, forming a tetramer responsible for oxygen transportation and storage. Few studies have elucidated fish hemoglobin immune functions. Megalobrama amblycephala is a freshwater-cultured fish prevalent in China. We identified two M. amblycephala hemoglobin subunits and analyzed their expression patterns and antibacterial activities. The respective full-length cDNA sequences of the M. amblycephala Hb α (MaHbα) and ß (MaHbß) subunits were 588 and 603 bp, encoding 143 and 148 amino acids. MaHbα and MaHbß were highly homologous to hemoglobins from other fish, displaying typical globin-like domains, most heme-binding sites, and tetramer interface regions highly conserved in teleosts. In phylogenetic analyses, the hemoglobin genes from M. amblycephala and other cypriniformes clustered into one branch, and those from other fishes and mammals clustered into other branches, revealing fish hemoglobin conservation. These M. amblycephala Hb subunits exhibit different expression patterns in various tissues and during development. MaHbα is mainly expressed in the blood and brain, while MaHbß gene expression is highest in the muscle. MaHbα expression was detectable and abundant post-fertilization, with levels fluctuating during the developmental stages. MaHbß expression began at 3 dph and gradually increased. Expression of both M. amblycephala Hb subunits was down-regulated in most examined tissues and time points post-Aeromonas hydrophila infection, which might be due to red blood cell (RBC) and hematopoietic organ damage. Synthetic MaHbα and MaHbß peptides showed excellent antimicrobial activities, which could inhibit survival and growth in five aquatic pathogens. Two M. amblycephala hemoglobin subunits were identified, and their expression patterns and antibacterial activities were analyzed, thereby providing a basis for the understanding of evolution and functions of fish hemoglobins.

Formalin Fixation Followed by Paraffin Embedding Allows Long-Term Storage of Proteins for Liquid Chromatography-Tandem Mass Spectrometry Analysis.

In an anatomical pathology laboratory, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to characterize amyloid deposits identified in formalin-fixed paraffin-embedded tissue (FFPET). However, the development of additional tests is partially limited by the lack of information the passage of time has on the proteins in FFPET. To investigate the reliability of LC-MS/MS in the analysis of old FFPET specimens, 1 bone marrow aspirate clot was analyzed by LC-MS/MS yearly from 2014 to 2018, in 3 consecutive months. Peptide-spectrum match, number of peptides identified, and percentage of the proteins covered were the parameters collected for the hemoglobin subunits alpha (HbA), beta (HbB), delta (HbD), and gamma (HbG). These proteins are constant components of the peripheral blood and are present in high and low abundance, allowing the monitorization of the performance of the test across varying protein concentrations. The hemoglobin subunits were stable over the years studied; 71% to 74% of HbA, 77% to 80% of HbB, 69% to 77% of HbD, and 57% to 63% of HbG were covered, with no statistical difference between 2014 and 2018. The number of peptides identified was also constant, 11 to 13 for HbA, 13 to 15 for HbB, 11 to 14 for HbD, and 7 to 9 for HbG. Peptide spectrum match was only slightly more variable: 209 to 327 for HbA, 569 to 1052 for HbB, 286 to 533 HbD, and 142 to 292 for HbG. In conclusion, high abundance hemoglobins, HbA and HbB, and relatively low abundance ones, HbD and HbG, are preserved in FFPET and confidently identified by LC-MS/MS for at least 5 years.

LC-MS/MS-Based Absolute Quantitation of Hemoglobin Subunits from Dried Blood Spots Reveals Novel Biomarkers for α-Thalassemia Silent Carriers.

Identification of α-thalassemia silent carriers is challenging with conventional phenotype-based screening methods. A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based approach may offer novel biomarkers to address this conundrum. In this study, we collected dried blood spot samples from individuals with three α-thalassemia subtypes for biomarker discovery and validation. We observed differential expression patterns of hemoglobin subunits among various α-thalassemia subtypes and normal controls through proteomic profiling of 51 samples in the discovery phase. Then, we developed and optimized a multiple reaction monitoring (MRM) assay to measure all detectable hemoglobin subunits. The validation phase was conducted in a cohort of 462 samples. Among the measured hemoglobin subunits, subunit µ was significantly upregulated in all the α-thalassemia groups with distinct fold changes. The hemoglobin subunit µ exhibits great potential as a novel biomarker for α-thalassemia, especially for silent α-thalassemia. We constructed predictive models based on the concentrations of hemoglobin subunits and their ratios to classify the various subtypes of α-thalassemia. In the binary classification problems of silent α-thalassemia vs normal, non-deletional α-thalassemia vs normal, and deletional α-thalassemia vs normal, the best performance of the models achieved average ROCAUCs of 0.9505, 0.9430, and 0.9976 in the cross-validation, respectively. In the multiclass model, the best performance achieved an average ROCAUC of 0.9290 in cross-validation. The performance of our MRM assay and models demonstrated that the hemoglobin subunit µ would play a vital role in screening silent α-thalassemia in clinical practice.

