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

Interactive analysis of SDN-driven defence against Distributed Denial of Service attacks

The Secure Autonomous Response Networks (SARNET) framework introduces a mechanism to respond autonomously to security attacks in Software Defined Networks (SDN). Still the range of responses possible and their effectiveness need to be properly evaluated such that the decision making process and the self-learning capability of such systems are optimized. To this purpose we developed a touch-table driven interactive SARNET prototype, named VNET, and we demonstrated its use through real-time monitoring and control of real and virtualised networks. By observing users interacting with the system at SC15 in Austin, we concluded that in a SDN it is possible to achieve high effectiveness of responses by carefully choosing a relatively minor number of actions

An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro

BACKGROUND: The standard in vitro protocol currently in use for drug testing against Plasmodium falciparum, based on the incorporation of the purine [(3)H]-hypoxanthine, has two serious drawbacks. Firstly it is unsuitable for the testing of drugs that directly or indirectly impact on purine salvage or metabolism. Secondly, it relies on the use of expensive radiolabelled material, with added issues concerning detection, storage and waste disposal that make it unsuitable for use in many disease-endemic areas. Recently, the use of fluorochromes has been suggested as an alternative, but quenching of the fluorescence signal by the haemoglobin present in cultures of Plasmodium falciparum-infected erythrocytes severely limits the usefulness of this approach. METHODS: In order to resolve this problem, a new PicoGreen(®)-based procedure has been developed which incorporates additional steps to remove the interfering haemoglobin. The 50% inhibitory concentration (IC(50)) values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 were determined using the new protocol. RESULTS: The IC(50 )values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 determined with the new fluorescence-based protocol were statistically identical to those obtained using the traditional (3)H-hypoxanthine incorporation method, and consistent with literature values. CONCLUSION: The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen(® )method has the potential to replace traditional in vitro drug resistance assay techniques

Auditable secure network overlays for multi-domain distributed applications

The push for data sharing and data processing across organisational boundaries creates challenges at many levels of the software stack. Data sharing and processing rely on the participating parties agreeing on the permissible operations and expressing them into actionable contracts and policies. Converting these contracts and policies into a operational infrastructure is still a matter of research and therefore begs the question how should a digital data market place infrastructure look like? In this paper we investigate how communication fabric and applications can be tightly coupled into a multi-domain overlay network which enforces accountability. We prove our concepts with a prototype which shows how a simple workflow can run across organisational boundaries

Developing Well Adapted Early to Midseason Cultivars of Trifoiloum subterraneum ssp. Brachycalycinum

Development and selection of early to midseason cultivars of subterranean clover (Trifolium subterraneum L.) ssp. brachycalycinum has mostly involved the use of early generation selection nursery swards (F2-progeny method). Four sites were sown in the mid-North region of South Australia. At the end of 1995, the top 20 lines from each site had burrs (seed pods) sampled at random. The burr will proceed into the final early generation selection phase (1 - 2 years). Dependent on the site, many lines have out-performed the standard checkplot cultivar, Clare. High hardseed levels have also been incorporated into 1989 crossbreds

Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters - the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol(107 cells)-1s-1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol(107 cells)-1 s-1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family

Preferred Islet Delivery Device Characteristics and Implantation Strategies of Patients With Type 1 Diabetes

Islet delivery devices (IDDs) offer potential benefits for islet transplantation and stem cell-based replacement in type 1 diabetes. Little is known about patient preferences regarding islet delivery device characteristics and implantation strategies. Patient preferences for IDDs and implantation strategies remain understudied. We invited patients, parents and caregivers to fill in an online questionnaire regarding IDDs. An online survey gathered responses from 809 type 1 diabetes patients and 47 caregivers. We also assessed diabetes distress in a subgroup of 412 patients. A significant majority (97%) expressed willingness to receive an IDD. Preferred IDD attributes included a 3.5 cm diameter for 37.7% of respondents, while when provided with all options, 30.4% found dimensions unimportant. Respondents were open to approximately 4 implants, each with a 5 cm incision. Many favored a device functioning for 12 months (33.4%) or 24 months (24.8%). Younger participants (16-30) were more inclined to accept a 6 months functional duration ( p < 0.001). Functional duration outweighed implant quantity and size ( p < 0.001) in device importance. This emphasizes patients' willingness to accommodate burdens related to IDD features and implantation methods, crucial for designing future beta cell replacement strategies