Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing

Conservative management versus open reduction and internal fixation for mid-shaft clavicle fractures in adults - The Clavicle Trial: Study protocol for a multicentre randomized controlled trial

Background: Clavicle fractures account for around 4% of all fractures and up to 44% of fractures of the shoulder girdle. Fractures of the middle third (or mid-shaft) account for approximately 80% of all clavicle fractures. Management of this group of fractures is often challenging and the outcome can be unsatisfactory. In particular it is not clear whether surgery produces better outcomes than non-surgical management. Currently there is much variation in the use of surgery and a lack of good quality evidence to inform our decision.Methods/Design: We aim to undertake a multicentre randomised controlled trial evaluating the effectiveness and safety of conservative management versus open reduction and internal fixation for displaced mid-shaft clavicle fractures in adults. Surgical treatment will be performed using the Acumed clavicle fixation system. Conservative management will consist of immobilisation in a sling at the side in internal rotation for 6 weeks or until clinical or radiological union. We aim to recruit 300 patients. These patients will be followed-up for at least 9 months. The primary endpoint will be the rate of non-union at 3 months following treatment. Secondary endpoints will be limb function measured using the Constant-Murley Score and the Disabilities of the Arm, Shoulder and Hand (DASH) Score at 3 and 9 months post-operatively.Discussion: This article presents the protocol for a multicentre randomised controlled trial. It gives extensive details of, and the basis for, the chosen methods, and describes the key measures taken to avoid bias and to ensure validity.Trial Registration: United Kingdom Clinical Research Network ID: 8665. The date of registration of the trial is 07/09/2006. The date the first patient was recruited is 18/12/2007. © 2011 Longo et al; licensee BioMed Central Ltd

Nedd8 hydrolysis by UCH proteases in Plasmodium parasites

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.Includes Wellcome and BBSRC

Integrative Genetic Manipulation of Plasmodium cynomolgi Reveals Multidrug Resistance-1 Y976F Associated With Increased In Vitro Susceptibility to Mefloquine

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species

Cell motility: the integrating role of the plasma membrane

The plasma membrane is of central importance in the motility process. It defines the boundary separating the intracellular and extracellular environments, and mediates the interactions between a motile cell and its environment. Furthermore, the membrane serves as a dynamic platform for localization of various components which actively participate in all aspects of the motility process, including force generation, adhesion, signaling, and regulation. Membrane transport between internal membranes and the plasma membrane, and in particular polarized membrane transport, facilitates continuous reorganization of the plasma membrane and is thought to be involved in maintaining polarity and recycling of essential components in some motile cell types. Beyond its biochemical composition, the mechanical characteristics of the plasma membrane and, in particular, membrane tension are of central importance in cell motility; membrane tension affects the rates of all the processes which involve membrane deformation including edge extension, endocytosis, and exocytosis. Most importantly, the mechanical characteristics of the membrane and its biochemical composition are tightly intertwined; membrane tension and local curvature are largely determined by the biochemical composition of the membrane and the biochemical reactions taking place; at the same time, curvature and tension affect the localization of components and reaction rates. This review focuses on this dynamic interplay and the feedbacks between the biochemical and biophysical characteristics of the membrane and their effects on cell movement. New insight on these will be crucial for understanding the motility process

Demand for Zn2+ in Acid-Secreting Gastric Mucosa and Its Requirement for Intracellular Ca2+

Recent work has suggested that Zn(2+) plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn(2+) in gastric mucosa under baseline conditions and its regulation during secretory stimulation.Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn(2+) were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, (70)Zn(2+), from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of (70)Zn(2+) from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn(2+) was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn(2+) {[Zn(2+)](i)} during exposure to standard extracellular concentrations of Zn(2+) (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn(2+) increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca(2+) {[Ca(2+)](i)}. Uptake of Zn(2+) was abolished following removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores, suggesting that demand for extracellular Zn(2+) increases and depends on influx of extracellular Ca(2+).This study is the first to characterize the content and distribution of Zn(2+) in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca(2+) integrates basolateral demand for Zn(2+) with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues