Association of the Degree of Erythroid Expansion and Maturation Arrest with the Clinical Severity of β0-Thalassemia/Hemoglobin E Patients

2021 ◽  
pp. 1-12
Author(s):  
Thunwarat Suriyun ◽  
Woratree Kaewsakulthong ◽  
Pinyaphat Khamphikham ◽  
Sukanya Chumchuen ◽  
Suradej Hongeng ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Underlying Disease ◽  
Clinical Severity ◽  
Disease Heterogeneity ◽  
Hemoglobin E ◽  
Maturation Arrest ◽  
Modifying Factors ◽  
Erythroid Maturation

<b><i>Introduction:</i></b> β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. <b><i>Methods:</i></b> To elucidate the correlation between ineffective erythropoiesis and β<sup>0</sup>-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. <b><i>Results:</i></b> The β<sup>0</sup>-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β<sup>0</sup>-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. <b><i>Discussion/Conclusion:</i></b> The degree of erythroid expansion and maturation arrest contributes to the severity of β<sup>0</sup>-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.

scholarly journals NMDA and AMPA Receptor Autoantibodies in Brain Disorders: From Molecular Mechanisms to Clinical Features

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Fabrizio Gardoni ◽  
Jennifer Stanic ◽  
Diego Scheggia ◽  
Alberto Benussi ◽  
Barbara Borroni ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Ampa Receptor ◽  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
In Vivo Studies ◽  
Ionotropic Glutamate Receptors ◽  
Brain Disorders ◽  
Functional Impairments ◽  
Different Types

The role of autoimmunity in central nervous system (CNS) disorders is rapidly expanding. In the last twenty years, different types of autoantibodies targeting subunits of ionotropic glutamate receptors have been found in a variety of patients affected by brain disorders. Several of these antibodies are directed against NMDA receptors (NMDAR), mostly in autoimmune encephalitis, whereas a growing field of research has identified antibodies against AMPA receptor (AMPAR) subunits in patients with different types of epilepsy or frontotemporal dementia. Several in vitro and in vivo studies performed in the last decade have dramatically improved our understanding of the molecular and functional effects induced by both NMDAR and AMPAR autoantibodies at the excitatory glutamatergic synapse and, consequently, their possible role in the onset of clinical symptoms. In particular, the method by which autoantibodies can modulate the localization at synapses of specific target subunits leading to functional impairments and behavioral alterations has been well addressed in animal studies. Overall, these preclinical studies have opened new avenues for the development of novel pharmacological treatments specifically targeting the synaptic activation of ionotropic glutamate receptors.

scholarly journals Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

2020 ◽  
Author(s):  
James A. Gregory ◽  
Emily Hoelzli ◽  
Rawan Abdelaal ◽  
Catherine Braine ◽  
Miguel Cuevas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Mouse Tissue ◽  
Cell Type ◽  
Primary Mouse ◽  
Cell Type Specific

AbstractGenetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease, but also challenge our ability to resolve cell-type specific perturbations. Here we report an extension of the RiboTag system, first developed to achieve cell-type restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables efficient depletion of off-target RNA in mixed species primary co-cultures and in hiPSC-derived neural progenitor cells, motor neurons, and GABAergic neurons. Nonetheless, depletion efficiency varies across independent experimental replicates. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

Expression Of Mutant Spliceosomal Protein SF3B1 Results In Dysregulated Hematopoietic Maturation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2773-2773
Author(s):  
Alexander C. Minella ◽  
Oscar Ramirez ◽  
Yanfei Xu ◽  
Tushar Murthy ◽  
Xiaodong Yang ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Peptide Sequence ◽  
Causative Role ◽  
Wild Type ◽  
Erythroid Maturation

Abstract Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cell Type-Specific In Vitro Gene Expression Profiling of Stem Cell-Derived Neural Models

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1406
Author(s):  
James A. Gregory ◽  
Emily Hoelzli ◽  
Rawan Abdelaal ◽  
Catherine Braine ◽  
Miguel Cuevas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Mouse Tissue ◽  
Cell Type ◽  
Primary Mouse ◽  
Cell Type Specific

