Evaluation of correlated studies using liquid cell and cryo-transmission electron microscopy: Hydration of calcium sulphate and the phase transformation pathways of bassanite to gypsum.

Insight into the nucleation, growth and phase transformations of calcium sulphate could improve the performance of construction materials, reduce scaling in industrial processes and aid understanding of its formation in the natural environment. Recent studies have suggested that the calcium sulphate pseudo polymorph, gypsum (CaSO4 ·2H2 O) can form in aqueous solution via a bassanite (CaSO4 ·0.5H2 O) intermediate. Some in situ experimental work has also suggested that the transformation of bassanite to gypsum can occur through an oriented assembly mechanism. In this work, we have exploited liquid cell transmission electron microscopy (LCTEM) to study the transformation of bassanite to gypsum in an undersaturated aqueous solution of calcium sulphate. This was benchmarked against cryogenic TEM (cryo-TEM) studies to validate internally the data obtained from the two microscopy techniques. When coupled with Raman spectroscopy, the real-time data generated by LCTEM, and structural data obtained from cryo-TEM show that bassanite can transform to gypsum via more than one pathway, the predominant one being dissolution/reprecipitation. Comparisons between LCTEM and cryo-TEM also show that the transformation is slower within the confined region of the liquid cell as compared to a bulk solution. This work highlights the important role of a correlated microscopy approach for the study of dynamic processes such as crystallisation from solution if we are to extract true mechanistic understanding.

Spherical-supported membranes as platforms for screening against membrane protein targets.

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

The Growing Role of Electron Microscopy in Anti-parasitic Drug Discovery.

BACKGROUND: Parasite diseases are a huge burden on human health causing significant morbidity and mortality. However, parasite structure based drug discovery programmes have been hindered by a lack of high resolution structural information from parasite derived proteins and have often relied upon homology models from mammalian systems. The recent renaissance in electron microscopy (EM) has caused a dramatic rise in the number of structures being determined at high resolution and subsequently enabled it to be thought of as a tool in drug discovery. RESULTS: In this review, we discuss the challenges associated with the structural determination of parasite proteins including the difficulties in obtaining sufficient quantities of protein. We then discuss the reasons behind the resurgence in EM, how it may overcome some of these challenges and provide examples of EM derived parasite protein structures. Finally, we discuss the challenges which EM needs to overcome before it is used as a mainstream technique in anti-parasite drug discovery. CONCLUSIONS: This review reports the progress that has been made in obtaining sufficient quantities of proteins for structural studies and the role EM may play in future structure based drug design programs. The outlook for future structure based drug design programs against some of the most devastating parasite diseases looks promising.

The potential use of single-particle electron microscopy as a tool for structure-based inhibitor design.

Recent developments in electron microscopy (EM) have led to a step change in our ability to solve the structures of previously intractable systems, especially membrane proteins and large protein complexes. This has provided new opportunities in the field of structure-based drug design, with a number of high-profile publications resolving the binding sites of small molecules and peptide inhibitors. There are a number of advantages of EM over the more traditional X-ray crystallographic approach, such as resolving different conformational states and permitting the dynamics of a system to be better resolved when not constrained by a crystal lattice. There are still significant challenges to be overcome using an EM approach, not least the speed of structure determination, difficulties with low-occupancy ligands and the modest resolution that is available. However, with the anticipated developments in the field of EM, the potential of EM to become a key tool for structure-based drug design, often complementing X-ray and NMR studies, seems promising.

Methods to account for movement and flexibility in cryo-EM data processing.

Recent advances in direct electron detectors and improved CMOS cameras have been accompanied by the development of a range of software to take advantage of the data they produce. In particular they allow for the correction of two types of motion in cryo electron microscopy samples: motion correction for movements of the sample particles in the ice, and differential masking to account for heterogeneity caused by flexibility within protein complexes. Here we provide several scripts that allow users to move between RELION and standalone motion correction and centring programs. We then compare the computational cost and improvements in data quality with each program. We also describe our masking procedures to account for conformational flexibility. For the different elements of this study we have used three samples; a high symmetry virus, flexible protein complex (∼1MDa) and a relatively small protein complex (∼550kDa), to benchmark four widely available motion correction packages. Using these as test cases we demonstrate how motion correction and differential masking, as well as an additional particle re-centring protocol can improve final reconstructions when used within the RELION image-processing package.

Type I and type II fatty acid biosynthesis in Eimeria tenella: enoyl reductase activity and structure.

