scholarly journals Human iPSC-Based Model Reveals NOX4 as Therapeutic Target in Duchenne Cardiomyopathy

2021 ◽  
Author(s):  
Robin Duelen ◽  
Domiziana Costamagna ◽  
Guillaume Gilbert ◽  
Liesbeth De Waele ◽  
Nathalie Goemans ◽  
...  
Keyword(s):  
Stress Responses ◽  
Early Death ◽  
Dystrophin Gene ◽  
Patient Specific ◽  
Ros Production ◽  
Atp Production ◽  
Protein Levels ◽  
Nadph Oxidase 4 ◽  
Cellular Stress Responses ◽  
Induced Pluripotent

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder, caused by mutations in the Dystrophin gene. Cardiomyopathy is one of the major causes of early death. In this study, we used DMD patient-specific induced pluripotent stem cells (iPSCs) to model cardiomyopathic features in DMD and unravel novel pathological mechanistic insights. Cardiomyocytes (CMs) differentiated from DMD iPSCs showed enhanced premature cell death, due to significantly elevated intracellular reactive oxygen species (ROS) concentrations, as a result of depolarized mitochondria and high NADPH oxidase 4 (NOX4) protein levels. Genetic correction of Dystrophin through CRISPR/Cas9 editing restored normal ROS levels. Application of ROS reduction by N-acetyl-L-cysteine (NAC), partial Dystrophin re-expression by ataluren (PTC124) and enhancing mitochondrial electron transport chain function by idebenone improved cell survival of DMD iPSC-CMs. We show applications that could counteract the detrimental oxidative stress environment in DMD iPSC-CMs by stimulating adenosine triphosphate (ATP) production. ATP could bind to the ATP-binding domain in the NOX4 enzyme, and we demonstrate that ATP resulted in partial inhibition of the NADPH-dependent ROS production of NOX4. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose to target ROS production and prevent the detrimental effects of NOX4 on DMD CMs as a promising therapeutic strategy.

scholarly journals Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells

2020 ◽  
Vol 21 (20) ◽  
pp. 7667
Author(s):  
Alexandra V. Jürs ◽  
Christin Völkner ◽  
Maik Liedtke ◽  
Katharina Huth ◽  
Jan Lukas ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Antioxidative Defense ◽  
Defense System ◽  
Protein Levels ◽  
Antioxidative Defense System ◽  
Induced Pluripotent

Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer’s disease and Parkinson’s disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

scholarly journals An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1262
Author(s):  
Nannan Duan ◽  
Shuqing Tang ◽  
Baitao Zeng ◽  
Zhiqing Hu ◽  
Qian Hu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Editing ◽  
High Efficiency ◽  
Expression Profiles ◽  
Rflp Analysis ◽  
Dystrophin Gene ◽  
Biomedical Science ◽  
Patient Specific ◽  
Editing Efficiency ◽  
Induced Pluripotent

(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.

scholarly journals Cellular Stress Responses of the Endemic Freshwater Fish Species Alburnus vistonicus Freyhof & Kottelat, 2007 in a Constantly Changing Environment

2021 ◽  
Vol 11 (22) ◽  
pp. 11021
Author(s):  
Emmanouil Tsakoumis ◽  
Thomais Tsoulia ◽  
Konstantinos Feidantsis ◽  
Foivos Alexandros Mouchlianitis ◽  
Panagiotis Berillis ◽  
...  
Keyword(s):  
Fish Species ◽  
Stress Responses ◽  
Cellular Stress ◽  
Chemical Parameters ◽  
Northern Greece ◽  
Protein Levels ◽  
Physico Chemical ◽  
Mitogen Activated Protein ◽  
Salinity Increase ◽  
Cellular Stress Responses

Herein we investigated the cellular responses of the endemic fish species Alburnus vistonicus Freyhof & Kottelat, 2007, under the variation of several physico-chemical parameters including temperature (°C), salinity (psu), dissolved oxygen (mg/L), pH and conductivity (μS/cm), which were measured in situ. Monthly fish samplings (October 2014–September 2015) were conducted in Vistonis Lake in northern Greece, a peculiar ecosystem with brackish waters in its southern part and high salinity fluctuations in its northern part. Fish gills and liver responses to the changes of the physico-chemical parameters were tested biochemically and histologically. Heat shock protein levels appeared to be correlated with salinity fluctuations, indicating the adaptation of A. vistonicus to the particular environment. The latter is also enhanced by increased Na+-K+ ATPase levels, in response to salinity increase during summer. The highest mitogen activated protein kinases phosphorylation levels were observed along with the maximum mean salinity values. A variety of histological lesions were also detected in the majority of the gill samples, without however securing salinity as the sole stress factor. A. vistonicus cellular stress responses are versatile and shifting according to the examined tissue, biomarker and season, in order for this species to adapt to its shifting habitat.

scholarly journals Cellular stress responses of long-lived and cancer-resistant naked mole-rats

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Perinur Bozaykut
Keyword(s):  
Stress Responses ◽  
Pluripotent Stem Cells ◽  
Target Gene ◽  
Cellular Mechanisms ◽  
Cellular Defense ◽  
Cellular Stress Responses ◽  
Mouse Cells ◽  
Cellular Stresses ◽  
Induced Pluripotent ◽  
Transcription Factor Nuclear Factor

