The Plasmodium falciparum, Nima-related kinase Pfnek-4: a marker for asexual parasites committed to sexual differentiation

<b>Background</b> Malaria parasites undergo, in the vertebrate host, a developmental switch from asexual replication to sexual differentiation leading to the formation of gametocytes, the only form able to survive in the mosquito vector. Regulation of the onset of the sexual phase remains largely unknown and represents an important gap in the understanding of the parasite's complex biology. <b>Methods:</b> The expression and function of the Nima-related kinase Pfnek-4 during the early sexual development of the human malaria parasite Plasmodium falciparum were investigated, using three types of transgenic Plasmodium falciparum 3D7 lines: (i) episomally expressing a Pfnek-4-GFP fusion protein under the control of its cognate pfnek-4 promoter; (ii) episomally expressing negative or positive selectable markers, yeast cytosine deaminase-uridyl phosphoribosyl transferase, or human dihydrofolate reductase, under the control of the pfnek-4 promoter; and (iii) lacking a functional pfnek-4 gene. Parasite transfectants were analysed by fluorescence microscopy and flow cytometry. In vitro growth rate and gametocyte formation were determined by Giemsa-stained blood smears. <b>Results:</b> The Pfnek-4-GFP protein was found to be expressed in stage II to V gametocytes and, unexpectedly, in a subset of asexual-stage parasites undergoing schizogony. Culture conditions stimulating gametocyte formation resulted in significant increase of this schizont subpopulation. Moreover, sorted asexual parasites expressing the Pfnek-4-GFP protein displayed elevated gametocyte formation when returned to in vitro culture in presence of fresh red blood cells, when compared to GFP- parasites from the same initial population. Negative selection of asexual parasites expressing pfnek-4 showed a marginal reduction in growth rate, whereas positive selection caused a marked reduction in parasitaemia, but was not sufficient to completely abolish proliferation. Pfnek-4- clones are not affected in their asexual growth and produced normal numbers of stage V gametocytes. <b>Conclusions:</b> The results indicate that Pfnek-4 is not strictly gametocyte-specific, and is expressed in a small subset of asexual parasites displaying high rate conversion to sexual development. Pfnek-4 is not required for erythrocytic schizogony and gametocytogenesis. This is the first study to report the use of a molecular marker for the sorting of sexually-committed schizont stage P. falciparum parasites, which opens the way to molecular characterization of this pre-differentiated subpopulation

Ecological influences on the behaviour and fertility of malaria parasites

BACKGROUND: Sexual reproduction in the mosquito is essential for the transmission of malaria parasites and a major target for transmission-blocking interventions. Male gametes need to locate and fertilize females in the challenging environment of the mosquito blood meal, but remarkably little is known about the ecology and behaviour of male gametes. METHODS: Here, a series of experiments explores how some aspects of the chemical and physical environment experienced during mating impacts upon the production, motility, and fertility of male gametes. RESULTS AND CONCLUSIONS: Specifically, the data confirm that: (a) rates of male gametogenesis vary when induced by the family of compounds (tryptophan metabolites) thought to trigger gamete differentiation in nature; and (b) complex relationships between gametogenesis and mating success exist across parasite species. In addition, the data reveal that (c) microparticles of the same size as red blood cells negatively affect mating success; and (d) instead of swimming in random directions, male gametes may be attracted by female gametes. Understanding the mating ecology of malaria parasites, may offer novel approaches for blocking transmission and explain adaptation to different species of mosquito vectors

Information use and plasticity in the reproductive decisions of malaria parasites

