The Dysferlin Transcript Containing the Alternative Exon 40a is Essential for Myocyte Functions

Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is a transmembrane protein involved in several muscle functions like T-tubule maintenance and membrane repair. In 2009, a study showed the existence of fourteen dysferlin transcripts generated from alternative splicing. We were interested in dysferlin transcripts containing the exon 40a, and among them the transcript 11 which contains all the canonical exons and exon 40a. This alternative exon encodes a protein region that is cleaved by calpains during the muscle membrane repair mechanism. Firstly, we tested the impact of mutations in exon 40a on its cleavability by calpains. We showed that the peptide encoded by the exon 40a domain is resistant to mutations and that calpains cleaved dysferlin in the first part of DYSF exon 40a. To further explore the implication of this transcript in cell functions, we performed membrane repair, osmotic shock, and transferrin assay. Our results indicated that dysferlin transcript 11 is a key factor in the membrane repair process. Moreover, dysferlin transcript 11 participates in other cell functions such as membrane protection and vesicle trafficking. These results support the need to restore the dysferlin transcript containing the alternative exon 40a in patients affected with dysferlinopathy.

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

10.21203/rs.2.19199/v1 ◽

2019 ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background: Dysferlin is a type-II transmembrane protein and the causative gene of dysferlinopathies, which are characterized by absence or marked reduction in dysferlin protein and muscle weakness. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. The muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Release of thrombospondin-1 (TSP-1) by dysferlin deficient muscle has been reported as a possible factor of the inflammation observed in the muscle of both human and mouse models of dysferlinopathy. It has also been reported that treatment with vitamin D3 enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic implication in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient carrying two missense mutationsMethods: Dysferlin deficient myotubes were treated with EB1089, a vitamin D3 analog, oprozomib and ixazomib to assess proteasome inhibition. Western blot was performed to analyze the effect of the different treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using Enzyme Linked Immunosorbent Assay to analyze the effect of these drugs on its release.A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. Data were analyzed using a one-way ANOVA test followed by by Tukey post hoc test and analysis of variance. Ap≤0.05 was considered statistically significant. Results : Treatment with proteasome inhibitors and EB1089 resulted in a slight increase of dysferlin expression which was accompanied by a low increase of myogenin expression. Also, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes from a dysferlinopathy patient. However, the increase of dysferlin had no effect on the repair of muscle membrane after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Proteasome inhibitors reduce thrombospondin-1 release in human dysferlin-deficient myotubes

BMC Musculoskeletal Disorders ◽

10.1186/s12891-020-03756-7 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44. Methods To assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p ≤ 0.05 was considered statistically significant. Results Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury. Conclusions Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

10.21203/rs.2.19199/v2 ◽

2020 ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background: Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy from patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of human and mouse models of dysferlinopathy suggest dysferlin-deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patient with two missense mutations in exon 44. Methods: To assess proteasome inhibition we treated dysferlin deficient myotubes with oprozomib, ixazomib, and EB1089, a vitamin D3 analogue. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury. We also calculated the fusion index in response to the different treatments. Data were analysed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p≤0.05 was considered statistically significant. Results: Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on muscle membrane repair after injury. Conclusions: Our findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Proteasome Inhibitors Reduce Thrombospondin-1 Release in Human Dysferlin-Deficient Myotubes

10.21203/rs.2.19199/v3 ◽

2020 ◽

Author(s):

Esther Fernández-Simón ◽

Cinta Lleixà ◽

Xavier Suarez-Calvet ◽

Jordi Diaz-Manera ◽

Isabel Illa ◽

...

Keyword(s):

Vitamin D3 ◽

Transmembrane Protein ◽

Proteasome Inhibitors ◽

Ubiquitin Proteasome System ◽

Muscle Membrane ◽

Enzyme Linked Immunosorbent Assay ◽

Missense Mutations ◽

Membrane Repair ◽

Thrombospondin 1 ◽

Ubiquitin Proteasome

Abstract Background Dysferlinopathies are a group of muscle disorders causing muscle weakness and absence or low levels of dysferlin, a type-II transmembrane protein and the causative gene of these dystrophies. Dysferlin is implicated in vesicle fusion, trafficking, and membrane repair. Muscle biopsy of patients with dysferlinopathy is characterized by the presence of inflammatory infiltrates. Studies in the muscle of both human and mouse models of dysferlinopathy suggest dysferlin deficient muscle plays a role in this inflammation by releasing thrombospondin-1. It has also been reported that vitamin D3 treatment enhances dysferlin expression. The ubiquitin-proteasome system recognizes and removes proteins that fail to fold or assemble properly and previous studies suggest that its inhibition could have a therapeutic effect in muscle dystrophies. Here we assessed whether inhibition of the ubiquitin proteasome system prevented degradation of dysferlin in immortalized myoblasts from a patients with two missense mutations in exon 44. MethodsTo assess proteasome inhibition we treated dysferlin deficient myotubes with EB1089, a vitamin D3 analog, oprozomib and ixazomib. Western blot was performed to analyze the effect of these treatments on the recovery of dysferlin and myogenin expression. TSP-1 was quantified using the enzyme-linked immunosorbent assay to analyze the effect of these drugs on its release. A membrane repair assay was designed to assess the ability of treated myotubes to recover after membrane injury and fusion index was also measured with the different treatments. Data were analyzed using a one-way ANOVA test followed by Tukey post hoc test and analysis of variance. A p≤0.05 was considered statistically significant. Results Treatment with proteasome inhibitors and EB1089 resulted in a trend towards an increase in dysferlin and myogenin expression. Furthermore, EB1089 and proteasome inhibitors reduced the release of TSP-1 in myotubes. However, no effect was observed on the repair of muscle membrane after injury. ConclusionsOur findings indicate that the ubiquitin-proteasome system might not be the main mechanism of mutant dysferlin degradation. However, its inhibition could help to improve muscle inflammation by reducing TSP-1 release.

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Competition Policy under Economic Crisis

Voprosy Ekonomiki ◽

10.32609/0042-8736-2009-3-54-69 ◽

2009 ◽

pp. 54-69 ◽

Cited By ~ 1

Author(s):

A. Shastitko ◽

S. Avdasheva ◽

S. Golovanova

Keyword(s):

Division Of Labor ◽

Economic Crisis ◽

Industrial Policy ◽

Competition Policy ◽

Transition Economy ◽

Economic Recession ◽

Price Rigidity ◽

Key Factor ◽

Policy Measures ◽

The Impact

The analysis of competition policy under economic crisis is motivated by the fact that competition is a key factor for the level of productivity. The latter, in its turn, influences the scope and length of economic recession. In many Russian markets buyers' gains decline because of the weakness of competition, since suppliers are reluctant to cut prices in spite of the decreasing demand. Data on prices in Russia and abroad in the second half of 2008 show asymmetric price rigidity. At least two questions are important under economic crisis: the 'division of labor' between pro-active and protective tools of competition policy and the impact of anti-crisis policy on competition. Protective competition policy is insufficient in transition economy, especially in the days of crisis it should be supplemented with the well-designed industrial policy measures which do not contradict the goals of competition. The preferable tools of anti-crisis policy are also those that do not restrain competition.

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Designing a CRM Framework for online & offline sports footwear retailers

"Carácter" Revista Cientifica de la Universidad Del Pacifico ISSN 2602-8476 ◽

10.35936/caracter.v3i1.41 ◽

2015 ◽

Vol 3 (1) ◽

Author(s):

Rodrigo Cueva ◽

Guillem Rufian ◽

Maria Gabriela Valdes

Keyword(s):

Environmental Changes ◽

Customer Relationship ◽

Business Strategies ◽

Economic Sectors ◽

Running Shoes ◽

Brick And Mortar ◽

Key Factor ◽

On Line ◽

The Impact

The use of Customer Relationship Managers to foster customers loyalty has become one of the most common business strategies in the past years. However, CRM solutions do not fill the abundance of happily ever-after relationships that business needs, and each client’s perception is different in the buying process. Therefore, the experience must be precise, in order to extend the loyalty period of a customer as much as possible. One of the economic sectors in which CRM’s have improved this experience is retailing, where the personalized attention to the customer is a key factor. However, brick and mortar experiences are not enough to be aware in how environmental changes could affect the industry trends in the long term. A base unified theoretical framework must be taken into consideration, in order to develop an adaptable model for constructing or implementing CRMs into companies. Thanks to this approximation, the information is complemented, and the outcome will increment the quality in any Marketing/Sales initiative. The goal of this article is to explore the different factors grouped by three main domains within the impact of service quality, from a consumer’s perspective, in both on-line and off-line retailing sector. Secondly, we plan to go a step further and extract base guidelines about previous analysis for designing CRM’s solutions focused on the loyalty of the customers for a specific retailing sector and its product: Sports Running Shoes.

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The Predominant microRNAs in β-cell Clusters for Insulin Regulation and Diabetic Control

Current Drug Targets ◽

10.2174/1389450121666191230145848 ◽

2020 ◽

Vol 21 (7) ◽

pp. 722-734

Author(s):

Adele Soltani ◽

Arefeh Jafarian ◽

Abdolamir Allameh

Keyword(s):