Evolutionary honing in and mutational replacement: how long-term directed mutational responses to specific environmental pressures are possible.

Recent results have shown that the human malaria-resistant hemoglobin S mutation originates de novo more frequently in the gene and in the population where it is of adaptive significance, namely, in the hemoglobin subunit beta gene compared to the nonresistant but otherwise identical 20A[Formula: see text]T mutation in the hemoglobin subunit delta gene, and in sub-Saharan Africans, who have been subject to intense malarial pressure for many generations, compared to northern Europeans, who have not. This finding raises a fundamental challenge to the traditional notion of accidental mutation. Here, we address this finding with the replacement hypothesis, according to which preexisting genetic interactions can lead directly and mechanistically to mutations that simplify and replace them. Thus, an evolutionary process under selection can gradually hone in on interactions of importance for the currently evolving adaptations, from which large-effect mutations follow that are relevant to these adaptations. We exemplify this hypothesis using multiple types of mutation, including gene fusion mutations, gene duplication mutations, A[Formula: see text]G mutations in RNA-edited sites and transcription-associated mutations, and place it in the broader context of a system-level view of mutation origination called interaction-based evolution. Potential consequences include that similarity of mutation pressures may contribute to parallel evolution in genetically related species, that the evolution of genome organization may be driven by mutational mechanisms, that transposable element movements may also be explained by replacement, and that long-term directed mutational responses to specific environmental pressures are possible. Such mutational phenomena need to be further tested by future studies in natural and artificial settings.

Saliva proteomic profile of early childhood caries and caries-free children.

OBJECTIVE: Saliva plays an important antimicrobial role and it is related to the pathogenesis of early childhood caries (ECC). The aim of this study was to compare the proteomic profile of unstimulated saliva of children aged 3-5 years who had ECC and caries-free (CF) children. MATERIALS AND METHODS: After the saliva collection from 20 children (ECC: n = 10; CF: n = 10), the samples were processed for proteomic analysis on a mass spectrometer. RESULTS: 1638 proteins were identified, of which 355 were present in both groups. A total of 579 proteins were exclusively identified in the CF group and included Leucine-rich alpha-2-glycoprotein, Protein S100-A5, Protein S100-A8 and Mucin-2. Moreover, 704 proteins were exclusively identified in the ECC group, including Enamelin. The differential expression analysis revealed that 112 proteins were up-regulated in the CF group. Among these proteins, we highlighted Hemoglobin subunit gamma-1 (343-fold increase), gamma-2 (336-fold increase) and alpha (40-fold increase). CONCLUSIONS: The proteomic profile of the saliva varied substantially between the groups. Hemoglobin subunit gamma-1, gamma-2 and alpha may play a protective role in children with ECC. These proteins should be evaluated in future studies, because they may be possible good candidates to be included in anti-caries dental products.

Experimental Bothropsatrox Envenomation: Blood Plasma Proteome Effects after Local Tissue Damage and Perspectives on Thromboinflammation.

The clinical manifestations of Bothrops atrox envenoming involve local and systemic changes, among which edema requires substantial attention due to its ability to progress to compartmental syndromes and sometimes cause tissue loss and amputations. However, the impact of edema on the poisoned body's system has not been explored. Thus, the present study aimed to explore the systemic pathological and inflammatory events that are altered by intraplantar injection of B. atrox venom in a mouse model through hematologic, lipidic, and shotgun proteomics analysis. Plasma samples collected showed a greater abundance of proteins related to complement, coagulation, lipid system, platelet and neutrophil degranulation, and pathways related to cell death and ischemic tolerance. Interestingly, some proteins, in particular, Prdx2 (peroxiredoxin 2), Hba (hemoglobin subunit alpha), and F9 (Factor IX), increased according to the amount of venom injected. Our findings support that B. atrox venom activates multiple blood systems that are involved in thromboinflammation, an observation that may have implications for the pathophysiological progression of envenomations. Furthermore, we report for the first time a potential role of Prdx2, Hba, and F9 as potential markers of the severity of edema/inflammation in mice caused by B. atrox.

G6PD and HBB polymorphisms in the Senegalese population: prevalence, correlation with clinical malaria.

