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CBMDT Members Publication List

The following is a list of recent open access journals publications by members of the Centre. Most papers are available directly as PDFs. The remainder can be obtained by following the link to the JCU Research Online page and clicking the "Request a copy" button.

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cover9 cover10 Journal of Organic Chemistry Nov 2004 cover IJP hookworm cover cover13 cover14 Dalton Transactions 28 Feb 2015 Richard Keene coverInternational Journal of Parasitology cover, July 2015

International Journal of Parasitology cover, January 2016 cover18 cover 19

Nature Reviews Disease Primer: Hookworm Infection. Alex Loukas et al 2016

Hookworms are soil-transmitted nematode parasites that can reside for many years in the small intestine of their human hosts; Necator americanus is the predominant infecting species. Adult worms feed on the blood of a host and can cause iron deficiency anaemia, especially in high-risk populations (children and women of childbearing age). Almost 500 million people in developing tropical countries are infected, and simulation models estimate that hookworm infection is responsible for >4 million disability-adjusted life years lost annually. Humans mount an immune response to hookworms, but it is mostly unsuccessful at removing adult worms from the bowel. Accordingly, the host switches to an immune-tolerant state that enables hookworms to reside in the gut for many years. Although anthelmintic drugs are available and widely used, their efficacy varies and the drugs do not prevent reinfection. Thus, other control strategies aimed at improving water quality, sanitation and hygiene are needed. In addition, efforts are underway to develop a human hookworm vaccine through public–private partnerships. However, hookworms could also be a resource; as hookworms have the capability to regulate the host’s inflammation, researchers are experimentally infecting patients to treat some inflammatory diseases as an approach to discover new anti-inflammatory molecules. This area of endeavour might well yield new biotherapeutics for autoimmune and allergic diseases.

Membrane Insertion of a Dinuclear Polypyridylruthenium(II) Complex Revealed by Solid-State NMR and Molecular Dynamics Simulation: Implications for Selective Antibacterial Activity. Weber et al, 2016

Dinuclear polypyridylruthenium(II) complexes bridged by a flexible methylene linker have received considerable interest as potential antibacterial agents. Their potency and uptake into bacterial cells is directly modulated by the length of the bridging linker, which has implicated membrane interactions as an essential feature of their mechanism of action. In this work, a combination of molecular dynamics (MD) simulations and solid-state NMR was used to present an atomistic model of a polypyridylruthenium(II) complex bound and incorporated into a bacterial membrane model. The results of 31P, 2H, 1H, and 13C NMR studies revealed that the antibacterial [{Ru(phen)2 }2(μ-bb12 )]4+ complex (Rubb12), where phen = 1,10-phenanthroline and bb12  = bis[4(4′ -methyl-2,2′ -bipyridyl)]-1,12-dodecane), incorporated into a negatively-charged model bacterial membrane, but only associated with the surface of a charge-neutral model of a eukaryotic membrane. Furthermore, an inactive [{Ir(phen)2}2(μ-bb12)]6+ (Irbb12) analogue, which is not taken up by bacterial cells, maintained only a surface-bound association with both bacterial and eukaryotic model membranes according to 31P and 2H NMR. The effects of Rubb12 on 31P chemical shift anisotropy and 2H acyl chain order parameters for negatively charged membranes correlated with a membrane-spanning state of the complex according to MD simulation – in which the metal centers embed in the lipid head group  region and the central void, created by the biconic shape of the complex, resulting in increasing disorder of lipid acyl chains and membrane-thinning. A transbilayer mechanism and membrane-spanning may be essential for the cellular uptake and antibacterial activity of this class of compounds.

Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma. Navarro et al, 2016

In the developed world, declining prevalence of some parasitic infections correlates with increased incidence of allergic and autoimmune disorders. Moreover, experimental human infection with some parasitic worms confers protection against inflammatory diseases in phase 2 clinical trials. Parasitic worms manipulate the immune system by secreting immunoregulatory molecules that offer promise as a novel therapeutic modality for inflammatory diseases. We identify a protein secreted by hookworms, anti-inflammatory protein-2 (AIP-2), that suppressed airway inflammation in a mouse model of asthma, reduced expression of costimulatory markers on human dendritic cells (DCs), and suppressed proliferation ex vivo of T cells from human subjects with house dust mite allergy. In mice, AIP-2 was primarily captured by mesenteric CD103+ DCs and suppression of airway inflammation was dependent on both DCs and Foxp3+ regulatory T cells (Tregs) that originated in the mesenteric lymph nodes (MLNs) and accumulated in distant mucosal sites. Transplantation of MLNs from AIP-2–treated mice into naïve hosts revealed a lymphoid tissue conditioning that promoted Treg induction and long-term maintenance. Our findings indicate that recombinant AIP-2 could serve as a novel curative therapeutic for allergic asthma and potentially other inflammatory diseases.

Biological processing of dinuclear ruthenium complexes in eukaryotic cells. Li et al, 2016

The biological processing – mechanism of cellular uptake, effects on the cytoplasmic and mitochondrial membranes, intracellular sites of localisation and induction of reactive oxygen species – of two dinuclear polypyridylruthenium(II) complexes has been examined in three eukaryotic cells lines. Flow cytometry was used to determine the uptake of [{Ru(phen)2}2{μ-bb12}]4+ (Rubb12) and [Ru(phen)2(μ-bb7)Ru(tpy)Cl]3+ {Rubb7-Cl, where phen = 1,10-phenanthroline, tpy = 2,2’:6’,2’’-terpyridine and bbn = bis[4(4’-methyl-2,2-bipyridyl)]-1,n-alkane} in baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (HepG2) cell lines. The results demonstrated that the major uptake mechanism for Rubb12 and Rubb7-Cl was active transport, although with a significant contribution from carrier-assisted diffusion for Rubb12 and passive diffusion for Rubb7-Cl. Flow cytometry coupled with Annexin V/TO-PRO-3 double-staining was used to compare cell death by membrane damage or apoptosis. Rubb12 induced significant direct membrane damage, particularly with HepG2 cells, while Rubb7-Cl caused considerably less membrane damage but induced greater levels of apoptosis. Confocal microscopy, coupled with JC-1 assays, demonstrated that Rubb12 depolarises the mitochondrial membrane, whereas Rubb7-Cl had a much smaller affect. Cellular localisation experiments indicated that Rubb12 did not accumulate in the mitochondria, whereas significant mitochondrial accumulation was observed for Rubb7-Cl. The effect of Rubb12 and Rubb7-Cl on intracellular superoxide dismutase activity showed that the ruthenium complexes could induce cell death via a reactive oxygen species-mediated pathway. The results of this study demonstrate that Rubb12 predominantly kills eukaryotic cells by damaging the cytoplasmic membrane. As this dinuclear ruthenium complex has been previously shown to exhibit greater toxicity towards bacteria than eukaryotic cells, the results of the present study suggest that metal-based cationic oligomers can achieve selective toxicity against bacteria, despite exhibiting a non-specific membrane damage mechanism of action. 
{This paper was selected as a Molecular BioSystems “Hot Article” by the journal}

Effect of different intensities of physical activity on cardiometabolic markers and vascular and cardiac function in adult rats fed with a high-fat high-carbohydrate diet, Romeo B. Batacan, Mitch J. Duncan, Vincent J. Dalbo, Geraldine L. Buitrago, Andrew S. Fenning, 2016

Background: Physical activity (PA) and diet are 2 lifestyle factors that affect cardiometabolic risk. However, data on how a high-fat high carbohydrate (HFHC) diet influences the effect of different intensities of PA on cardiometabolic health and cardiovascular function in a controlled setting are yet to be fully established. This study investigated the effect of sedentary behavior, light-intensity training, and high-intensity interval training on cardiometabolic markers and vascular and cardiac function in HFHC-fed adult rats. Methods: Twelve-week-old Wistar rats were randomly allocated to 4 groups (12 rats/group): control (CTL), sedentary (SED), light-intensity training (LIT), and high-intensity interval training (HIIT). Biometric indices, glucose and lipid control, inflammatory and oxidative stress markers, vascular reactivity, and cardiac electrophysiology of the experimental groups were examined after 12 weeks of HFHC-diet feeding and PA interventions. Results: The SED group had slower cardiac conduction (p = 0.0426) and greater thoracic aortic contractile responses (p < 0.05) compared with the CTL group. The LIT group showed improved cardiac conduction compared with the SED group (p = 0.0003), and the HIIT group showed decreased mesenteric artery contractile responses compared with all other groups and improved endothelium-dependent mesenteric artery relaxation compared with the LIT group (p < 0.05). The LIT and HIIT groups had lower visceral (p = 0.0057 for LIT, p = 0.0120 for HIIT) and epididymal fat (p < 0.0001 for LIT, p = 0.0002 for HIIT) compared with the CTL group. Conclusion: LIT induced positive adaptations on fat accumulation and cardiac conduction, and HIIT induced a positive effect on fat accumulation, mesenteric artery contraction, and endothelium-dependent relaxation. No other differences were observed between groups. These findings suggest that few positive health effects can be achieved through LIT and HIIT when consuming a chronic and sustained HFHC diet.

Occupational Exposure to Bioaerosols in Norwegian Crab Processing Plants. Marte R. Thomassen, Sandip D. Kamath, Andreas L. Lopata, Anne Mette Madsen, Wijnand Eduard, Berit E. Bang and Lisbeth Aasmoe, 2016

Aerosolization of components when processing king crab (Paralithodes camtschaticusand edible crab (Cancer pagurus) may cause occupational health problems when inhaled by workers. Methods: A cross-sectional study was carried out in three king crab plants and one edible crab plant. Personal exposure measurements were performed throughout work shifts. Air was collected for measurement of tropomyosin, total protein, endotoxin, trypsin, and N-acetyl-β-d-glucosaminidase (NAGase). T-tests and ANOVAs were used to compare the levels of exposure in the different plants and areas in the plants. Results: Total protein and tropomyosin levels were highest in the edible crab plant, endotoxin levels were highest in king crab plants. King crab exposure levels were highest during raw processing. Tropomyosin levels were highest during raw king crab processing with geometric mean (GM) 9.6 versus 2.5 ng m−3 during cooked processing. Conversely, edible crab tropomyosin levels were highest during cooked processing with GM 45.4 versus 8.7 ng m−3 during raw processing. Endotoxin levels were higher in king crab plants than in the edible crab plant with GM = 6285.5 endotoxin units (EU) m−3 versus 72 EU m−3. In the edible crab plant, NAGase levels were highest during raw processing with GM = 853 pmol4-methylumbelliferone (MU) m−3 versus 422 pmol4-MU m−3 during cooked processing. Trypsin activity was found in both king crab and edible crab plants and levels were higher in raw than cooked processing. Differences in exposure levels between plants and worker groups (raw and cooked processing) were identified. Conclusions: Norwegian crab processing workers are exposed to airborne proteins, tropomyosin, endotoxins, trypsin, and NAGase in their breathing zone. Levels vary between worker groups and factories.

Oligonuclear polypyridylruthenium(II) complexes: selectivity between bacteria and eukaryotic cells. Anil Gorle, Xin Li, Sabastian, Fangfei Li, Marshall Feterl, Robert Kinobe, Jeffrey Warner, Richard Keene, and Grant Collins, 2016.

