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    Ploidy variation in Rhododendron subsection Maddenia and its implications for conservation.
    (Oxford University Press on behalf of the Annals of Botany Company, 2023-06-01) Hu L; Tate JA; Gardiner SE; MacKay M; Loureiro J
    Polyploidy, which is common in plants, can confound taxon recognition and hence conservation assessments. In the taxonomically complex genus Rhododendron, 25 % of the over 1,300 taxa are considered under threat and 27 % Near Threatened or Data Deficient, with their taxonomy needing to be resolved urgently. Although ploidy levels of Rhododendron taxa range from diploid (2x) to dodecaploid (12x) according to previous reports, the extent of polyploidy across the genus has not been examined. We first summarized the taxonomic distribution of polyploids in the genus based on the literature. Then as a case study, we estimated ploidy levels of 47 taxa in subsection Maddenia (subgenus Rhododendron, section Rhododendron) using flow cytometry, together with verification of meiotic chromosome counts for representative taxa. The summary of reported ploidy in Rhododendron indicates that polyploidy is most common in subgenera Pentanthera and Rhododendron. In subsection Maddenia, all examined taxa are diploids except for the R. maddenii complex that shows a high ploidy variation (2-8x, 12x). We investigated ploidy level of 12 taxa in subsection Maddenia for the first time, and estimated genome sizes of two Rhododendron species. Knowledge of ploidy levels will inform phylogenetic analysis of unresolved species complexes. Overall, our study of subsection Maddenia provides a model for examining multiple issues including taxonomic complexity, ploidy variation and geographic distribution in relation to biodiversity conservation.
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    A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.
    (Frontiers Media S.A., 2023-05-22) Ogbuigwe P; Roberts JM; Knox MA; Heiser A; Pita A; Haack NA; Garcia-Ramirez JC; Velathanthiri N; Biggs PJ; French NP; Hayman DTS; Xu R
    Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.