| 1. |
- Dapena, Carlos, et al.
(författare)
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Nuclear and Cell Morphological Changes during the Cell Cycle and Growth of the Toxic Dinoflagellate Alexandrium minutum.
- 2015
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Ingår i: Protist. - : Elsevier BV. - 1434-4610. ; 166:1, s. 146-160
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Tidskriftsartikel (refereegranskat)abstract
- Elucidation of the cell cycle of dinoflagellates is essential to understand the processes leading to their massive proliferations, known as harmful algal blooms. In this study, we used imaging flow cytometry (IFC) to monitor the changes in DNA content and nuclear and cell morphology that occur during clonal growth of the toxic species Alexandrium minutum Halim. Our results indicate that the population was in S phase (C→2C DNA content) during the light period, whereas haploid cells with a C DNA content peaked only during a short interval of the dark period. The timing of the phases, identified based on the nuclear morphology and cytoplasmic-to-nuclear (CNR) ratio of the cells, suggests that the length of the G2/M phase is regulated by nutrient levels whereas the beginning of S phase is clock controlled. In addition we found that up to 7% of individual cells achieved a DNA content higher than 2C, indicative of either zygote formation and replication (homothallism), or of double-haploid cells able to divide (polyploid forms). Cells belonging to different cell cycle phases (G1-S-G2/M) could be readily discriminated based on nuclear size. Our study provides evidence of cell-cycle plasticity during clonal growth and unambiguously characterizes the cell-cycle phases of this dinoflagellate species.
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| 2. |
- Figueroa, Rosa, et al.
(författare)
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The Hidden Sexuality of Alexandrium Minutum: An Example of Overlooked Sex in Dinoflagellates.
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- Dinoflagellates are haploid eukaryotic microalgae in which rapid proliferation causes dense blooms, with harmful health and economic effects to humans. The proliferation mode is mainly asexual, as the sexual cycle is believed to be rare and restricted to stressful environmental conditions. However, sexuality is key to explaining the recurrence of many dinoflagellate blooms because in many species the fate of the planktonic zygotes (planozygotes) is the formation of resistant cysts in the seabed (encystment). Nevertheless, recent research has shown that individually isolated planozygotes in the lab can enter other routes besides encystment, a behavior of which the relevance has not been explored at the population level. In this study, using imaging flow cytometry, cell sorting, and Fluorescence In Situ Hybridization (FISH), we followed DNA content and nuclear changes in a population of the toxic dinoflagellate Alexandrium minutum that was induced to encystment. Our results first show that planozygotes behave like a population with an "encystment-independent" division cycle, which is light-controlled and follows the same Light:Dark (L:D) pattern as the cycle governing the haploid mitosis. Resting cyst formation was the fate of just a small fraction of the planozygotes formed and was restricted to a period of strongly limited nutrient conditions. The diploid-haploid turnover between L:D cycles was consistent with two-step meiosis. However, the diel and morphological division pattern of the planozygote division also suggests mitosis, which would imply that this species is not haplontic, as previously considered, but biphasic, because individuals could undergo mitotic divisions in both the sexual (diploid) and the asexual (haploid) phases. We also report incomplete genome duplication processes. Our work calls for a reconsideration of the dogma of rare sex in dinoflagellates.
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| 3. |
- Spinozzi, Daniele, et al.
(författare)
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Evaluation of the Suitability of Biocompatible Carriers as Artificial Transplants Using Cultured Porcine Corneal Endothelial Cells
- 2019
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Ingår i: Current Eye Research. - : TAYLOR & FRANCIS INC. - 0271-3683 .- 1460-2202. ; 44:3, s. 243-249
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Tidskriftsartikel (refereegranskat)abstract
- Purpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK). Materials and Methods: pCECs were isolated, cultured up to P2 and seeded onto LinkCell (TM) bioengineered matrices of 20 mu m (LK20) or 100 mu m (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM). Results: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 +/- 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a "taco" for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. Conclusions: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.
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| 4. |
- Spinozzi, Daniele, et al.
(författare)
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In Vitro Evaluation and Transplantation of Human Corneal Endothelial Cells Cultured on Biocompatible Carriers
- 2020
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Ingår i: Cell Transplantation. - : SAGE PUBLICATIONS INC. - 0963-6897 .- 1555-3892. ; 29
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Tidskriftsartikel (refereegranskat)abstract
- Corneal transplantation is currently the only effective treatment option for dysfunctional corneal endothelial cells (CEC). In this study, we test in vitro the surgical potential of cultivated human corneal endothelial cells (hCEC) on human anterior lens capsule (HALC), LinkCell (TM) bioengineered collagen sheets of 20-mu m thickness (LK20), and denuded Descemet membrane (dDM) as tissue-engineered grafts for Descemet membrane (DM) endothelial keratoplasty (DMEK) to bypass the problem of donor tissue availability. Primary hCEC cultured on all carriers formed a monolayer of tightly packed cells with a high cell viability rate (96% +/- 4%). hCEC on HALC and LK20 showed unremarkable expression of zonula occludens-1 (ZO-1) and Na+/K+-adenosine triphosphatase (ATPase), while Na+/K+-ATPase expression of cells seeded on dDM was mainly cytoplasmic. All hCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using a human donor cornea without DM mounted on an artificial anterior chamber (AC) and a regular DMEK-graft used as a surgical reference model. During in vitro surgery, hCEC-HALC constructs behaved most similarly to a DMEK-graft during implantation and unfolding, showing good adhesion to the bare stroma. On the other hand, hCEC-LK20 and hCEC-dDM constructs required some additional handling because of challenges related to the surgical procedure, although they were both successfully unfolded and implanted in the artificial AC. The hCEC-dDM constructs showed similar graft adherence as hCEC-HALC constructs, while adherence of hCEC-LK20 constructs was less effective. After the in vitro surgery, the estimated area populated by viable cells on the hCEC-HALC and hCEC-LK20 constructs was similar to 83% and similar to 67%, respectively. Overall, hCEC-HALC constructs behaved most similarly to a DMEK-graft during in vitro DMEK surgery, while graft adhesion and surgical handling, respectively, are parameters still requiring optimization for hCEC-LK20 and hCEC-dDM constructs.
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