Mirna Expression ◽

Expression Profiles ◽

Expression Patterns ◽

Diabetic Control ◽

In Vitro Proliferation ◽

Cell Functions ◽

Mirna Expression Profiles ◽

Multiple Processes ◽

The Impact

micro (mi)-RNAs are vital regulators of multiple processes including insulin signaling pathways and glucose metabolism. Pancreatic β-cells function is dependent on some miRNAs and their target mRNA, which together form a complex regulative network. Several miRNAs are known to be directly involved in β-cells functions such as insulin expression and secretion. These small RNAs may also play significant roles in the fate of β-cells such as proliferation, differentiation, survival and apoptosis. Among the miRNAs, miR-7, miR-9, miR-375, miR-130 and miR-124 are of particular interest due to being highly expressed in these cells. Under diabetic conditions, although no specific miRNA profile has been noticed, the expression of some miRNAs and their target mRNAs are altered by posttranscriptional mechanisms, exerting diverse signs in the pathobiology of various diabetic complications. The aim of this review article is to discuss miRNAs involved in the process of stem cells differentiation into β-cells, resulting in enhanced β-cell functions with respect to diabetic disorders. This paper will also look into the impact of miRNA expression patterns on in vitro proliferation and differentiation of β-cells. The efficacy of the computational genomics and biochemical analysis to link the changes in miRNA expression profiles of stem cell-derived β-cells to therapeutically relevant outputs will be discussed as well.

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Correlation of Altmetric Attention Scores with Citations in Major Pharmacy Journals (Preprint)

10.2196/preprints.13129 ◽

2018 ◽

Author(s):

Dave L Dixon ◽

William L Baker

Keyword(s):

Citation Count ◽

Cross Sectional Study ◽

Scientific Output ◽

Cross Sectional ◽

Promotion And Tenure ◽

Article Type ◽

Key Factor ◽

Number Of Citations ◽

The Impact

BACKGROUND The impact and quality of a faculty members publications is a key factor in promotion and tenure decisions and career advancement. Traditional measures, including citation counts and journal impact factor, have notable limitations. Since 2010, alternative metrics have been proposed as another means of assessing the impact and quality of scholarly work. The Altmetric Attention Score is an objective score frequently used to determine the immediate reach of a published work across the web, including news outlets, blogs, social media, and more. Several studies evaluating the correlation between the Altmetric Attention Score and number of citations have found mixed results and may be discipline-specific. OBJECTIVE To determine the correlation between higher Altmetric Attention Scores and citation count for journal articles published in major pharmacy journals. METHODS This cross-sectional study evaluated articles from major pharmacy journals ranked in the top 10% according to the Altmetric Attention Score. Sources of attention that determined the Altmetric Attention Score were obtained, as well each articles open access status, article type, study design, and topic. Correlation between journal characteristics, including the Altmetric Attention Score and number of citations, was assessed using the Spearman’s correlation test. A Kruskal-Wallis 1-way analysis of variance (ANOVA) was used to compare the Altmetric Attention Scores between journals. RESULTS Six major pharmacy journals were identified. A total of 1,376 articles were published in 2017 and 137 of these represented the top 10% with the highest Altmetric Attention Scores. The median Altmetric Attention Score was 19 (IQR 15-28). Twitter and Mendeley were the most common sources of attention. Over half (56.2%) of the articles were original investigations and 49.8% were either cross-sectional, qualitative, or cohort studies. No significant correlation was found between the Altmetric Attention Score and citation count (rs=0.07, P = 0.485). Mendeley was the only attention source that correlated with the number of citations (rs=0.486, P<0.001). The median Altmetric Attention Score varied widely between each journal (P<0.001). CONCLUSIONS The overall median Altmetric Attention score of 19 suggests articles published in major pharmacy journals are near the top 5% of all scientific output. However, we found no correlation between the Altmetric Attention Score and number of citations for articles published in major pharmacy journals in the year 2017.

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Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

International Journal of Molecular Sciences ◽

10.3390/ijms22052476 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2476

Author(s):

Kento Fujiwara ◽

Masaki Kitaura ◽

Ayaka Tsunei ◽

Hotaka Kusabuka ◽

Erika Ogaki ◽

...

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

Second Generation ◽

Independent Manner ◽

Cell Functions ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

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Building Information Modeling Methods for Post-Earthquake Retrofitting Visualization of Buildings Using Augmented Reality

Applied Sciences ◽

10.3390/app11125739 ◽

2021 ◽

Vol 11 (12) ◽

pp. 5739

Author(s):

Zhansheng Liu ◽

Wenyan Bai

Keyword(s):

Augmented Reality ◽

Building Information Modeling ◽

Repair Process ◽

Information Modeling ◽

Three Dimensions ◽

Visualization Process ◽

Building Information ◽

Key Factor ◽

Technical Solutions ◽

Seismic Resilience

The post-earthquake retrofitting and repair process of a building is a key factor in improving its seismic capability. A thorough understanding of retrofitting methods and processes will aid in repairing post-earthquake buildings and improving seismic resilience. This study aims to develop a visualization framework for the post-earthquake retrofitting of buildings which builds models based on building information modeling (BIM) and realizes visualization using augmented reality (AR). First, multi-level representation methods and coding criteria are used to process the models for a damaged member. Then, an information collection template is designed for integrating multi-dimensional information, such as damage information, retrofitting methods, technical solutions, and construction measures. Subsequently, a BIM model is presented in three dimensions (3D) using AR. Finally, the visualization process is tested through experiments, which demonstrate the feasibility of using the framework to visualize the post-earthquake retrofitting of a building.

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