<i>P. berghei</i> telomerase subunit TERT is essential for parasite survival

Telomeres define the ends of chromosomes protecting eukaryotic cells from chromosome instability and eventual cell death. The complex regulation of telomeres involves various proteins including telomerase, which is a specialized ribonucleoprotein responsible for telomere maintenance. Telomeres of chromosomes of malaria parasites are kept at a constant length during blood stage proliferation. The 7-bp telomere repeat sequence is universal across different Plasmodium species (GGGTTT/CA), though the average telomere length varies. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is present in all sequenced Plasmodium species and is approximately three times larger than other eukaryotic TERTs. The Plasmodium RNA component of TERT has recently been identified in silico. A strategy to delete the gene encoding TERT via double cross-over (DXO) homologous recombination was undertaken to study the telomerase function in P. berghei. Expression of both TERT and the RNA component (TR) in P. berghei blood stages was analysed by Western blotting and Northern analysis. Average telomere length was measured in several Plasmodium species using Telomere Restriction Fragment (TRF) analysis. TERT and TR were detected in blood stages and an average telomere length of ~950 bp established. Deletion of the tert gene was performed using standard transfection methodologies and we show the presence of tert− mutants in the transfected parasite populations. Cloning of tert- mutants has been attempted multiple times without success. Thorough analysis of the transfected parasite populations and the parasite obtained from extensive parasite cloning from these populations provide evidence for a so called delayed death phenotype as observed in different organisms lacking TERT. The findings indicate that TERT is essential for P. berghei cell survival. The study extends our current knowledge on telomere biology in malaria parasites and validates further investigations to identify telomerase inhibitors to induce parasite cell death

A calorimetric investigation of doxorubicin-polymer bead interactions

Isothermal titration calorimetry (ITC) was utilised to investigate suitability of the technique to determine the stoichiometry and thermodynamics of the interactions that occur between a commonly used chemotherapeutic drug, namely doxorubicin, and a polymer bead-based drug delivery embolisation system (DC Bead™). Six temperatures were selected for drug-polymer titrations (293 to 313 K) and in all cases an initially exothermic signal reverted to an endothermic response upon the saturation of the beads with drug. From these experiments, and subsequent calculations, the molar ratio of drug to SO3- (polymer) was found to be 0.4:1 at all temperatures studied. Enthalpic data was calculated from the raw ITC data with an average enthalpy of drug-polymer binding of – 14.8 kJmol-1 at 293 K through to – 19.4 kJmol-1 at 313 K implying the process is enthalpically-driven yet only affected by an increase in experimental temperature to a limited extent whereby an increase in experimental temperature results in a small increase in the negativity in change in enthalpy recorded. The application of ITC in this study (with its unique ability to monitor real-time interactions and facilitate stoichiometric calculations) resolves the lack of knowledge regarding the thermodynamics of this specific drug-polymer interaction. This study confirms that ITC is not only useful for this specific system, but also highlights the potential use of ITC for more general studies in this area

Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites

Are variations in rates of attending cultural activities associated with population health in the United States?

<p>Abstract</p> <p>Background</p> <p>Population studies conducted in Sweden have revealed an association between attendance at cultural activities and health. Using data from US residents, we examined whether the association could be observed in the US.</p> <p>Methods</p> <p>Participants in the current study included 1,244 individuals who participated in the 1998 General Social Survey.</p> <p>Results</p> <p>A significant association between cultural activities and self-reported health (SRH) was observed, even after controlling for age, gender, marital status, race, number of children, subjective social class, employment status, household income, and educational attainment. Specifically, the more cultural activities people reported attending, the better was their SRH.</p> <p>Conclusion</p> <p>The data confirm that an association between cultural activity and health is present in a US sample. The data do not mean that the association is causal, but they suggest that further longitudinal research is warranted.</p

A unique Kelch domain phosphatase in Plasmodium regulates ookinete morphology, motility and invasion