Background: Host genetic factors contribute to the variability of malaria phenotypes and can allow a better understanding of mechanisms involved in susceptibility and/or resistance to Plasmodium falciparum infection outcomes. Several genetic polymorphisms were reported to be prevalent among populations living in tropical malaria-endemic regions and induce protection against malaria. The present study aims to investigate the prevalence of HBB (chr11) and G6PD (chrX) deficiencies polymorphisms among Senegalese populations and their associations with the risk for severe Plasmodium falciparum malaria occurrence. Methods: We performed a retrospective study with 437 samples, 323 patients recruited in hospitals located in three different endemic areas where malaria episodes were confirmed and 114 free malaria controls. The patients enrolled were classified into two groups: severe malaria (SM) (153 patients) and uncomplicated malaria (UM) (170 patients). PCR and DNA sequencing assessed host genetic polymorphisms in HBB and G6PD. Using a multivariate regression and additive model, estimates of the impact of human HBB and G6PD polymorphisms on malaria incidence were performed. Results: Six frequent SNPs with minor allele frequencies (MAF) > 3% were detected in the HBB gene (rs7946748, rs7480526, rs10768683, rs35209591, HbS (rs334) and rs713040) and two in the G6PD gene (rs762515 and rs1050828 (G6PD-202 G > A). Analysis of selected HbS polymorphism showed significant association with protective effect against severe malaria with a significant p-value = 0.033 (OR 0.38, 95% CI [0.16-0.91]) for SM vs. UM comparison. Surprisingly, our study did not identify the protective effect of variant HbC polymorphism against severe malaria. Finally, we found some of the polymorphisms, like HbS (rs334), are associated with age and biological parameters like eosinophils, basophils, lymphocytes etc. Conclusion: Our data report HBB and G6PD polymorphisms in the Senegalese population and their correlation with severe/mild malaria and outcome. The G6PD and HBB deficiencies are widespread in West Africa endemic malaria regions such as The Gambia, Mali, and Burkina Faso. The study shows the critical role of genetic factors in malaria outcomes. Indeed, genetic markers could be good tools for malaria endemicity prognosis.

Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits.

TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.

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.

lncRNA Neat1 regulates neuronal dysfunction post-sepsis via stabilization of hemoglobin subunit beta.

Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.

The possible role of hypoxia in the affected tissue of relapsed clubfoot.

Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2, FN1, TGFB1, HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.

A comprehensive and comparative proteomic analysis of horse serum proteins in colitis.

BACKGROUND: Equine colitis is a diarrhoeal disease caused by inflammation of the large bowel and can potentially be life-threatening due to its rapid progression. Pathogenesis is multifactorial and pathophysiology is highly complicated, therefore, reliable diagnostic biomarkers are needed in the veterinary field. OBJECTIVE: Serum is one of the most commonly used diagnostic tools in equine clinical investigation. To discover diagnostic or prognostic protein markers for colitis in horse serum, comprehensive and comparative proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS). STUDY DESIGN: Case-control study. METHODS: Serum samples were collected from 36 healthy Thoroughbreds and 12 Thoroughbreds with colitis. Serum from each horse suffering from colitis was collected daily until death or recovery. Collected sera were digested with trypsin. Peptides obtained from serum proteins were measured by Q-Exactive HF Orbitrap mass spectrometer. The identification and quantification of peptides were performed using Proteome Discoverer version 2.2. RESULTS: On day 1 of treatment, eight proteins in the colitis group were upregulated (P < .05, more than a twofold change) compared with the healthy group. Among the eight proteins, biliverdin reductase B was significantly upregulated (P < .05) in the non-survivor group (n = 5) compared with the survivor group (n = 7). On the last day of the treatment, haemoglobin subunit alpha, clusterin, glyceraldehyde-3-phosphate dehydrogenase, lipopolysaccharide-binding protein, and biliverdin reductase B showed significant increases (P < .05) in the non-survivor group. MAIN LIMITATIONS: The number of the identified proteins is limited due to the existence of abundant proteins. CONCLUSIONS: Measuring the changes of these proteins together may enable a potential prognosis or early diagnosis of horses suffering from colitis.

The Role of Hemoglobin Subunit Delta in the Immunopathy of Multiple Sclerosis: Mitochondria Matters.

