Transport proteins determine drug sensitivity and resistance in a protozoan parasite, Trypanosoma brucei

Drug resistance in pathogenic protozoa is very often caused by changes to the ‘transportome’ of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (Berenil®), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (MPXR) is the result of loss of a separate High Affinity Pentamidine Transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the “selectivity region” of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability

r-Java 2.0: the nuclear physics

[Aims:] We present r-Java 2.0, a nucleosynthesis code for open use that performs r-process calculations as well as a suite of other analysis tools. [Methods:] Equipped with a straightforward graphical user interface, r-Java 2.0 is capable of; simulating nuclear statistical equilibrium (NSE), calculating r-process abundances for a wide range of input parameters and astrophysical environments, computing the mass fragmentation from neutron-induced fission as well as the study of individual nucleosynthesis processes. [Results:] In this paper we discuss enhancements made to this version of r-Java, paramount of which is the ability to solve the full reaction network. The sophisticated fission methodology incorporated into r-Java 2.0 which includes three fission channels (beta-delayed, neutron-induced and spontaneous fission) as well as computation of the mass fragmentation is compared to the upper limit on mass fission approximation. The effects of including beta-delayed neutron emission on r-process yield is studied. The role of coulomb interactions in NSE abundances is shown to be significant, supporting previous findings. A comparative analysis was undertaken during the development of r-Java 2.0 whereby we reproduced the results found in literature from three other r-process codes. This code is capable of simulating the physical environment of; the high-entropy wind around a proto-neutron star, the ejecta from a neutron star merger or the relativistic ejecta from a quark nova. As well the users of r-Java 2.0 are given the freedom to define a custom environment. This software provides an even platform for comparison of different proposed r-process sites and is available for download from the website of the Quark-Nova Project: http://quarknova.ucalgary.ca/Comment: 26 pages, 18 figures, 1 tabl

The Effects of Automation on Crew Coordination in the Corporate Cockpit

The implementation of automated systems in the cockpit has somewhat changed pilots\u27 working environment. The present study, which focus is on corporate aviation, looks at the influence automation has on crew coordination. A questionnaire including both Likert scale and open-ended questions was sent to 880 qualified pilots. A descriptive as well as an inferential analysis of the data provided by the Likert scale questions was conducted. Out of the five variables tested, the only one to generate any significant differences (p Overall, pilots pointed out the importance of verbal communication and cockpit resource management training, emphasizing the strong need for crew coordination. They also cited automation as contributing to some changes in the role of pilots and some improvements in situational awareness

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics

Trypanosoma brucei methylthioadenosine phosphorylase protects the parasite from the antitrypanosomal effect of deoxyadenosine

Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mM deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (kcat) and Km values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors

The drugs of sleeping sickness: their mechanisms of action and resistance, and a brief history

With the incidence of sleeping sickness in decline and genuine progress being made towards the WHO goal of eliminating sleeping sickness as a major public health concern, this is a good moment to evaluate the drugs that ‘got the job done’: their development, their limitations and the resistance that the parasites developed against them. This retrospective looks back on the remarkable story of chemotherapy against trypanosomiasis, a story that goes back to the very origins and conception of chemotherapy in the first years of the 20 century and is still not finished today

Drug resistance in protozoan parasites

As with all other anti-infectives (antibiotics, anti-viral drugs, and anthelminthics), the limited arsenal of anti-protozoal drugs is being depleted by a combination of two factors: increasing drug resistance and the failure to replace old and often shamefully inadequate drugs, including those compromised by (cross)-resistance, through the development of new anti-parasitics. Both factors are equally to blame: a leaking bathtub may have plenty of water if the tap is left open; if not, it will soon be empty. Here, I will reflect on the factors that contribute to the drug resistance emergency that is unfolding around us, specifically resistance in protozoan parasites