Deorphanization of biogenic amine-responsive G protein-coupled receptors in various model protozoa and platyhelminthes

This dissertation explores the relevance of G protein-coupled receptors in disease causing protozoans and free living flatworms. It explored the biological importance of a catecholamine responsive GPCR in Tetrahymena and the physiological importance of serotonergic receptors in the locomotory events of two species of flatworms; Girardia tigrina and Schmidtea mediterranea. During the preliminary studies, a protozoan GPCR was obtained by genome mining, cloned and heterologously expressed in a yeast expression system, subjected to agonist treatment, and hence, de-orphanized. The serotonin GPCRs in both Schmidtea mediterranea and Girardia tigrina were cloned into Gateway vector with which double stranded RNA production was induced in an RNAse III deficient bacterial strain for RNAi studies. We demonstrated the biological relevance of a novel Tetrahymena receptor (TetEPI-1) as related to bacterial engulfment. Further, in the presence of serotonin, TetEPI-1 was shown to have been stabilized in inactive state, giving the course to suspect serotonin as its potential inverse agonist or antagonist. TetEPI-1 is a potential therapeutic target for selective manipulation of related pathogenic protozoa, especially considering the inverse agonism is the paradigm for antihistamine drugs that stabilize H1 receptors in the absence of histamine during chronic allergic responses of atopy. By means of alternative loss-of-function technique, this dissertation also described the successful characterization of two novel serotonergic GPCR mediating locomotory events in two species of flatworms, Girardia tigrina (Dtig.ser-85) and Schmidtea mediterranea (smed-ser85, smed-ser39). Serotonin, by virtue of its dual effects, depending on its targets, demonstrated both cilio-excitatory and moderate cilio-inhibitory effect in these organisms. In the process of characterizing these receptors, this work established flatworm species differences in their response to serotonin. The suppression of these GPCRs demonstrated significant impairment in the motility of S. mediterranea while a decrease in cAMP also lessens the moderate inhibitory effects of serotonin on G. tigrina locomotion, a clear indication of the relevance of these receptors to these worms. These putative GPCRs compare with characterized 5HT4 receptors, hence, could be classified as planarian 5HT4 GPCRs, namely Dtig-ser85, Smed-ser85 and Smed-ser39. The homologs of these GPCRs will be pursued in the human parasite Schistosoma mansoni

Evidence for a Bacterial Lipopolysaccharide-Recognizing G-ProteinCoupled Receptor in the Bacterial Engulfment by Entamoeba histolytica

Entamoeba histolytica is the causative agent of amoebic dysentery, a worldwide protozoal disease that results in approximately 100,000 deaths annually. The virulence of E. histolytica may be due to interactions with the host bacterial flora, whereby trophozoites engulf colonic bacteria as a nutrient source. The engulfment process depends on trophozoite recognition of bacterial epitopes that activate phagocytosis pathways. E. histolytica GPCR-1 (EhGPCR-1) was previously recognized as a putative G-protein-coupled receptor (GPCR) used by Entamoeba histolytica during phagocytosis. In the present study, we attempted to characterize EhGPCR-1 by using heterologous GPCR expression in Saccharomyces cerevisiae. We discovered that bacterial lipopolysaccharide (LPS) is an activator of EhGPCR-1 and that LPS stimulates EhGPCR-1 in a concentration-dependent manner. Additionally, we demonstrated that Entamoeba histolytica prefers to engulf bacteria with intact LPS and that this engulfment process is sensitive to suramin, which prevents the interactions of GPCRs and G-proteins. Thus, EhGPCR-1 is an LPS-recognizing GPCR that is a potential drug target for treatment of amoebiasis, especially considering the well-established drug targeting to GPCRs

Effects of intravenous administration of polymyxin B in neonatal foals with experimental endotoxemia

Objective—To evaluate the effect of IV administration of polymyxin B on clinical and serum biochemical variables in foals with experimental endotoxemia. Design—Prospective experimental study. Animals—14 healthy neonatal foals. Procedures—Foals were randomly assigned to a treatment or control group and were administered a single dose of lipopolysaccharide (0.5 μg/kg [0.23 μg/lb]) IV over 30 minutes. The treatment group received polymyxin B (6,000 U/kg [2,727 U/lb], IV) immediately after completion of lipopolysaccharide infusion; the control group was administered an equal volume of saline (0.9% NaCl) solution. Subsequent doses of polymyxin B or saline solution were administered IV at 8 and 16 hours. Blood was collected at various time points, and outcome variables, including heart rate, respiratory rate, rectal temperature, attitude score, WBC count, neutrophil count, lymphocyte count, monocyte count, platelet count, Hct, blood lactate concentration, blood glucose concentration, serum tumor necrosis factor-α concentration, and plasma thromboxane B2 concentration, were measured. Urine was collected prior to and after experimentation to determine whether nephrotoxicosis was associated with treatment. Results—The treatment group had significantly lower blood lactate concentration and serum tumor necrosis factor-α and plasma thromboxane B2 concentrations and had higher blood glucose concentrations and better attitude scores, compared with the control group, at various time points during the study. No other significant differences and no evidence of overt nephrotoxicosis were detected. Conclusions and Clinical Relevance—Administration of polymyxin B IV in healthy neonatal foals challenged with lipopolysaccharide attenuated some clinical and serum biochemical derangements associated with endotoxemia

Novel RNAi-Mediated Approach to G Protein-Coupled Receptor Deorphanization: Proof of Principle and Characterization of a Planarian 5-HT Receptor

G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gαi/o. The method was then extended to deorphanize a novel Gαs-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla

Functional analysis of Girardia tigrina transcriptome seeds pipeline for anthelmintic target discovery

Background Neglected diseases caused by helminth infections impose a massive hindrance to progress in the developing world. While basic research on parasitic flatworms (platyhelminths) continues to expand, researchers have yet to broadly adopt a free-living model to complement the study of these important parasites. Methods We report the high-coverage sequencing (RNA-Seq) and assembly of the transcriptome of the planarian Girardia tigrina across a set of dynamic conditions. The assembly was annotated and extensive orthology analysis was used to seed a pipeline for the rational prioritization and validation of putative anthelmintic targets. A small number of targets conserved between parasitic and free-living flatworms were comparatively interrogated. Results 240 million paired-end reads were assembled de novo to produce a strictly filtered predicted proteome consisting of over 22,000 proteins. Gene Ontology annotations were extended to 16,467 proteins. 2,693 sequences were identified in orthology groups spanning flukes, tapeworms and planaria, with 441 highlighted as belonging to druggable protein families. Chemical inhibitors were used on three targets in pharmacological screens using both planaria and schistosomula, revealing distinct motility phenotypes that were shown to correlate with planarian RNAi phenotypes. Conclusions This work provides the first comprehensive and annotated sequence resource for the model planarian G. tigrina, alongside a prioritized list of candidate drug targets conserved among parasitic and free-living flatworms. As proof of principle, we show that a simple RNAi and pharmacology pipeline in the more convenient planarian model system can inform parasite biology and serve as an efficient screening tool for the identification of lucrative anthelmintic targets

Release of Small RNA-containing Exosome-like Vesicles from the Human Filarial Parasite Brugia malayi

Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30–120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers

Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs) as transducers of Salmonella engulfment by the model protozoanTetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena) engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates byTetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymenawas assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule

Gendered analysis of ICT-enabled agricultural extension and advisory services : the case of Agro-Tech Smart Ex Model

This work was carried out with the aid of a grant from Canada’s International Development Research Centre (IDRC), and with financial support from the Government of Canada, provided through Global Affairs Canada (GAC)This paper deals with strategies to overcome barriers to agricultural extension and advisory services for women in agriculture. In targeting extension services towards women in the agricultural value chain, specific challenges cited in the study surveys are: small farm sizes due to lack of access to land; limited access to credit, input supply, and assets; and inadequate access to tractor services. Men have main access to silos and warehouses for grain storage. Other barriers are lack of access to radio and smart phones, lack of farmer organizations for women, and heavy workloads relating to farm work, care giving, and household chores

Digital platforms for agro-advisory and business service delivery : lessons from scaling-up of AgroTech in Ghana

This work was carried out with the aid of a grant from Canada’s International Development Research Centre (IDRC), and with financial support from the Government of Canada, provided through Global Affairs Canada (GAC)The aim is to test the scale up of AgroTech (an ICT platform) in the delivery of agricultural extension and agribusiness advisory services to smallholders through private sector value chain players. AgroTech combines interactive radio broadcast and customized field coaching, bringing together information, inputs and markets through private businesses. The goal of the project is to contribute to policy reform of the agricultural extension system with the view of improving responsiveness, accountability and operational sustainability

Appraisal of the Agro-Tech Smart Extension Model in Ghana : payment options and challenges in ICT-enabled extension services delivery

This work was carried out with the aid of a grant from Canada’s International Development Research Centre (IDRC), and with financial support from the Government of Canada, provided through Global Affairs Canada (GAC)The paper presents an overview of agricultural extension services in terms of providers, financing options and advisory services delivery. Farmer demand is high for ICT- enabled extension services delivery. The Grameen Foundation (GFUSA) and Farm Radio International have developed a mobile software application (AgroTech SmartEx) for smallholder farmer management purposes and to support better decision-making towards increased productivity. The AgroTech Model combines ICT-enabled extension and advisory services with direct to farmer interactive radio. Survey data from 400 farmers indicates a high performance rating for the AgroTech Smart Ex Model, where over 70% of respondents consider it to be excellent