Background: Although the exact pathophysiology of MS has not been identified, mitochondrial stress can be one of the culprits in MS development. Herein, we have applied microarray analysis, single-cell sequencing analysis, and ex vivo study to elucidate the role of mitochondrial stress in PBMCs of MS patients. Methods: For this purpose, we analyzed the GSE21942 and GSE138266 datasets to identify the DEGs and hub genes in the PBMCS of MS patients and describe the expression of shared genes in the different immune cells. The GO pathway analysis of DEGs and turquoise module genes were conducted to shed light on their biological significance. To validate the obtained results, the gene expression of HBD, as the most remarkable DEG in the PBMCS of affected patients, was measured in the PBMCS of healthy donors, treatment-naïve MS patients, and MS patients treated with GA, fingolimod, DMF, and IFNß-1α. Results: Based on WGCNA and DEGs analysis, HBD, HBM, SLC4A1, LILRA5, SLC25A37, SELENBP1, ALYREF, SNRNP40, and HINT3 are the identified common genes in the PMBCS. Using single-cell sequencing analysis on PBMCS, we have characterized various cell populations in MS and illustrated the common gene expression on the different immune cells. Furthermore, GO pathway analysis of DEGs, and turquoise module genes have indicated that these genes are involved in immune responses, myeloid cell activation, leukocyte activation, oxygen carrier activity, and replication fork processing bicarbonate transport pathways. Our ex vivo investigation has shown that HBD expression in the treatment-naïve RRMS patients is significantly increased compared to healthy donors. Of interest, immunomodulatory therapies with fingolimod, DMF, and IFNß-1α have significantly decreased HBD expression. Conclusion: HBD is one of the remarkably up-regulated genes in the PBMCS of MS patients. HBD is substantially up-regulated in treatment-naïve MS patients, and immunomodulatory therapies with fingolimod, DMF, and IFNß-1α can remarkably down-regulate HBD expression. Based on the currently available evidence, the cytoprotective nature of HBD against oxidative stress can be the underlying reason for HBD up-regulation in MS. Nevertheless, further investigations are needed to shed light on the molecular mechanisms of HBD in the oxidative stress of MS patients.

Drug Repurposing: Hydroxyurea Therapy Improves the Transfusion-Free Interval in HbE/Beta-Thalassemia-Major Patients with the XmnI Polymorphism.

Aims: HbE/ß-thalassemia is the most prevalent form of severe ß-thalassemia in Asian countries. Hydroxyurea (HU) is the most common drug used for the management of sickle-cell anemia but not thalassemia. In this study, we aimed to assess clinical HU response among the Bengali HbE/ß-thalassemia patients with respect to the XmnI γGglobin polymorphism and elucidate the association between this polymorphism and HU response efficacy. Materials and Methods: We enrolled 49 transfusion-dependent patients with HbE/ß-thalassemia. Fetal hemoglobin levels were measured using high-performance liquid chromatography and complete blood counts were determined pre- and post-HU therapy. Polymerase chain reaction-restriction fragment length polymorphism analyses were performed for genotyping the XmnI γGglobin polymorphism. Results: A total of 30 (61.22%) patients were found to be responders, whereas the remaining 19 (38.78%) were nonresponders. We found 33 patients with the heterozygous (C/T) and three with the homozygous mutant (T/T) genotype status. We obtained a statistically significant correlation (p < 0.001) between the XmnI polymorphism genotype and transfusion-free interval. Patients with the XmnI polymorphism were found to be good responders for HU therapy and showed increased hemoglobin levels. Conclusions: Our findings indicate that HU is a potential drug candidate for thalassemia management, particularly for HbE/ß-thalassemia. These results hold implications in repurposing HU as an effective and efficient therapy for HbE/ß-thalassemia.

Advances in mass spectrometric methods for detection of hemoglobin disorders.

Hemoglobin disorders are caused due to alterations in the hemoglobin molecules. These disorders are categorized in two broad classes - hemoglobin variants and thalassemias. The hemoglobin variants arise due to point mutations in the alpha (α), beta (ß), gamma (γ), delta (δ), or epsilon (ε) globin chains of these proteins, while thalassemias are caused due to the under-production of α or ß globin chain. Hemoglobin disorders account for 7 % of the major health issues globally. Mass Spectrometry is an extensively used analytical tool in the field of protein identification, protein-protein interaction, biomarker discovery and diagnosis of several impairments including hemoglobin related disorders. The remarkable advancements in the technology and method development have enormously augmented the clinical significance of mass spectrometry in these fields. The present review describes hemoglobin disorders and the recent advancements in mass spectrometry in the detection of such disorders, including its advantages, lacunae, and future directions. The literature evidence concludes that mass spectrometry can be potentially used as a 'First Line Screening Assay' for the detection of hemoglobin disorders in the near future.

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.