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      Establishment and characterization of pygmy killer whale ( Feresa attenuata) dermal fibroblast cell line

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          Abstract

          The pygmy killer whale ( Feresa attenuata) (PKW) is a tropical and subtropical marine mammal commonly found in the Atlantic, Indian and Pacific oceans. Since the PKWs live in offshore protected territories, they are rarely seen onshore. Hence, PKW are one of the most poorly understood oceanic species of odontocetes. The dermal tissue comes primarily from stranding events that occur along the coast of the Shantou, Guangdong, China. The sampled tissues were immediately processed and attached on collagen-coated 6-well tissue culture plate. The complete medium (DMEM and Ham’s F12, fetal bovine serum, antibiotic and essential amino acids) was added to the culture plates. The primary culture (PKW-LWH) cells were verified as fibroblast by vimentin and karyotype analyses, which revealed 42 autosomes and two sex chromosomes X and Y. Following transfection of PKW-LWH cells with a plasmid encoding, the SV40 large T-antigens and the transfected cells were isolated and expanded. Using RT-PCR, western blot, immunofluorescence analysis and SV40 large T-antigen stability was confirmed. The cell proliferation rate of the fibroblast cells, PKW-LWHT was faster than the primary cells PKW-LWH with the doubling time 68.9h and 14.4h, respectively. In this study, we established PKW dermal fibroblast cell line for the first time, providing a unique opportunity for in vitro studies on the effects of environmental pollutants and pathogens that could be determined in PKW and/or Cetaceans.

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          Enumeration of the simian virus 40 early region elements necessary for human cell transformation.

          While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.
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            Primary Skin Fibroblasts as a Model of Parkinson's Disease

            Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues.
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              Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.

              The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.
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                Author and article information

                Contributors
                Role: InvestigationRole: Writing – original draft
                Role: ConceptualizationRole: Formal analysisRole: Writing – original draft
                Role: Investigation
                Role: Investigation
                Role: Writing – review & editing
                Role: Software
                Role: Software
                Role: ResourcesRole: SupervisionRole: Validation
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                29 March 2018
                2018
                : 13
                : 3
                : e0195128
                Affiliations
                [1 ] Marine Biology Institute, College of Science Shantou University, Shantou, Guangdong, P.R. China
                [2 ] Faculty of Veterinary and Animal Sciences, Lasbela University of Agriculture, Water and Marine Sciences, Uthal, Balochistan, Pakistan
                [3 ] Ocean College, Qinzhou University, Qinzhou, Guangxi, P.R. China
                University of Rochester School of Medicine and Dentistry, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-3172-4541
                Article
                PONE-D-17-39373
                10.1371/journal.pone.0195128
                5875847
                29596530
                977fd8ea-6bdc-4c83-983c-2dbdae5e6295
                © 2018 Yajing et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 November 2017
                : 16 March 2018
                Page count
                Figures: 7, Tables: 1, Pages: 15
                Funding
                Funded by: National Natural Science Foundation
                Award ID: 41676166
                Award Recipient :
                Funded by: National Natural Science Foundation of China (CN)
                Award ID: 41776174
                Award Recipient :
                This research was supported by the National Natural Science Foundation of China (grant numbers 41676166 and 41776174) to LW, Ministry of Agriculture (Chinese White Dolphin Conservation Action) and CNOOC Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Medicine and Health Sciences
                Anatomy
                Biological Tissue
                Connective Tissue
                Connective Tissue Cells
                Fibroblasts
                Biology and Life Sciences
                Biochemistry
                Proteins
                Cytoskeletal Proteins
                Vimentin
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Molecular Biology Techniques
                Transfection
                Research and Analysis Methods
                Biological Cultures
                Cell Lines
                Cultured Fibroblasts
                Biology and life sciences
                Organisms
                Viruses
                DNA viruses
                Polyomaviruses
                Sv40
                Biology and Life Sciences
                Microbiology
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                Microbial Pathogens
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                Medicine and Health Sciences
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                Physical Sciences
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                Earth Sciences
                Marine and Aquatic Sciences
                Marine Biology
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