52 research outputs found

    Cell-Type–Specific Transcriptional Profiles of the Dimorphic Pathogen Penicillium marneffei Reflect Distinct Reproductive, Morphological, and Environmental Demands

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    Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify “phase or cell-state–specific” gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase–encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity

    PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model

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    Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org

    Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool.

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    MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans -Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans -Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general

    Erythroid enucleation requires intracellular calcium signaling.

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    <p><b>(A-B)</b> Orthochromatic erythroblasts were FACS sorted and incubated in the presence of the compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated inhibitors at the indicated concentrations. Data are means (+/- SD) of 3 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). <b>(C-E)</b> Orthochromatic erythroblasts were incubated in media containing DMSO (vehicle control) or the indicated compounds at the indicated concentration for 5h and subsequently cytospun. For quantitative analysis cells were manually examined (Olympus BX-51 microscope; 100x/1.40 NA oil objective) using the Spot Advanced software (version 4.7)) and assigned a morphological class as per illustration. Data are means (+/- SD) of 3 independent experiments. Scalebar = 10μm.</p

    Erythroid enucleation requires uptake of extracellular calcium.

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    <p><b>(A-C)</b> Left panels: Orthochromatic erythroblasts were incubated in the presence of the indicated compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated compounds at the indicated concentrations. Data are means (+/- SD) of 3–4 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). Middle and right panels: Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated compounds. Data are means (+/- SD) of 3 independent experiments. Scale bar = 10μm.</p

    Model of potential roles for calcium in erythroid enucleation.

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    <p>Model of potential actions of calcium during nuclear extrusion. Inhibitors that target calcium-dependent signaling pathways and resulted in arrest of enucleation are shown in red.</p

    Orthochromatic erythroblasts display transient calcium bursts prior to enucleation.

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    <p>Orthochromatic erythroblasts were isolated by FACS and incubated in the presence of Fluo-3 for 30min. Orthochromatic erythroblasts were then washed and allowed to settle on a microgrid for live cell imaging using the Leica Confocal Sp5 microscope. Images were taken every minute for 2h. <b>(A)</b> Images of the same orthochromatic erythroblast at various time points prior (-), during (0) and after (+) nuclear extrusion. For the cell demonstrated here, Fluo-3 fluorescence is most intense between 12 and 8 min prior to nuclear extrusion. <b>(B)</b> Analysis of Fluo-3 fluorescence intensity in different enucleating cells compared to non-enucleating ones.</p

    Erythroid enucleation requires calmodulin signaling.

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    <p><b>(A)</b> Immunofluorescence staining for calmodulin (CaM) in orthochromatic erythroblasts at different stages of the enucleation process. Scalebar = 5μm. <b>(B-C)</b> Left panels: Orthochromatic erythroblasts were incubated in the presence of the indicated compounds for 5h. Graphs showing percentages of enucleation in the presence of the indicated compounds at the indicated concentrations. Data are means (+/- SD) of 3–4 independent experiments analyzed using FACS LSR II (*P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (paired student’s t-test)). Middle and right panels: Cytospins and quantitative analysis of orthochromatic erythroblasts treated with the indicated compounds. Data are means (+/- SD) of 3 independent experiments. Scale bar = 10μm.</p

    Examining the cognitive effects of a special extract of Bacopa monniera (CDRI 08: Keenmind): a review of ten years of research at Swinburne University

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    Bacopa monniera (EBm), an Indian aquatic herb, has been used in traditional Ayurvedic medicine for centuries for indications related to memory and inflammation. More recently specific extracts of EBm have emerged that have been subjected to rigorous in vitro, animal and now human clinical trials. In this paper we discuss some of these studies with special reference to mechanisms and efficacy of a special extract of Bacopa (CDRI08). Studies using this extract indicate that CDRI08 has several modes of action on the human brain. Promising indications for use in humans include improving cognition in the elderly and in patients with neurodegenerative disorders
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