Genetic and genomic studies of brain disease increasingly demonstrate disease-associated interactions between the cell types of the brain. Increasingly complex and more physiologically relevant human-induced pluripotent stem cell (hiPSC)-based models better explore the molecular mechanisms underlying disease but also challenge our ability to resolve cell type-specific perturbations. Here, we report an extension of the RiboTag system, first developed to achieve cell type-restricted expression of epitope-tagged ribosomal protein (RPL22) in mouse tissue, to a variety of in vitro applications, including immortalized cell lines, primary mouse astrocytes, and hiPSC-derived neurons. RiboTag expression enables depletion of up to 87 percent of off-target RNA in mixed species co-cultures. Nonetheless, depletion efficiency varies across independent experimental replicates, particularly for hiPSC-derived motor neurons. The challenges and potential of implementing RiboTags in complex in vitro cultures are discussed.

scholarly journals Cellular and Molecular Mechanisms Mediating Methylmercury Neurotoxicity and Neuroinflammation

2021 ◽  
Vol 22 (6) ◽  
pp. 3101
Author(s):  
João P. Novo ◽  
Beatriz Martins ◽  
Ramon S. Raposo ◽  
Frederico C. Pereira ◽  
Reinaldo B. Oriá ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Environmental Concern ◽  
In Vivo Studies ◽  
Molecular Alterations ◽  
Starting Point ◽  
The Central Nervous System ◽  
Cell Death Mechanisms

Methylmercury (MeHg) toxicity is a major environmental concern. In the aquatic reservoir, MeHg bioaccumulates along the food chain until it is consumed by riverine populations. There has been much interest in the neurotoxicity of MeHg due to recent environmental disasters. Studies have also addressed the implications of long-term MeHg exposure for humans. The central nervous system is particularly susceptible to the deleterious effects of MeHg, as evidenced by clinical symptoms and histopathological changes in poisoned humans. In vitro and in vivo studies have been crucial in deciphering the molecular mechanisms underlying MeHg-induced neurotoxicity. A collection of cellular and molecular alterations including cytokine release, oxidative stress, mitochondrial dysfunction, Ca2+ and glutamate dyshomeostasis, and cell death mechanisms are important consequences of brain cells exposure to MeHg. The purpose of this review is to organize an overview of the mercury cycle and MeHg poisoning events and to summarize data from cellular, animal, and human studies focusing on MeHg effects in neurons and glial cells. This review proposes an up-to-date compendium that will serve as a starting point for further studies and a consultation reference of published studies.

scholarly journals Applicability of a clinical scoring criteria for disease severity of ß-thalassemia/hemoglobin E in Indonesia

2018 ◽  
Vol 27 (1) ◽  
pp. 26-32 ◽  
Author(s):  
Pustika A. Wahidiyat ◽  
Sudigdo Sastroasmoro ◽  
Suthat Fucharoen ◽  
Iswari Setianingsih ◽  
Siti A. Putriasih
Keyword(s):  
Classification System ◽  
Clinical Symptoms ◽  
Descriptive Study ◽  
Clinical Severity ◽  
Thalassemia Patient ◽  
Hemoglobin E ◽  
Stunted Growth ◽  
Indonesian Population ◽  
Severe Group ◽  
Clinical Scoring

Background: β-thalassemia/HbE presents with a variety of clinical symptoms, from asymptomatic to severe, requiring routine transfusion. However, there is currently no agreed classification system to stratify patients based on clinical severity of β-thalassemia/HbE in the Indonesian population. Thailand has already established a classification system, and this study aimed to identify the applicability of the Thailand clinical scoring criteria to determine the severity of β-thalassemia/HbE in the Indonesian population.Methods: This descriptive study was conducted by evaluating patients with β-thalassemia/HbE, who were classified into mild, moderate, or severe groups based on the Thailand clinical scoring criteria.Results: A total of 293 subjects with β-thalassemia/HbE were included. Based on this clinical scoring criteria, it was found that only 21.5% of patients were classified as mild, and the remaining 35.5% and 44% were classified as moderate and severe respectively. Approximately 68.2% of the subjects in the severe group received transfusion at <4 years old, while only 10% of those in the mild group were transfused at the same age. In the mild group, only 10% of the subjects underwent routine transfusion, compared to 98.4% of the subjects in the severe group. In addition, only 27% of the subjects in the mild group showed stunted growth, while that in the moderate and severe groups were 54.5% and 86.8%, respectively.Conclusion: Thailand clinical scoring criteria is able to determine the severity of Indonesia thalassemia patient which needs further management, i.e. transfusion and observation of stunted growth. This scoring system will help provide the provision of the most ideal management for the groups of patients based on their requirements.

scholarly journals Peroxisome proliferator-activated receptors in reproductive tissues: from gametogenesis to parturition

2006 ◽  
Vol 189 (2) ◽  
pp. 199-209 ◽  
Author(s):  
P Froment ◽  
F Gizard ◽  
D Defever ◽  
B Staels ◽  
J Dupont ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Polycystic Ovary ◽  
Mammalian Species ◽  
Peroxisome Proliferator ◽  
Toxic Substances ◽  
Placental Development ◽  
Insulin Sensitizer ◽  
Peroxisome Proliferator Activated Receptors

Peroxisome proliferator-activated receptors (PPARα, PPARβ/δ and PPARγ) are a family of nuclear receptors that are activated by binding of natural ligands, such as polyunsaturated fatty acids or by synthetic ligands. Synthetic molecules of the glitazone family, which bind to PPARγ, are currently used to treat type II diabetes and also to attenuate the secondary clinical symptoms frequently associated with insulin resistance, including polycystic ovary syndrome (PCOS). PPARs are expressed in different compartments of the reproductive system (hypothalamus, pituitary, ovary, uterus and testis). Conservative functions of PPARs in mammalian species could be suggested through several in vivo and in vitro studies, especially in the ovary and during placental development. Several groups have described a strong expression of PPARγ in ovarian granulosa cells, and glitazones modulate granulosa cell proliferation and steroidogenesis in vitro. All these recent data raise new questions about the biologic actions of PPARs in reproduction and their use in therapeutic treatments of fertility troubles such as PCOS or endometriosis. In this review, we first describe the roles of PPARs in different compartments of the reproductive axis (from male and female gametogenesis to parturition), with a focus on PPARγ. Secondly, we discuss the possible molecular mechanisms underlying the effect of glitazones on PCOS. Like other ‘insulin sensitizer’ molecules, such as metformin, glitazones may in fact act directly on ovarian cells. Finally, we discuss the eventual actions of PPARs as mediators of environmental toxic substances for reproductive function.

scholarly journals NSP2 Is Important for Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus to Trigger High Fever-Related COX-2-PGE2 Pathway in Pigs

2021 ◽  
Vol 12 ◽  
Author(s):  
Li Du ◽  
Honglei Wang ◽  
Fang Liu ◽  
Zeyu Wei ◽  
Changjiang Weng ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Nonstructural Protein ◽  
High Fever ◽  
Pge2 Production ◽  
Respiratory Syndrome Virus ◽  
Highly Pathogenic ◽  
Cox 2

In 2006, atypical porcine reproductive and respiratory syndrome (PRRS) caused by a highly pathogenic PRRSV (HP-PRRSV) strain broke out in China. Atypical PRRS is characterized by extremely high fever and high mortality in pigs of all ages. Prostaglandin E2 (PGE2) derived from arachidonic acid through the activation of the rate-limiting enzyme cyclooxygenase type 1/2 (COX-1/2) plays an important role in fever. Here, we showed that HP-PRRSV infection increased PGE2 production in microglia via COX-2 up-regulation depending on the activation of MEK1-ERK1/2-C/EBPβ signaling pathways. Then, we screened HP-PRRSV proteins and demonstrated that HP-PRRSV nonstructural protein 2 (NSP2) activated MEK1-ERK1/2-C/EBPβ signaling pathways by interacting with 14-3-3ζ to promote COX-2 expression, leading to PGE2 production. Furthermore, we identified that the amino acid residues 500-596 and 658-777 in HP-PRRSV NSP2 were essential to up-regulate COX-2 expression and PGE2 production. Finally, we made mutant HP-PRRS viruses with the deletion of residues 500-596 and/or 658-777, and found out that these viruses had impaired ability to up-regulate COX-2 and PGE2 production in vitro and in vivo. Importantly, pigs infected with the mutant viruses had relieved fever, clinical symptoms, and mortality. These data might help us understand the molecular mechanisms underlying the high fever and provide clues for the development of HP-PRRSV attenuated vaccines.

scholarly journals Molecular mechanisms and clinical significance of fibrinolysis

2013 ◽  
Vol 94 (5) ◽  
pp. 711-718 ◽  
Author(s):  
R I Litvinov
Keyword(s):  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Serum Levels ◽  
Fibrinolytic System ◽  
Laboratory Diagnostics ◽  
Fibrinolytic Enzymes ◽  
Pathological Conditions

Fibrinolysis is the process of proteolytic digestion of fibrin aimed in vivo at dissolving clots or thrombi to restore the blood flow. In blood, the fibrinolytic system comprises a network of interrelated biochemical reactions that occur predominantly on the surface of fibrin fibers, the structural scaffold of hemostatic clots and obstructive thrombi. This review provides a brief description of the fibrinolytic system components and of the most important fibrinolytic reactions, including their modulation. The central enzyme in fibrinolysis is plasmin, a serine protease formed from its inactive precursor, plasminogen, upon the action of proteins and enzymes, whose formation and/or activation is triggered by various pathological stimuli. Plasmin cleaves a variety of substrates other than fibrin and therefore is involved in a number of biological processes other than hemostasis and thrombosis. There are several mechanisms moderating the activity of fibrinolytic enzymes that may become altered in various diseases. When the ratio of blood pro-and antifibrinolytic compounds is altered, hyper-or hypofibrinolysis might develop that causes and/or exacerbates hemorrhage or thrombosis, respectively. The paper contains an original pathogenic classification of fibrinolytic disorders and describes mechanisms of the primary and secondary hypo-and hyperfibrinolysis in various pathological conditions. Diagnosis of fibrinolytic defects builds on clinical symptoms along with defining the time of serum clots dissolving in vitro and serum levels of molecular markers. The principles of laboratory diagnostics of pathological fibrinolysis, based on the comprehension of the molecular mechanisms of normal and impaired fibrinolytic reactions, are reviewed. Treatment of pathological fibrinolysis implies the correction of either hyper-or hypofibrinolytic conditions and, therefore, is based on the administration of either antifibrinolytics (ε-eminocaproic and tranexamic acids) or thrombolytics (mainly, plasminogen activators) in combination with other therapies against bleeding and thrombosis.

scholarly journals Vitamin B12 Deficiency Induces Imbalance in Melanocytes Homeostasis—A Cellular Basis of Hypocobalaminemia Pigmentary Manifestations

2018 ◽  
Vol 19 (9) ◽  
pp. 2845 ◽  
Author(s):  
Zuzanna Rzepka ◽  
Michalina Respondek ◽  
Jakub Rok ◽  
Artur Beberok ◽  
Keith ó Proinsias ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Molecular Mechanisms ◽  
Clinical Symptoms ◽  
Scientific Evidence ◽  
Neuropsychiatric Symptoms ◽  
Skin Pigmentation ◽  
Vitamin B12 Deficiency ◽  
Image Cytometry ◽  
B12 Deficiency

Vitamin B12 deficiency causes significant changes in cellular metabolism leading to various clinical symptoms, such as hematological, psychiatric, and neurological disorders. We hypothesize that skin pigmentation disorders may be a diagnostically important manifestation of vitamin B12 deficiency, however the cellular and molecular mechanisms underlying these effects remain unknown. The aim of this study was to examine the effect of vitamin B12 deficiency on melanocytes homeostasis. Hypocobalaminemia in vitro model was developed by treating epidermal melanocytes with synthesized vitamin B12 antagonist—hydroxycobalamin(c-lactam). The cells were examined using immunoenzymatic, spectrophotometric, and fluorimetric assays as well as image cytometry. Significant melanogenesis stimulation—the increase of relative melanin content and tyrosinase activity up to 131% and 135%, respectively—has been indicated. Cobalamin-deficient cells displayed the elevation (by 120%) in reactive oxygen species level. Moreover, the redox status imbalance was stated. The study provided a scientific evidence for melanocytes homeostasis disturbance under hypocobalaminemia, thus indicating a significant element of the hyperpigmentation mechanism due to vitamin B12 deficiency. Furthermore, the implication between pigmentary and hematological and/or neuropsychiatric symptoms in cobalamin-deficient patients may be an important issue.

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