Ancestral Diversity in Lipoprotein(a) Studies Helps Address Evidence Gaps

INTRODUCTION: The independent and causal cardiovascular disease risk factor lipoprotein(a) (Lp(a)) is elevated in \u3e1.5 billion individuals worldwide, but studies have prioritised European populations. METHODS: Here, we examined how ancestrally diverse studies could clarify Lp(a)\u27s genetic architecture, inform efforts examining application of Lp(a) polygenic risk scores (PRS), enable causal inference and identify unexpected Lp(a) phenotypic effects using data from African (n=25 208), East Asian (n=2895), European (n=362 558), South Asian (n=8192) and Hispanic/Latino (n=8946) populations. RESULTS: Fourteen genome-wide significant loci with numerous population specific signals of large effect were identified that enabled construction of Lp(a) PRS of moderate (R CONCLUSIONS: Our results emphasise the merits of prioritising ancestral diversity when addressing Lp(a) evidence gaps

mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

Background: mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling. Methodology/Principal Findings: Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1b, IFN-c and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways. Conclusions/Significance: We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation an

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus

Branched chain amino acids harbor distinct and often opposing effects on health and disease

Abstract Background The branched chain amino acids (BCAA) leucine, isoleucine, and valine are essential nutrients that have been associated with diabetes, cancers, and cardiovascular diseases. Observational studies suggest that BCAAs exert homogeneous phenotypic effects, but these findings are inconsistent with results from experimental human and animal studies. Methods Hypothesizing that inconsistencies between observational and experimental BCAA studies reflect bias from shared lifestyle and genetic factors in observational studies, we used data from the UK Biobank and applied multivariable Mendelian randomization causal inference methods designed to address these biases. Results In n = 97,469 participants of European ancestry (mean age = 56.7 years; 54.1% female), we estimate distinct and often opposing total causal effects for each BCAA. For example, of the 117 phenotypes with evidence of a statistically significant total causal effect for at least one BCAA, almost half (44%, n = 52) are associated with only one BCAA. These 52 associations include total causal effects of valine on diabetic eye disease [odds ratio = 1.51, 95% confidence interval (CI) = 1.31, 1.76], valine on albuminuria (odds ratio = 1.14, 95% CI = 1.08, 1.20), and isoleucine on angina (odds ratio = 1.17, 95% CI = 1.31, 1.76). Conclusions Our results suggest that the observational literature provides a flawed picture of BCAA phenotypic effects that is inconsistent with experimental studies and could mislead efforts developing novel therapeutics. More broadly, these findings motivate the development and application of causal inference approaches that enable ‘omics studies conducted in observational settings to account for the biasing effects of shared genetic and lifestyle factors

Biphasic zinc compartmentalisation in a human fungal pathogen

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2 Delta growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrtl/Zrt2-cellular import, followed by Zrcl-dependent intracellular compartmentalisation

Zrc1 is required for virulence in a <i>Galleria</i> infection model.

<p><i>Galleria</i> larvae (10 per group) were infected with 10⁵ <i>C</i>. <i>albicans</i> cells and monitored every 12 h. Note that whilst wild type result in high mortality, only one <i>zrc1</i>Δ-infected larvae died. Experiment performed twice—here, and in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.s009" target="_blank">S8 Fig</a>. <i>zrc1</i>Δ is significantly attenuated compared to wild type (P = 0.0001) and <i>zrc1</i>Δ+<i>ZRC1</i> (P = 0.0009), but not compared to PBS control (P = 0.3173); Log-rank (Mantel-Cox) test.</p

Relationship between Zrc1, zincosomes and zinc tolerance.

<p>(A) Cells were challenged with potentially toxic zinc (1 mM), stained with zinquin and fluorescence determined. <i>P</i> < 0.0001 compared to wild type and revertant. (B) Micrographs of cells treated as in A. Note that <i>zrc1</i>Δ is highly defective for zincosome formation in response to 1 mM ZnSO₄ –a condition under which wild type, but not <i>zrc1</i>Δ cells can grow (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.s008" target="_blank">S7 Fig</a>).</p

Kinetics of zincosome formation in <i>C</i>. <i>albicans</i>.

<p>Cells were incubated overnight in YNB-zinc-dropout medium (SD0) to deplete zincosomes and pulsed with 25 μM ZnSO₄ for indicated time points. Cells were then stained with zinquin to probe for zincosomal zinc and the cell wall stained with Concanavalin A conjugated to Alexa-647. Left hand column shows false colour overlay of cell wall (cyan) and zincosomes (magenta). Right hand column shows DIC; Experiment performed three times and representative images shown.</p

Zinc uptake by <i>C</i>. <i>albicans</i> is mediated by Zrt1 and Zrt2.

<p>(<b>A</b>) Indicated strains were cultured in low zinc medium (SD0, pH ~4.7), exposed to 25 μM ZnSO₄ and zinc acquisition determined at indicated time points by measuring how much zinc remained in the cell free supernatant. <i>C</i>. <i>albicans</i> wild type acquires all measurable zinc within 60 minute; <i>zrt2</i>Δ does not; complementation restored zinc acquisition to 68%. Experiment performed three times (<b>B</b>) Indicated strains were incubated in RPMI without zinc for 24 h, exposed to 25 μM ZnSO₄ and zinc acquisition determined as in panel A. Wild type cells acquire 74% of zinc by three hours; uptake is reduced by approximately 50% in <i>zrt1</i>Δ and <i>zrt2</i>Δ. <i>zrt1</i>Δ/<i>zrt2</i>Δ fails to take up zinc. Experiment performed twice. Data points have been shifted to the right to make them visible amongst strains.</p

Zincosome formation is Zrc1 dependent.

<p>(A) Zincosome screen. Wild type, ZnT deletion mutants and <i>zrc1</i>Δ+<i>ZRC1</i> strains were pulsed with 25 μM zinc for 20 minutes and zincosome fluorescence determined by staining with zinquin. Prepulsed cells were also stained as control. Experiment was performed at least twice in duplicates and all data normalised to the post-pulse value of wild type. ANOVA was first performed on initial (pre-normalised data). Asterisks indicate statistical significance compared to wild type and to relevant deletion mutant ** P <0.01. (B) As panel A, except zinquin fluorescence kinetics was determined by flow cytometry. Experiment performed three times. <i>zrc1</i>Δ exhibits significantly reduced zinquin fluorescence compared to wild type and revertant at 20 minutes <i>P</i> < 0.001, ANOVA.</p