Signalling through post-translational modification (PTM) of proteins is a process central to cell homeostasis, development and responses to external stimuli. The best characterised PTM is protein phosphorylation which is reversibly catalysed at specific residues through the action of protein kinases (addition) and phosphatases (removal). Here, we report characterisation of an orphan protein phosphatase that possesses a domain architecture previously only described in Plantae. Through gene disruption and the production of active site mutants, the enzymatically active Protein Phosphatase containing Kelch-Like domains (PPKL, PBANKA_132950) is shown to play an essential role in the development of an infectious ookinete. PPKL is produced in schizonts and female gametocytes, is maternally inherited where its absence leads to the development of a malformed, immotile, non-infectious ookinete with an extended apical protrusion. The distribution of PPKL includes focussed localization at the ookinete apical tip implying a link between its activity and the correct deployment of the apical complex and microtubule cytoskeleton. Unlike wild type parasites, ppkl(-) ookinetes do not have a pronounced apical distribution of their micronemes yet secretion of microneme cargo is unaffected in the mutant implying that release of microneme cargo is either highly efficient at the malformed apical prominence or secretion may also occur from other points of the parasite, possibly the pellicular pores

Development and application of a positive–negative selectable marker system for use in reverse genetics in Plasmodium

A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems

Phenylcyanamidocopper(I) and Silver(I) Complexes: Synthetic and Structural Studies

Phenylcyanamidocopper(I) and silver(I) complexes of the type, [{M(PPh3)2L}2] (M = Cu, L = 4-NO2pcyd or 4-Me2Npcyd; M = Ag, L = 4-Me2Npcyd), [Cu(PPh3)3L] (L = pcyd or 4-NO2-pcyd), [Ag-(PPh3)3L] (L = pcyd, 2-Clpcyd, 4-Clpcyd, 4-Brpcyd, 4-MeOpcyd, 4-NO2pcyd or 4-Me2Npcyd), [Ag(Me2phen)(2-Clpcyd)] (Me2phen = 2,9-dimethyl-1,10-phenanthroline) and [Ag(dppm)(4-Brpcyd)] (dppm = bis(diphenylphosphino)methane) have been synthesised and characterised and the crystal structures of four of the complexes determined. For both [{Cu(PPh3)2(4-Me2Npcyd)}2] ⋅ CH2Cl2 and [{Ag(PPh3)2(4-Me2Npcyd)}2], the cyanamide ligands bridge the metal atoms in a &mu;-1,3-fashion through the cyano and amido nitrogens. Each metal atom has a distorted tetrahedral geometry, being bound to two triphenylphosphine phosphorus atoms and two nitro-gen atoms from 4-Me2Npcyd ligands to give a \u27P2N2\u27 coordination sphere. In the case of the Cu complex the dimer is centrosymmetric but for the Ag complex the metal atoms are not equivalent. The complexes, [Ag(PPh3)3(4-Brpcyd)] and [Ag(PPh3)3(4-Me-Opcyd)], are discrete monomers, in which each of the Ag atoms adopts a distorted tetrahedral geometry, being bound to three triphenylphosphine phosphorus atoms and one phenylcyanamide ligand binding in a terminal fashion through the cyano nitrogen

Zygote morphogenesis but not the establishment of cell polarity in plasmodium berghei Is controlled by the small GTPase, RAB11A

Plasmodium species are apicomplexan parasites whose zoites are polarized cells with a marked apical organisation where the organelles associated with host cell invasion and colonization reside. Plasmodium gametes mate in the mosquito midgut to form the spherical and presumed apolar zygote that morphs during the following 24 hours into a polarized, elongated and motile zoite form, the ookinete. Endocytosis-mediated protein transport is generally necessary for the establishment and maintenance of polarity in epithelial cells and neurons, and the small GTPase RAB11A is an important regulator of protein transport via recycling endosomes. PbRAB11A is essential in blood stage asexual of Plasmodium. Therefore, a promoter swap strategy was employed to down-regulate PbRAB11A expression in gametocytes and zygotes of the rodent malaria parasite, Plasmodium berghei which demonstrated the essential role of RAB11A in ookinete development. The approach revealed that lack of PbRAB11A had no effect on gamete production and fertility rates however, the zygote to ookinete transition was almost totally inhibited and transmission through the mosquito was prevented. Lack of PbRAB11A did not prevent meiosis and mitosis, nor the establishment of polarity as indicated by the correct formation and positioning of the Inner Membrane Complex (IMC) and apical complex. However, morphological maturation was prevented and parasites remained spherical and immotile and furthermore, they were impaired in the secretion and distribution of microneme cargo. The data are consistent with the previously proposed model of RAB11A endosome mediated delivery of plasma membrane in Toxoplasma gondii if not its role in IMC formation and implicate it in microneme function