Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification

Role of Mesenchymal Stem Cells on Cornea Wound Healing Induced by Acute Alkali Burn

The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×106 MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68+ cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68+ cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68+ cells and the down-regulation of MIP-1α, TNF-α and VEGF

Crystal Structures of T. b. rhodesiense Adenosine Kinase Complexed with Inhibitor and Activator: Implications for Catalysis and Hyperactivation

Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) and its derivatives exhibit specific antitrypanosomal activity toward T. b. rhodesiense, the causative agent of the acute form of HAT. We found that compound 1 would target the parasite adenosine kinase (TbrAK), an important enzyme of the purine salvage pathway, by acting via hyperactivation of the enzyme. This represents a novel and hitherto unexplored strategy for the development of trypanocides. These findings prompted us to investigate the mechanism of action at the molecular level. The present study reports the first three-dimensional crystal structures of TbrAK in complex with the bisubstrate inhibitor AP5A, and in complex with the activator (compound 1). The subsequent structural analysis sheds light on substrate and activator binding, and gives insight into the possible mechanism leading to hyperactivation. Further structure-activity relationships in terms of TbrAK activation properties support the observed binding mode of compound 1 in the crystal structure and may open the field for subsequent optimization of this compound series

Post Eclosion Age Predicts the Prevalence of Midgut Trypanosome Infections in Glossina

The teneral phenomenon, as observed in Glossina sp., refers to the increased susceptibility of the fly to trypanosome infection when the first bloodmeal taken is trypanosome-infected. In recent years, the term teneral has gradually become synonymous with unfed, and thus fails to consider the age of the newly emerged fly at the time the first bloodmeal is taken. Furthermore, conflicting evidence exists of the effect of the age of the teneral fly post eclosion when it is given the infected first bloodmeal in determining the infection prevalence. This study demonstrates that it is not the feeding history of the fly but rather the age (hours after eclosion of the fly from the puparium) of the fly when it takes the first (infective) bloodmeal that determines the level of fly susceptibility to trypanosome infection. We examine this phenomenon in male and female flies from two distinct tsetse clades (Glossina morsitans morsitans and Glossina palpalis palpalis) infected with two salivarian trypanosome species, Trypanosoma (Trypanozoon) brucei brucei and Trypanosoma (Nannomonas) congolense using Fisher's exact test to examine differences in infection rates. Teneral tsetse aged less than 24 hours post-eclosion (h.p.e.) are twice as susceptible to trypanosome infection as flies aged 48 h.p.e. This trend is conserved across sex, vector clade and parasite species. The life cycle stage of the parasite fed to the fly (mammalian versus insect form trypanosomes) does not alter this age-related bias in infection. Reducing the numbers of parasites fed to 48 h.p.e., but not to 24 h.p.e. flies, increases teneral refractoriness. The importance of this phenomenon in disease biology in the field as well as the necessity of employing flies of consistent age in laboratory-based infection studies is discussed

Genetic Engineering of Trypanosoma (Dutonella) vivax and In Vitro Differentiation under Axenic Conditions

Trypanosoma vivax is one of the most common parasites responsible for animal trypanosomosis, and although this disease is widespread in Africa and Latin America, very few studies have been conducted on the parasite's biology. This is in part due to the fact that no reproducible experimental methods had been developed to maintain the different evolutive forms of this trypanosome under laboratory conditions. Appropriate protocols were developed in the 1990s for the axenic maintenance of three major animal Trypanosoma species: T. b. brucei, T. congolense and T. vivax. These pioneer studies rapidly led to the successful genetic manipulation of T. b. brucei and T. congolense. Advances were made in the understanding of these parasites' biology and virulence, and new drug targets were identified. By contrast, challenging in vitro conditions have been developed for T. vivax in the past, and this per se has contributed to defer both its genetic manipulation and subsequent gene function studies. Here we report on the optimization of non-infective T. vivax epimastigote axenic cultures and on the process of parasite in vitro differentiation into metacyclic infective forms. We have also constructed the first T. vivax specific expression vector that drives constitutive expression of the luciferase reporter gene. This vector was then used to establish and optimize epimastigote transfection. We then developed highly reproducible conditions that can be used to obtain and select stably transfected mutants that continue metacyclogenesis and are infectious in immunocompetent rodents

What Constitutes a Natural Fire Regime? Insight from the Ecology and Distribution of Coniferous Forest Birds in North America

Bird species that specialize in the use of burned forest conditions can provide insight into the prehistoric fire regimes associated with the forest types that they have occupied over evolutionary time. The nature of their adaptations reflects the specific post-fire conditions that occurred prior to the unnatural influence of humans after European settlement. Specifically, the post-fire conditions, nest site locations, and social systems of two species (Bachman\u27s sparrow [Aimophila aestivalis] and red-cockaded woodpecker [Picoides borealis]) suggest that, prehistorically, a frequent, low-severity fire regime characterized the southeastern pine system in which they evolved. In contrast, the patterns of distribution and abundance for several other bird species (black-backed woodpecker [Picoides arcticus], buff-breasted flycatcher [Empidonax fulvifrons], Lewis\u27 woodpecker [Melanerpes lewis], northern hawk owl [Surnia ulula], and Kirtland\u27s warbler [Dendroica kirtlandii]) suggest that severe fire has been an important component of the fire regimes with which they evolved. Patterns of habitat use by the latter species indicate that severe fires are important components not only of higher-elevation and high-latitude conifer forest types, which are known to be dominated by such fires, but also of mid-elevation and even low-elevation conifer forest types that are not normally assumed to have had high-severity fire as an integral part of their natural fire regimes. Because plant and animal adaptations can serve as reliable sources of information about what constitutes a natural fire regime, it might be wise to supplement traditional historical methods with careful consideration of information related to plant and animal adaptations when attempting to restore what are thought to be natural fire regimes