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      High-Throughput Yeast Aging Analysis for Cryptococcus (HYAAC) microfluidic device streamlines aging studies in Cryptococcus neoformans

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          Abstract

          Cryptococcus neoformans (Cn) is a deadly fungal pathogen responsible for ~ 180,000 deaths per year and despite effective antifungals, treatment failure and resistance to antifungals are increasingly problematic. Aging and age-related phenotypes are prominent virulence traits that contribute to the resilience of Cn to host responses and antifungals. Traditional methods to study aging in Cn are expensive, inefficient and in need of improvement. Here, we demonstrate the development and use of a High-Throughput Yeast Aging Analysis for Cryptococcus (HYAAC) microfluidic device to better study aging and age-associated genes in Cn. Compared to traditional methods, the HYAAC is superior in its efficiency to isolate, manipulate and observe old cells for analysis. It allows for the trapping and tracking of individual cells over the course of their lifespan, allowing for more precise measurements of lifespan, tracking of age-related phenotypes with age, and a more high-throughput ability to investigate genes associated with aging.

          Abstract

          Orner et al. develops a microfluidic device called High-Throughput Yeast Aging Analysis for Cryptococcus (HYAAC) that can be used to measure lifespan, cell death, and dynamics of gene expression changes across age. This device may streamline aging studies in Cryptococcus neoformans and facilitate investigations in yeast pathogens.

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          Most cited references17

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          A Comprehensive Analysis of Replicative Lifespan in 4,698 Single-Gene Deletion Strains Uncovers Conserved Mechanisms of Aging.

          Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is nonadditive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging.
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            Gene Duplication Associated with Increased Fluconazole Tolerance in Candida auris cells of Advanced Generational Age

            Candida auris is an emerging multi-drug resistant yeast that causes systemic infections. Here we show that C. auris undergoes replicative aging (RA) that results from asymmetric cell division and causes phenotypic differences between mother and daughter cells similar to other pathogenic yeasts. Importantly, older C. auris cells (10 generations) exhibited higher tolerance to fluconazole (FLC), micafungin, 5- flucytosine and amphotericin B compared to younger (0–3 generation) cells. Increased FLC tolerance was associated with increased Rhodamine 6G (R6G) efflux and therapeutic failure of FLC in a Galleria infection model. The higher efflux in the older cells correlated with overexpression of the efflux pump encoding gene CDR1 (4-fold). In addition, 8-fold upregulation of the azole target encoding gene ERG11 was noted in the older cells. Analysis of genomic DNA from older cells by qPCR indicates that transient gene duplication of CDR1 and ERG11 causes the observed age-dependent enhanced FLC tolerance in C. auris strains. Furthermore, older cells exhibited a thickened cell wall, decreased neutrophil killing (24% vs 50%), increased epithelial cell adhesion (31.6% vs 17.8%) and upregulation of adhesin protein Als5p. Thus, this study demonstrates that transient gene duplication can occur during RA, causing increased FLC tolerance in old C. auris cells.
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              Identification of evolutionarily conserved genetic regulators of cellular aging

              To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.
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                Author and article information

                Contributors
                Bettina.fries@stonybrookmedicine.edu
                Journal
                Commun Biol
                Commun Biol
                Communications Biology
                Nature Publishing Group UK (London )
                2399-3642
                10 July 2019
                10 July 2019
                2019
                : 2
                : 256
                Affiliations
                [1 ]ISNI 0000 0001 2216 9681, GRID grid.36425.36, Department of Molecular Genetics and Microbiology, , Stony Brook University, ; Stony Brook, NY 11794 USA
                [2 ]ISNI 0000 0004 0445 0041, GRID grid.63368.38, Department of Nanomedicine, , Houston Methodist Research Institute, ; Houston, TX 77030 USA
                [3 ]ISNI 000000041936877X, GRID grid.5386.8, Department of Cell and Developmental Biology, , Weill Cornell Medical College, ; New York, NY 10065 USA
                [4 ]ISNI 0000 0001 2216 9681, GRID grid.36425.36, Department of Medicine, , Stony Brook University, ; Stony Brook, NY 11794 USA
                [5 ]ISNI 0000 0004 0420 1678, GRID grid.413840.a, Department of Medicine, , Northport VA Medical Center, ; Northport, NY 11794 USA
                Author information
                http://orcid.org/0000-0002-6253-7298
                http://orcid.org/0000-0002-8562-3877
                http://orcid.org/0000-0002-6847-5518
                Article
                504
                10.1038/s42003-019-0504-5
                6620289
                31312725
                d426c8f4-7c1b-405d-bd3a-1c24c8a48829
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 17 September 2018
                : 14 June 2019
                Funding
                Funded by: FundRef https://doi.org/10.13039/100000060, U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID);
                Award ID: R01AI127704-03
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000738, U.S. Department of Veterans Affairs (Department of Veterans Affairs);
                Award ID: 1I01BX003741-01A2
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100007259, SUNY | Stony Brook University (SBU);
                Award ID: Start-Up grant
                Award Recipient :
                Categories
                Article
                Custom metadata
                © The Author(s) 2018

                lab-on-a-chip,fungal pathogenesis,antimicrobial resistance

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