Apicomplexan parasites of the genus Eimeria are the major causative agent of avian coccidiosis, leading to high economic losses in the poultry industry. Recent results show that Eimeria tenella harbours an apicoplast organelle, and that a key biosynthetic enzyme, enoyl reductase, is located in this organelle. In related parasites, enoyl reductase is one component of a type II fatty acid synthase (FAS) and has proven to be an attractive target for antimicrobial compounds. We cloned and expressed the mature form of E. tenella enoyl reductase (EtENR) for biochemical and structural studies. Recombinant EtENR exhibits NADH-dependent enoyl reductase activity and is inhibited by triclosan with an IC50 value of 60 nm. The crystal structure of EtENR reveals overall similarity with other ENR enzymes; however, the active site of EtENR is unoccupied, a state rarely observed in other ENR structures. Furthermore, the position of the central beta-sheet appears to block NADH binding and would require significant movement to allow NADH binding, a feature not previously seen in the ENR family. We analysed the E. tenella genomic database for orthologues of well-characterized bacterial and apicomplexan FAS enzymes and identified 6 additional genes, suggesting that E. tenella contains a type II FAS capable of synthesizing saturated, but not unsaturated, fatty acids. Interestingly, we also identified sequences that appear to encode multifunctional type I FAS enzymes, a feature also observed in Toxoplasma gondii, highlighting the similarity between these apicomplexan parasites.

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella.

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.

Delivery of antimicrobials into parasites.

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Inhalation of an alkaline aerosol by subjects with mild asthma does not result in bronchoconstriction.

Although it is recognized that inhalation of acid aerosols by subjects with asthma can cause bronchoconstriction, the effects of the inhalation of an alkaline aerosol are unknown. When supplemental inflatable restraints (automobile air bags) are deployed an alkaline aerosol is released. This aerosol is composed of particles of sodium carbonate and sodium bicarbonate with some sodium hydroxide. The mass median aerodynamic diameter (MMAD) of the aerosol is approximately 1 micron, and the pH of the aerosol is 9.8 to 10.3. A group of 14 volunteer male subjects with mild asthma inhaled increasing concentrations of this aerosol for 20-min periods of mouth-only tidal ventilation. Pulmonary function tests were performed at baseline (preexposure), after inhalation of room air alone (control), and after each period of inhalation of the aerosol. A total of 5 subjects inhaled aerosols at nominal concentrations of 10, 20, and 40 mg/m3, whereas 11 subjects inhaled aerosols concentrations of approximately 30, 60, and 120 mg/m3. The mean changes in FEV1 and specific airways resistance (SRaw) for the 11 subjects who inhaled the higher concentrations (average highest concentration 126.6 +/- 7.5 mg/m3, mean +/- SEM) were -1.4 +/- 1.9 and +17.5 +/- 8.5%, respectively. Neither change in lung function was clinically or statistically significant. We conclude that the inhalation of relatively high concentrations of this alkaline aerosol by subjects with mild asthma does not result in bronchoconstriction.

Evidence for a localized conversion of endogenous tetrahydrofolate cofactors to dihydrofolate as an important element in antifolate action in murine leukemia cells.