BACKGROUND: Investment in the production of transmissible stages (gametocytes) and their sex ratio are malaria parasite traits that underpin mosquito infectivity and are therefore central to epidemiology. Malaria parasites adjust their levels of investment into gametocytes and sex ratio in response to changes in the in-host environment (including red blood cell resource availability, host immune responses, competition from con-specific genotypes in mixed infections, and drug treatment). This plasticity appears to be adaptive (strategic) because parasites prioritize investment (in sexual versus asexual stages and male versus female stages) in manners predicted to maximize fitness. However, the information, or ‘cues’ that parasites use to detect environmental changes and make appropriate decisions about investment into gametocytes and their sex ratio are unknown. METHODS: Single genotype Plasmodium chabaudi infections were exposed to ‘cue’ treatments consisting of intact or lysed uninfected red blood cells, lysed parasitized RBCs of the same clone or an unrelated clone, and an unmanipulated control. Infection dynamics (proportion of reticulocytes, red blood cell and asexual stage parasite densities) were monitored, and changes in gametocyte investment and sex ratio in response to cue treatments, applied either pre- or post-peak of infection were examined. RESULTS AND CONCLUSIONS: A significant reduction in gametocyte density was observed in response to the presence of lysed parasite material and a borderline significant increase in sex ratio (proportion of male gametocytes) upon exposure to lysed red blood cells (both uninfected and infected) was observed. Furthermore, the changes in gametocyte density and sex ratio in response to these cues depend on the age of infection. Demonstrating that variation in gametocyte investment and sex ratio observed during infections are a result of parasite strategies (rather than the footprint of host physiology), provides a foundation to investigate the fitness consequences of plasticity and explore whether drugs could be developed to trick parasites into making suboptimal decisions

Transcriptomic and Proteomic Analysis of the Epicardial Adipose Tissue

The study of epicardial adipose tissue (EAT) has been limited by its accessibility due to its proximity to the heart. Moreover, many common animal models do not have EAT, leaving its functional role underestimated and poorly elucidated. Recent advances in medicine and science have allowed for better studies that provide a more comprehensive understanding of its physiological role. One way to dissect its function is the study of its gene expression. In this chapter, we summarize transcriptomic and proteomic analyses which show that EAT expresses a unique set of genes setting it apart from other adipose tissues in the body. This distinctive set of genes modulates a feedback mechanism that has direct interaction with the myocardium. The EAT shares its blood supply with the coronary arteries and innervation with the cardiac muscle, provides physical protection, and regulates energetic metabolites needed by the myocardium. Transcriptomic and proteomic studies show that it is a local source of adipokines with paracrine influence on the myocardium due to the intimate microcirculation shared by both tissues. These analyses also show that it has a role in the immune and endocrine systems affecting the rest of the body. Furthermore, regulation of EAT gene expression is not monolithic and can be affected by multiple factors such as sex, age, underling disease, medication, etc. Gene expression studies can therefore provide great insight into the function of EAT and its role in health and disease

Optical Coherence Tomography (OCT): Imaging the Visual Pathway as a Model for Neurodegeneration

Axonal and neuronal degeneration are important features of multiple sclerosis (MS) and other neurologic disorders that affect the anterior visual pathway. Optical coherence tomography (OCT) is a non-invasive technique that allows imaging of the retinal nerve fiber layer (RNFL), a structure which is principally composed of ganglion cell axons that form the optic nerves, chiasm, and optic tracts. Since retinal axons are nonmyelinated until they penetrate the lamina cribrosa, the RNFL is an ideal structure (no other central nervous system tract has this unique arrangement) for visualizing the processes of neurodegeneration, neuroprotection and, potentially, even neuro-repair. OCT is capable of providing high-resolution reconstructions of retinal anatomy in a rapid and reproducible fashion and permits objective analysis of the RNFL (axons) as well as ganglion cells and other neurons in the macula. In a systematic OCT examination of multiple sclerosis (MS) patients, RNFL thickness and macular volumes are reduced when compared to disease-free controls. Conspicuously, these changes, which signify disorganization of retinal structural architecture, occur over time even in the absence of a history of acute demyelinating optic neuritis. RNFL axonal loss in MS is most severe in those eyes with a corresponding reduction in low-contrast letter acuity (a sensitive vision test involving the perception of gray letters on a white background) and in those patients who exhibit the greatest magnitude of brain atrophy, as measured by validated magnetic resonance imaging techniques. These unique structure–function correlations make the anterior visual pathway an ideal model for investigating the effects of standard and novel therapies that target axonal and neuronal degeneration. We provide an overview of the physics of OCT, its unique properties as a non-invasive imaging technique, and its potential applications toward understanding mechanisms of brain tissue injury in MS, other optic neuropathies, and neurologic disorders