The Toxoplasma gondii plastid replication and repair enzyme complex, PREX

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome

Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway

<p>Abstract</p> <p>Background</p> <p>Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of <it>Plasmodium falciparum </it>CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2.</p> <p>Results</p> <p>We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α <it>in vitro</it>.</p> <p>Conclusions</p> <p>Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.</p

Shrinking Bouma's window: How to model crowding in dense displays

Contains fulltext : 236043.pdf (Publisher’s version ) (Open Access)In crowding, perception of a target deteriorates in the presence of nearby flankers. Traditionally, it is thought that visual crowding obeys Bouma's law, i.e., all elements within a certain distance interfere with the target, and that adding more elements always leads to stronger crowding. Crowding is predominantly studied using sparse displays (a target surrounded by a few flankers). However, many studies have shown that this approach leads to wrong conclusions about human vision. Van der Burg and colleagues proposed a paradigm to measure crowding in dense displays using genetic algorithms. Displays were selected and combined over several generations to maximize human performance. In contrast to Bouma's law, only the target's nearest neighbours affected performance. Here, we tested various models to explain these results. We used the same genetic algorithm, but instead of selecting displays based on human performance we selected displays based on the model’s outputs. We found that all models based on the traditional feedforward pooling framework of vision were unable to reproduce human behaviour. In contrast, all models involving a dedicated grouping stage explained the results successfully. We show how traditional models can be improved by adding a grouping stage.14 p

The Toxoplasma gondii Plastid replication and Repair Enzyme Complex, PREX

A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genom

Impact of depth of propofol anaesthesia on functional residual capacity and ventilation distribution in healthy preschool children

Background Propofol is commonly used in children undergoing diagnostic interventions under anaesthesia or deep sedation. Because hypoxaemia is the most common cause of critical deterioration during anaesthesia and sedation, improved understanding of the effects of anaesthetics on pulmonary function is essential. The aim of this study was to determine the effect of different levels of propofol anaesthesia on functional residual capacity (FRC) and ventilation distribution. Methods In 20 children without cardiopulmonary disease mean age (sd) 49.75 (13.3) months and mean weight (sd) 17.5 (3.9)kg, anaesthesia was induced by a bolus of i.v. propofol 2mgkg−1 followed by an infusion of propofol 120µgkg−1min−1 (level I). Then, a bolus of propofol 1mgkg−1 was given followed by a propofol infusion at 240µgkg−1min−1 (level II). FRC and lung clearance index (LCI) were calculated at each level of anaesthesia using multibreath analysis. Results The FRC mean (sd) decreased from 20.7 (3.3)mlkg−1 at anaesthesia level I to 17.7 (3.9)mlkg−1 at level II (P < 0.0001). At the same time, mean (sd) LCI increased from 10.4 (1.1) to 11.9 (2.2) (P = 0.0038), whereas bispectral index score values decreased from mean (sd) 57.5 (7.2) to 35.5 (5.9) (P < 0.0001). Conclusions Propofol elicited a deeper level of anaesthesia that led to a significant decrease of the FRC whereas at the same time the LCI, an index for ventilation distribution, increased indicating an increased vulnerability to hypoxaemi

Self-expanding metal stent should be considered in patients with cirrhosis and uncontrolled variceal bleeding.

SCOPUS: le.jFLWINinfo:eu-repo/semantics/publishe

Expression in Yeast Links Field Polymorphisms in PfATP6 to in Vitro Artemisinin Resistance and Identifies New Inhibitor Classes

Background. The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. Results. Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. Conclusions. The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcom

PNAS plus: plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state

The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided

Host-directed therapy, an untapped opportunity for antimalarial intervention

Host-directed therapy (HDT) is gaining traction as a strategy to combat infectious diseases caused by viruses and intracellular bacteria, but its implementation in the context of parasitic diseases has received less attention. Here, we provide a brief overview of this field and advocate HDT as a promising strategy for antimalarial intervention based on untapped targets. HDT provides a basis from which repurposed drugs could be rapidly deployed and is likely to strongly limit the emergence of resistance. This strategy can be applied to any intracellular pathogen and is particularly well placed in situations in which rapid identification of treatments is needed, such as emerging infections and pandemics, as starkly illustrated by the current COVID-19 crisis.Ling Wei, Jack Adderley, Didier Leroy, David H. Drewry, Danny W. Wilson, Alexis Kaushansky and Christian Doeri

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodiumfalciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal “sterile α-motif” domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening