One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a highaffinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membranepermeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development

Background: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite. Methodology/Principal Findings: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 29deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-52/ 2 trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line. Conclusions/Significance: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the py

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes.

The aim was to build an educational website to teach preschoolers aged 2-5 years old some skills; related to reading, writing and pronouncing alphabet, numbers, animal names, shapes and other common words. Also, assist parents and teachers by providing digital game-based learning environment to “edutainment” the children. The study illustrated the usability and usefulness of this educational game-based website and provided future insights for researchers and developers to take into account in future work.

The classic problem for a logistically evolving single species population being harvested involves three parameters: rate constant, carrying capacity and harvesting rate, which are taken to be positive constants. However, in real world situations, these parameters may vary with time. This paper considers the situation where these vary on a time scale much longer than that intrinsic to the population evolution itself. Application of a multiple time scale approach gives approximate explicit closed form expressions for the changing population, that compare favorably with those generated from numerical solutions.

This paper proposes an estimation method based on the reflection of the (manifest) explanatory variable to estimate the parameters of a simple linear regression model when both response and explanatory variables are subject to measurement error (ME). The reflection method (RM) uses all observed data points, and does not exclude or ignore part of the data or replace them by their ranks. The RM is straightforward, and easy to implement. We show that the RM is equivalent or asymptotically equivalent to the orthogonal regression (OR) method. Simulation studies show that the RM produces estimators that are nearly asymptotically unbiased and efficient under the assumption that the ratio of the error variances equals one. Moreover, it allows to define the sum of squares of errors uniquely, the same way as in the case of no measurement error. Simulation based numerical comparisons of the RM with the ordinary least square (OLS) and OR methods are also included.

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1–10 μM.

Few studies have systematically addressed evolutionary changes in olfactory neuron assemblies, either by genetic drift or as an

adaptation to specific odor environments. We have studied the sense of olfaction in 2 congeneric scarab beetles, Pachnoda

interrupta Olivier and Pachnoda marginata Drury (Coleoptera: Scarabaeidae: Cetoniinae), which are both opportunistic

polyphages, feeding mainly on fruit and flowers. The 2 species occur in dissimilar habitats: P. interrupta is found in dry

savannah, and P. marginata in tropical parts of equatorial Africa. To study how these species may have adapted their sense of

olfaction to their odor environments, we utilized single-unit electrophysiology on olfactory sensilla with a wide selection of

food-related compounds. Despite the differences in habitat, we found that the species shared most of the physiological types

of olfactory receptor neurons (ORNs) encountered, although their proportions frequently varied between the species. The high

degree of conservation in olfaction between the species implies that a similar sensory strategy is efficient for food search in

both habitats. However, shifts in proportions of receptor neuron classes, and slight shifts in response profiles and/or presence

of some ORN classes unique to either species, may reflect adaptation to a different set of hosts.

Key words: comparative study, electrophysiology, olfaction, polyphagous herbivore, single sensillum recordings

Few studies have addressed how olfactory systems may be adapted to different odour environments. I have performed the largest study to date, comparing olfactory receptor neurons in the two congeneric species of scarab beetle, P. marginata and P. interrupta. Both species are fruit- and flower-eaters but geographically separated (equatorial versus sub-Saharan Africa, resp.). They have similar lamellate antennae covered mostly with numerous olfactory sensilla placodea (plate sensilla) and a lesser number of other types, mainly sensilla coeloconica (grooved peg), and smooth peg sensilla of unknown function. By means of single sensillum recordings with tungsten microelectrodes I screened a great number of olfactory sensilla with a large array of odorants. I compared 456 sensilla placodea, containing 212 responding cells, over the whole antennae in both species. The olfactory systems of these two species displayed an amazing degree of conservation, with 20 identified olfactory neuron classes, all except two of which were found in both species with no detectable difference in response profiles. The exceptions were two olfactory receptor neuron classes: methyl benzoate and gamma-nonalactone, which were only found in Pachnoda marginata. In general, the two species showed an almost total overlap in their receptor neuron assemblies. One aim of the study was also to test whether the arrangement of neurons within sensilla was conserved between the species, but this could not be tested as very few combinations of characterized neurons were encountered during the study.

Key words: olfaction, electrophysiology, scarab beetles, Pachnoda marginata, Pachnoda interrupta, single sensillum recording, olfactory receptor neurons