Objectives: To determine the in vitro susceptibility and cellular uptake for a series of dinuclear ruthenium(II) complexes, [{Ru(phen)2}2{m-bbn}]4+ (Rubbn), and the mononuclear complexes [Ru(Me4phen)3]2+ and [Ru(phen)2(bb7)]2+, against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa

Methods: The in vitro susceptibility was determined by MIC and MBC assays and time-kill curve experiments, while the cellular uptake was evaluated by monitoring the fluorescence of the complexes remaining in the supernatant of the cultures after incubation for various periods of time and confocal microscopy.

Results: Rubb12 and Rubb16 are highly active, with MIC and MBC values of 1-2 mg/L (0.5-1 µM) against the two Gram positive strains and 2-4 mg/L against E. coli, and 16-32 mg/L against P. aeruginosa.  The relative MBC to MIC values indicated that Rubb12 and Rubb16 are bactericidal, and from the time-kill curve experiments, the ruthenium complexes can kill the bacteria within 2-6 hours. The cellular uptake studies demonstrated that the observed antimicrobial activity is correlated with the level of uptake of the ruthenium complexes, across the range of bacteria and within the class of metal complex. Confocal microscopy confirmed the cellular uptake of Rubb16, and tentatively suggested that the ruthenium complex is localised in the bacteria.

Conclusions: The inert dinuclear ruthenium(II) complexes Rubb12 and Rubb16 have potential as new antimicrobial agents.  The structure of the dinuclear ruthenium complexes can be readily further modified (in terms of charge, size and lipophilicity) in order to achieve the optimal biological profile.

Dinuclear ruthenium(II) complexes containing one inert metal centre and one coordinatively-labile metal centre: syntheses and biological activities.   Xin Li, Kirsten Heimann, Fangfei Li, Jeffrey Warner, Richard Keene, and Grant Collins, 2016.

A series of non-symmetric dinuclear polypyridylruthenium(II) complexes (Rubbn-Cl) that contain one inert metal centre and one coordinatively-labile metal centre, linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ligand (“bbn” for n = 7, 12 and 16), have been synthesised and their potential as antimicrobial agents examined.  The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa).  The Rubbn-Cl complexes displayed good antimicrobial activity, with Rubb12-Cl being the most active complex against both Gram-positive and Gram-negative strains.  Interestingly, Rubb7-Cl was found to be eight- and sixteen-fold more active towards E. coli than against S. aureus and MRSA, respectively.  The cytotoxicities of the Rubbn-Cl complexes against three eukaryotic cell lines  two kidney cell lines (BHK and HEK-293) and one liver cell line (HepG2)  were examined.  The Rubbn-Cl complexes were found to be considerably less toxic towards eukaryotic cells than S. aureus, MRSA and E. coli, with Rubb12-Cl being thirty- to eighty-times more toxic to the bacteria than to BHK, HEK-293 or HepG2 cells.  Unexpectedly, Rubb7-Cl was far more toxic to HepG2 cells (24h-IC50 = 3.7 mM) and far less toxic to BHK cells (24h-IC50 = 238 mM) than the Rubb12-Cl and Rubb16-Cl complexes.  In order to understand the unexpected large differences in the cytotoxicities of the Rubbn-Cl complexes towards eukaryotic cells, a confocal microscopic study of their intracellular localisation was undertaken.  The results suggest that the observed cytotoxicity might be related to the extent of DNA binding.

Disulfide Bridges: Bringing Together Frustrated Structure in a Bioactive Peptide - Yi Zhang, Klaus Schulten, Martin Gruebele, Paramjit S. Bansal, David Wilson and Norelle L. Daly, April 2016

