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Analysis of the role of the secretory pathway Ca2+/Mn2+- ATPase, PMR1/ATP2C1, in calcium homeostasis: from oxidative stress to skin disease
Full text linkDuring my PhD, the genetically tractable Kluyveromyces lactis yeast has been used to study the molecular basis of Hailey–Hailey disease (HHD). This pathology, also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, characterized by acantholysis. The genetics and pathophysiology of HHD have been linked to mutations in ATP2C1. The gene encodes for an adenosine triphosphate (ATP)-powered calcium channel pump and PMR1 is the yeast orthologue. Similarly to human HHD-cells, in yeast the loss of PMR1 promotes cellular toxicity caused by increased oxidative stress linked to the alteration of calcium homeostasis. By a functional suppression screening of yeast mutant with a cDNA library, we found that Glutathione S-transferase (GST), an important detoxifying enzyme, could be a candidate gene associated with Hailey-Hailey disease. Expression of mammalian GST in Klpmr1 alleviated several defects; these include oxidative-stress toxicity, calcium alteration and mitochondrial dysfunction. Additionally, the discoveries made in yeast were validated in HHD-derived cells. Indeed, HHD-lesional derived keratinocytes showed a decreased expression of GST gene when compared to non-lesional skin derived cells from the same patients.
In parallel to the genetic screening, a drug screen was performed to find compounds with potential therapeutic effects for HHD treatment. Indeed, currently, there is no reported cure for HHD. For this reason, 131 FDA-approved natural compounds were screened for their ability to alleviate the calcium alterations and the oxidative stress of Kluyveromyces lactis pmr1cells. The drug collection includes inhibitors, activators and antagonists acting on molecular targets involved in different signaling pathways. Initially, for the sake of simplicity, the initial screening system was based on the cell wall alterations of the pmr1∆ mutant, utilizing the chitin binding stilbene fluorescent brightener calcofluor white. In the first part of the screening 12 molecules resulted toxic, while 71% of the library molecules were ineffective and the remaining appeared to have a partial effect. Therefore, in a second round of the screening, these last compounds were evaluated at higher concentrations. Based on this, six molecules were selected for further analysis. Their effects on other defects of pmr1 mutant strain were also evaluated, ranging from alteration of Ca2+ homeostasis to sensitivity to the ROS-generator menadione, as well as the mitochondrial functionality
Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease
Full text linkThe term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1. We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis. These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1. In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD
Glutathione S-transferase Θ-subunit as a phenotypic suppressor of pmr1Δ strain, the Kluyveromyces lactis model for Hailey-Hailey disease
No full textBackground: Hailey-Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1, the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump.
Methods: In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1, the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells.
Results: We have identified the Glutathione S-transferase Θ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δmutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca2+) homeostasis by decreasing the high cytosolic Ca2+ levels in pmr1Δstrain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart (GSTT1/M1) was observed in lesion-derived keratinocytes from HHD patients.
Conclusions: These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease.
General significance: Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology
Pmr-1 gene affects susceptibility of Caenorhabditis elegans to Staphylococcus aureus infection through glycosylation and stress response pathways alterations
Full text linkCalcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca 2+ -ATPase, encoded by pmr-1 gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the Caenorhabditis elegans model system to gain insight into the downstream response elicited by loss of pmr-1 gene. We found that pmr-1 knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of C. elegans surface glycoproteins by microbial agents, we provide genetic evidence that pmr-1 interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate pmr-1 as a candidate gene implicated in mediating the worm’s innate immune response
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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