Using Flow Cytometry to Analyze Cryptococcus Infection of Macrophages.

Flow cytometry is a powerful analytical technique, which is increasingly being used to study the interaction between host cells and intracellular pathogens. Flow cytometry is capable of measuring a greater number of infected cells within a sample compared to alternative techniques such as fluorescence microscopy. This means that robust quantification of rare events during infection is possible. Our lab and others have developed flow cytometry methods to study interactions between host cells and intracellular pathogens, such as Cryptococcus neoformans, to quantify phagocytosis, intracellular replication, and non-lytic expulsion or "vomocytosis" from the phagosome. Herein we describe these methods and how they can be applied to the study of C. neoformans as well as other similar intracellular pathogens

Automated Analysis of Cryptococcal Macrophage Parasitism Using GFP-Tagged Cryptococci

The human fungal pathogens Cryptococcus neoformans and C. gattii cause life-threatening infections of the central nervous system. One of the major characteristics of cryptococcal disease is the ability of the pathogen to parasitise upon phagocytic immune effector cells, a phenomenon that correlates strongly with virulence in rodent models of infection. Despite the importance of phagocyte/Cryptococcus interactions to disease progression, current methods for assaying virulence in the acrophage system are both time consuming and low throughput. Here, we introduce the first stable and fully characterised GFP–expressing derivatives of two widely used cryptococcal strains: C. neoformans serotype A type strain H99 and C. gattii serotype B type strain R265. Both strains show unaltered responses to environmental and host stress conditions and no deficiency in virulence in the macrophage model system. In addition, we report the development of a method to effectively and rapidly investigate macrophage parasitism by flow cytometry, a technique that preserves the accuracy of current approaches but offers a four-fold improvement in speed

Laser Time-of-Flight Mass Spectrometry for Future In Situ Planetary Missions

Laser desorption/ionization time-of-flight mass spectrometry (LD-TOF-MS) is a versatile, low-complexity instrument class that holds significant promise for future landed in situ planetary missions that emphasize compositional analysis of surface materials. Here we describe a 5kg-class instrument that is capable of detecting and analyzing a variety of analytes directly from rock or ice samples. Through laboratory studies of a suite of representative samples, we show that detection and analysis of key mineral composition, small organics, and particularly, higher molecular weight organics are well suited to this instrument design. A mass range exceeding 100,000 Da has recently been demonstrated. We describe recent efforts in instrument prototype development and future directions that will enhance our analytical capabilities targeting organic mixtures on primitive and icy bodies. We present results on a series of standards, simulated mixtures, and meteoritic samples

"A little theatrical but mostly athletic": The mutable erotics of Miranda July's The First Bad Man

By attending to the inherent flux of sexual fantasy, Miranda July’s first novel The First Bad Man reveals a mobile and mutable erotics capable of generating an enlarged range of self-identification and relational intimacy, far from any essentialist assumptions of stable or coherent sexual identity. July focuses specifically upon role-play as the means to unpack the normative categories of hetero/homosexuality, masculinity and femininity, celebrating rather than pathologising qualities like superficiality and inconsistency. The novel touches upon many polarising issues (for example, sexual violence, sadomasochism and assisted reproductive technology), deftly avoiding the conventional language which colours perception. Both erotic and humorous, The First Bad Man helps to redefine the often highly charged discourse around sex and sexuality

A Sensitive High-Throughput Assay for Evaluating Host-Pathogen Interactions in Cryptococcus neoformans Infection

Background: Cryptococcus neoformans causes serious disease in immunocompromised individuals, leading to over 600,000 deaths per year worldwide. Part of this impact is due to the organism’s ability to thwart what should be the mammalian hosts ’ first line of defense against cryptococcal infection: internalization by macrophages. Even when C. neoformans is engulfed by host phagocytes, it can survive and replicate within them rather than being destroyed; this ability is central in cryptococcal virulence. It is therefore critical to elucidate the interactions of this facultative intracellular pathogen with phagocytic cells of its mammalian host. Methodology/Principal Findings: To accurately assess initial interactions between human phagocytic cells and fungi, we have developed a method using high-throughput microscopy to efficiently distinguish adherent and engulfed cryptococci and quantitate each population. This method offers significant advantages over currently available means of assaying hostfungal cell interactions, and remains statistically robust when implemented in an automated fashion appropriate for screening. It was used to demonstrate the sensitivity of human phagocytes to subtle changes in the cryptococcal capsule, a major virulence factor of this pathogen. Conclusions/Significance: Our high-throughput method for characterizing interactions between C. neoformans and mammalian phagocytic cells offers a powerful tool for elucidating the relationship between these cell types durin

Division of labour in response to host oxidative burst drives a fatal Cryptococcus gattii outbreak

Cryptococcus gattii is an emerging intracellular pathogen and the cause of the largest primary outbreak of a life-threatening fungal disease in a healthy population. Outbreak strains share a unique mitochondrial gene expression profile and an increased ability to tubularize their mitochondria within host macrophages. However, the underlying mechanism that causes this lineage of C. gattii to be virulent in immunocompetent individuals remains unexplained. Here we show that a subpopulation of intracellular C. gattii adopts a tubular mitochondrial morphology in response to host reactive oxygen species. These fungal cells then facilitate the rapid growth of neighbouring C. gattii cells with non-tubular mitochondria, allowing for effective establishment of the pathogen within a macrophage intracellular niche. Thus, host reactive oxygen species, an essential component of the innate immune response, act as major signalling molecules to trigger a ‘division of labour’ in the intracellular fungal population, leading to increased pathogenesis within this outbreak lineage