The inhibition of de novo nucleotide, serine, and methionine biosynthesis in mammalian cells treated with antifolates has been attributed generally to a reduction in the levels of tetrahydrofolate cofactors. In L1210 leukemia cells grown in tritiated folic acid (1 microM), most of the endogenous radiolabeled folates were present as formyl-substituted tetrahydrofolates (60-73%, including 10- and 5-formyl and 5,10-methenyl tetrahydrofolate), with lower levels of tetrahydrofolate (including 5,10-methylene tetrahydrofolate), 5-methyl tetrahydrofolate, and non-metabolized folic acid. Trimetrexate (1 microM) caused an elevation of dihydrofolate levels within 5 min following drug addition, from approximately 1 to 20% of the total folates. Whereas total reduced folates were preserved, losses in the levels of individual forms ranged from minor changes in the formyl tetrahydrofolates (approx. 10% decrease), to significant losses in the levels of tetrahydrofolate (approx. 60%) and 5-methyl tetrahydrofolate (95%). Under these conditions, the incorporations of [3H]deoxyuridine into TMP and [14C]glycine into purines or of [14C]formate into biosynthetic products were inhibited (69-95%). The majority (59-100%) of the endogenous radiolabeled folates in L1210 cells grown in various concentrations (0.2 to 3 microM) of [3H]folic acid was bound to soluble intracellular proteins when cell-free extracts were fractionated by rapid gel filtration or charcoal adsorption. Total intracellular folate levels increased in proportion to the changes in medium folic acid concentration; however, cofactor binding was saturable. At low concentrations, below that which supported maximal growth (less than 0.75 microM), all of the intracellular folates were protein-bound; only when maximal growth was achieved, could unbound folates be detected. Incubation with trimetrexate (1 or 10 microM), methotrexate (10 microM), or calcium leuvovorin (50 microM) did not alter significantly the levels of total and protein-bound [3H]folates in cells grown in 1 microM [3H]folic acid. Under all conditions, formyl tetrahydrofolates were the major intracellular derivatives; however, these forms were poorly represented in the bound fraction. Conversely, all of the other intracellular folate forms were completely bound. Tetrahydrofolate was the predominant protein-bound derivative in control cells; in antifolate-treated cells, both bound tetrahydrofolate and 5-methyl tetrahydrofolate were largely replaced by protein-bound dihydrofolate. This interconversion in drug-treated cells was independent of (i) sustained levels of [3H]formyl tetrahydrofolates, or (ii) high extracellular concentrations of unlabeled calcium leucovorin (50 microM). Hence, protein-bound tetrahydrofolates must not only be substrates for enzyme mediated reactions (i.e. TMP synthesis) but also must slowly equilibrate with unbound cofactor. In this fashion, binding of endogenous folates to soluble proteins may function to "segregate' intracellular cofactor pools.(ABSTRACT TRUNCATED AT 400 WORDS)

Role for cytosolic folate-binding proteins in the compartmentation of endogenous tetrahydrofolates and the 5-formyl tetrahydrofolate-mediated enhancement of 5-fluoro-2'-deoxyuridine antitumor activity in vitro.

A major portion of the intracellular folates in L1210 cells grown in (6R,S)-5-formyltetrahydrofolate (leucovorin) was bound to cytosolic proteins when cell extracts were fractionated by rapid gel filtration or adsorption with activated charcoal. Only low levels of intracellular folates were associated with mitochondria (less than 5%). Protein-bound folates comprised 37-100% of the cytosolic cofactors following growth in 2-600 nM 5-formyltetrahydrofolate. Total intracellular folates increased in proportion to the changes in media folate concentration; however, binding was saturable. The maximum level of protein-bound folates in L1210 cells was 66 pmol/mg protein. Protein-bound folates were also detected in HT29 human colon adenocarcinoma cells grown in 5-formyltetrahydrofolate (maximum, 11 pmol/mg protein). For both lines, folate binding was specific for the tetrahydrofolate and 5,10-methylenetetrahydrofolate pool, and, to a lesser extent, 5-methyltetrahydrofolate. Extremely low levels of protein-bound 5-formyl-, 10-formyl-, and 5,10-methenyltetrahydrofolates were measured, even though considerable amounts were detected intracellularly. Pentaglutamyl folates were the predominant cofactor forms in L1210 cells; conversely, the tetraglutamates were the most abundant protein-bound folate derivatives. Increasing media concentrations of 5-formyltetrahydrofolate potentiated 5-fluoro-2'-deoxyuridine cytotoxicity. For L1210 cells, essentially all of the intracellular tetrahydrofolate and 5,10-methylenetetrahydrofolate fraction was protein bound over the concentration range of 5-formyltetrahydrofolate which maximally augmented fluoropyrimidine cytotoxicity. The relative changes in the 50% inhibitory concentrations for fluorodeoxyuridine directly approximated the increases in the levels of protein-bound tetrahydrofolates in L1210 cells. There was no direct relationship between the levels of unbound folates and fluorodeoxyuridine cytotoxicity. Similar results were obtained with HT29 cells. The major folate-binding protein in L1210 cells eluted during Sephacryl S-300 chromatography with a molecular weight of approximately 200,000; a small amount of a higher molecular weight folate-binding protein (Mr 450,000) was also detected. These findings support the concept of a compartmentation of endogenous folates involving specific binding to cytosolic proteins. These associations may regulate reduced folate availability for metabolic processes, and also mediate utilization of 5,10-methylenetetrahydrofolate for ternary complex formation with thymidylate synthase in cells treated with fluoropyrimidines. In this fashion, the levels of protein-bound tetrahydrofolates could represent an additional, previously unrecognized, determinant of fluoropyrimidine pharmacological activity toward mammalian cells.