Abstract Objectives Some organisms are long-lived and naturally resistant to cancer such as naked mole-rats (NMRs). Studies have shown that these animals can better tolerate stress due to mechanisms, such as upregulation of antioxidant pathways and improved proteostasis. In the present study, we aimed to analyze the tolerance against stress and the cellular mechanisms related to the stress response in NMR cells comparative to mouse cells. Materials and methods NMR and mouse fibroblasts were exposed to cellular stresses including H2O2 and/or NaNO3 and the viability of the cells were analyzed. In addition messanger RNA (mRNA) expression of antioxidant transcription factor Nuclear factor erythroid-derived 2-like 2 (Nrf2) and its target gene NAD(P)H quinone dehydrogenase 1 (NQO1) were determined by qRT-PCR and comprehensive analysis of stress-related gene expression was performed by RNA-Sequencing in fibroblasts and induced pluripotent stem cells (iPSC). Results Surprisingly, NMR fibroblasts were found to be more sensitive than mouse cells to H2O2 and NaNO3. Furthermore, it was shown that fibroblasts and iPSCs mainly aren’t identical in the expression pattern of cellular defense signaling and several factors are mainly downregulated in NMR iPSCs. Conclusions Collectively, the data gained from the present study help to improve the understanding of evolved mechanisms that contribute to stress resistance, aging and cancer.

Uncovering the Diversification of Tissue Engineering on the Emergent Areas of Stem Cells, Nanotechnology and Biomaterials

2020 ◽  
Vol 15 (3) ◽  
pp. 187-201 ◽  
Author(s):  
Sunil K. Dubey ◽  
Amit Alexander ◽  
Munnangi Sivaram ◽  
Mukta Agrawal ◽  
Gautam Singhvi ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Immune System ◽  
Clinical Application ◽  
Cell Types ◽  
Patient Specific ◽  
Potential Strategy ◽  
Induced Pluripotent ◽  
Treat All ◽  
The Impact

Damaged or disabled tissue is life-threatening due to the lack of proper treatment. Many conventional transplantation methods like autograft, iso-graft and allograft are in existence for ages, but they are not sufficient to treat all types of tissue or organ damages. Stem cells, with their unique capabilities like self-renewal and differentiate into various cell types, can be a potential strategy for tissue regeneration. However, the challenges like reproducibility, uncontrolled propagation and differentiation, isolation of specific kinds of cell and tumorigenic nature made these stem cells away from clinical application. Today, various types of stem cells like embryonic, fetal or gestational tissue, mesenchymal and induced-pluripotent stem cells are under investigation for their clinical application. Tissue engineering helps in configuring the stem cells to develop into a desired viable tissue, to use them clinically as a substitute for the conventional method. The use of stem cell-derived Extracellular Vesicles (EVs) is being studied to replace the stem cells, which decreases the immunological complications associated with the direct administration of stem cells. Tissue engineering also investigates various biomaterials to use clinically, either to replace the bones or as a scaffold to support the growth of stemcells/ tissue. Depending upon the need, there are various biomaterials like bio-ceramics, natural and synthetic biodegradable polymers to support replacement or regeneration of tissue. Like the other fields of science, tissue engineering is also incorporating the nanotechnology to develop nano-scaffolds to provide and support the growth of stem cells with an environment mimicking the Extracellular matrix (ECM) of the desired tissue. Tissue engineering is also used in the modulation of the immune system by using patient-specific Mesenchymal Stem Cells (MSCs) and by modifying the physical features of scaffolds that may provoke the immune system. This review describes the use of various stem cells, biomaterials and the impact of nanotechnology in regenerative medicine.

scholarly journals Cinnamon Oil Inhibits Penicillium expansum Growth by Disturbing the Carbohydrate Metabolic Process

2021 ◽  
Vol 7 (2) ◽  
pp. 123
Author(s):  
Tongfei Lai ◽  
Yangying Sun ◽  
Yaoyao Liu ◽  
Ran Li ◽  
Yuanzhi Chen ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Metabolic Process ◽  
Penicillium Expansum ◽  
Economic Losses ◽  
Pome Fruit ◽  
Cinnamon Oil ◽  
Atp Production ◽  
Protein Levels ◽  
Regulatory Enzymes ◽  
Expression Of Genes

Penicillium expansum is a major postharvest pathogen that mainly threatens the global pome fruit industry and causes great economic losses annually. In the present study, the antifungal effects and potential mechanism of cinnamon oil against P. expansum were investigated. Results indicated that 0.25 mg L−1 cinnamon oil could efficiently inhibit the spore germination, conidial production, mycelial accumulation, and expansion of P. expansum. In addition, it could effectively control blue mold rots induced by P. expansum in apples. Cinnamon oil could also reduce the expression of genes involved in patulin biosynthesis. Through a proteomic quantitative analysis, a total of 146 differentially expressed proteins (DEPs) involved in the carbohydrate metabolic process, most of which were down-regulated, were noticed for their large number and functional significance. Meanwhile, the expressions of 14 candidate genes corresponding to DEPs and the activities of six key regulatory enzymes (involving in cellulose hydrolyzation, Krebs circle, glycolysis, and pentose phosphate pathway) showed a similar trend in protein levels. In addition, extracellular carbohydrate consumption, intracellular carbohydrate accumulation, and ATP production of P. expansum under cinnamon oil stress were significantly decreased. Basing on the correlated and mutually authenticated results, we speculated that disturbing the fungal carbohydrate metabolic process would be partly responsible for the inhibitory effects of cinnamon oil on P. expansum growth. The findings would provide new insights into the antimicrobial mode of cinnamon oil.

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