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      Different Stress-Induced Calcium Signatures Are Reported by Aequorin-Mediated Calcium Measurements in Living Cells of Aspergillus fumigatus

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          Abstract

          Aspergillus fumigatus is an inhaled fungal pathogen of human lungs, the developmental growth of which is reliant upon Ca 2+-mediated signalling. Ca 2+ signalling has regulatory significance in all eukaryotic cells but how A. fumigatus uses intracellular Ca 2+ signals to respond to stresses imposed by the mammalian lung is poorly understood. In this work, A. fumigatus strains derived from the clinical isolate CEA10, and a non-homologous recombination mutant ΔakuB KU80, were engineered to express the bioluminescent Ca 2+-reporter aequorin. An aequorin-mediated method for routine Ca 2+ measurements during the early stages of colony initiation was successfully developed and dynamic changes in cytosolic free calcium ([Ca 2+] c) in response to extracellular stimuli were measured. The response to extracellular challenges (hypo- and hyper-osmotic shock, mechanical perturbation, high extracellular Ca 2+, oxidative stress or exposure to human serum) that the fungus might be exposed to during infection, were analysed in living conidial germlings. The ‘signatures’ of the transient [Ca 2+] c responses to extracellular stimuli were found to be dose- and age-dependent. Moreover, Ca 2+-signatures associated with each physico-chemical treatment were found to be unique, suggesting the involvement of heterogeneous combinations of Ca 2+-signalling components in each stress response. Concordant with the involvement of Ca 2+-calmodulin complexes in these Ca 2+-mediated responses, the calmodulin inhibitor trifluoperazine (TFP) induced changes in the Ca 2+-signatures to all the challenges. The Ca 2+-chelator BAPTA potently inhibited the initial responses to most stressors in accordance with a critical role for extracellular Ca 2+ in initiating the stress responses.

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          Calcium signaling.

          Calcium ions (Ca(2+)) impact nearly every aspect of cellular life. This review examines the principles of Ca(2+) signaling, from changes in protein conformations driven by Ca(2+) to the mechanisms that control Ca(2+) levels in the cytoplasm and organelles. Also discussed is the highly localized nature of Ca(2+)-mediated signal transduction and its specific roles in excitability, exocytosis, motility, apoptosis, and transcription.
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            Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET).

            Invasive fungal infections (IFIs) are a major cause of morbidity and mortality among organ transplant recipients. Multicenter prospective surveillance data to determine disease burden and secular trends are lacking. The Transplant-Associated Infection Surveillance Network (TRANSNET) is a consortium of 23 US transplant centers, including 15 that contributed to the organ transplant recipient dataset. We prospectively identified IFIs among organ transplant recipients from March, 2001 through March, 2006 at these sites. To explore trends, we calculated the 12-month cumulative incidence among 9 sequential cohorts. During the surveillance period, 1208 IFIs were identified among 1063 organ transplant recipients. The most common IFIs were invasive candidiasis (53%), invasive aspergillosis (19%), cryptococcosis (8%), non-Aspergillus molds (8%), endemic fungi (5%), and zygomycosis (2%). Median time to onset of candidiasis, aspergillosis, and cryptococcosis was 103, 184, and 575 days, respectively. Among a cohort of 16,808 patients who underwent transplantation between March 2001 and September 2005 and were followed through March 2006, a total of 729 IFIs were reported among 633 persons. One-year cumulative incidences of the first IFI were 11.6%, 8.6%, 4.7%, 4.0%, 3.4%, and 1.3% for small bowel, lung, liver, heart, pancreas, and kidney transplant recipients, respectively. One-year incidence was highest for invasive candidiasis (1.95%) and aspergillosis (0.65%). Trend analysis showed a slight increase in cumulative incidence from 2002 to 2005. We detected a slight increase in IFIs during the surveillance period. These data provide important insights into the timing and incidence of IFIs among organ transplant recipients, which can help to focus effective prevention and treatment strategies.
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              Aspergillus fumigatus and aspergillosis.

              J P Latgé (1999)
              Aspergillus fumigatus is one of the most ubiquitous of the airborne saprophytic fungi. Humans and animals constantly inhale numerous conidia of this fungus. The conidia are normally eliminated in the immunocompetent host by innate immune mechanisms, and aspergilloma and allergic bronchopulmonary aspergillosis, uncommon clinical syndromes, are the only infections observed in such hosts. Thus, A. fumigatus was considered for years to be a weak pathogen. With increases in the number of immunosuppressed patients, however, there has been a dramatic increase in severe and usually fatal invasive aspergillosis, now the most common mold infection worldwide. In this review, the focus is on the biology of A. fumigatus and the diseases it causes. Included are discussions of (i) genomic and molecular characterization of the organism, (ii) clinical and laboratory methods available for the diagnosis of aspergillosis in immunocompetent and immunocompromised hosts, (iii) identification of host and fungal factors that play a role in the establishment of the fungus in vivo, and (iv) problems associated with antifungal therapy.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                24 September 2015
                2015
                : 10
                : 9
                : e0138008
                Affiliations
                [1 ]Manchester Fungal Infection Group, Institute of Inflammation and Repair, University of Manchester, Manchester, United Kingdom
                [2 ]Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
                [3 ]Centre for Molecular Bacteriology and Infection, Department of Medicine, Imperial College London, London, United Kingdom
                Universidade de Sao Paulo, BRAZIL
                Author notes

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

                Conceived and designed the experiments: AM MB JB NI EMB NDR. Performed the experiments: AM MB JB NI. Analyzed the data: AM MB JB NI EMB NDR. Contributed reagents/materials/analysis tools: EMB NDR. Wrote the paper: AM MB EMB NDR.

                [¤a]

                Current address: The Sainsbury Laboratory, Norwich Research Park, Norwich, United Kingdom

                [¤b]

                Current address: Division of Nanoscopy, M4I (Maastricht multimodal molecular imaging institute), Maastricht University, Maastricht, Netherlands

                Article
                PONE-D-15-23401
                10.1371/journal.pone.0138008
                4581630
                26402916
                ae163515-4f9d-46b2-974a-010f16a818ca
                Copyright @ 2015

                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
                : 29 May 2015
                : 24 August 2015
                Page count
                Figures: 5, Tables: 3, Pages: 23
                Funding
                This work was supported by grants to EMB and NDR from the Wellcome Trust (WT093596/A/10/Z and WT093596/C/10/Z, respectively), and in part by grants to EMB from the Medical Research Council (G0501164) and the BBSRC (BB/G009619/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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                Research Article
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                All relevant data are within the paper and its Supporting Information files.

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