Disulfide bridges are commonly found covalent bonds that are usually believed to maintain structural stability of proteins. Here, we investigate the influence of disulfide bridges on protein dynamics through molecular dynamics simulations on the cysteine-rich trypsin inhibitor MCoTI-II with three disulfide bridges. Correlation analysis of the reduced cyclic peptide shows that two of the three disulfide distances (Cys11-Cys23 and Cys17-Cys29) are anticorrelated within ~1 ms of bridge formation or dissolution: when the peptide is in nativelike structures and one of the distances shortens to allow bond formation, the other tends to lengthen. Simulations over longer timescales, when the denatured state is less structured, do not show the anticorrelation. e propose that the native state contains structural elements that frustrate one another’s folding, and that the two bridges are critical for snapping the frustrated native structure into place. In contrast, the Cys4-Cys21 bridge is predicted to form together with either of the other two bridges. Indeed, experimental chromatography and nuclear magnetic resonance data show that an engineered peptide with the Cys4-Cys21 bridge deleted can still fold into its near-native structure even in its noncyclic form, confirming the lesser role of the Cys4-Cys21 bridge. The results highlight the importance of disulfide bridges in a small bioactive peptide to bring together frustrated structure in addition to maintaining protein structural stability.

Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates - Javier Sotillo, Mark Pearson, Luke Becker, Jason Mulvenna, Alex Loukas, 2016

Herein we show for the first time that Schistosoma mansoni adult worms secrete exosome-like extracellular vesicles ranging from 50 to 130 nm in size. Extracellular vesicles were collected from the excretory/secretory products of cultured adult flukes and purified by Optiprep density gradient, resulting in highly pure extracellular vesicle preparations as confirmed by transmission electron microscopy and Nanosight tracking analysis. Extracellular vesicle proteomic analysis showed numerous known vaccine candidates, potential virulence factors and molecules implicated in feeding. These findings provide new avenues for the exploration of host–schistosome interactions and offer a potential mechanism by which some vaccine antigens exert their protective efficacy.oukas, 2015

Differential anticancer activities of the geometric isomers of dinuclear iridium(III) complexes.  Mallesh Pandrala, Madhu Sundaraneedi, Alaina Ammit, Lynne Wallace, Richard Keene and Grant Collins, 2015. 

The anticancer activities of the dinuclear iridium(III) complexes [{Ir(tpy)Cl}2{μ-bbn}]4+ {Cl-Irbbn; tpy = 2,2':6',2"-terpyridine, bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 and 16)} against MCF-7 and MDA-MB-231 breast cancer cell lines have been determined.  The activities of the Cl-Irbbn complexes increased with increasing chain length, with the Cl-Irbb16 complex showing the best anticancer properties.  However, while the Cl-Irbb16 was active against the metastatic MDA-MB-231 cells (3 mM), it was relatively inactive against the non-metastatic MCF-7 line (29 mM).  The three geometric isomers of Cl-Irbb16 were isolated and characterised by NMR and mass spectroscopy, and their anticancer activities, lipophilicities (log P) and DNA binding abilities examined.  The trans,trans (t,t) isomer (IC50 = 2 mM against MDA-MB-231) showed considerably greater anticancer activity than the cis,trans (c,t) and cis,cis (c,c) isomers (8 31 mM, respectively).  From log P measurements, the t,t isomer of Cl-Irbb16 (log P = -0.62) was found to be slightly more lipophilic than the other geometric isomers (-0.95 and -0.98 for the c,c and c,t isomers, respectively).  Calf-thymus DNA binding experiments demonstrated that the t,t isomer did induce a different alteration to the helix conformation compared to the other isomers.   The results of this study also highlight the significance of the differences in three-dimensional shape that can be obtained through geometric isomerism for octahedral coordination complexes.

Wilson, D. T. R. The venom of Australian spiders. In P. Gopalakrishnakone, G. A. Corzo, E. Diego-Garcia, & M. E. de Lima (Eds.),  Toxinology: Spider Venoms. Dordrecht: Springer Netherlands, 2015

Australia is home to an estimated 10,000 species of spider, including species from the Latrodectus genera and Atracinae family, two of the four widely recognized medically significant spider groups. It is predicted in excess of 5,000 spider bite cases occurring annually in Australia, predominantly by spiders that have not shown any medical relevance. Bites by medically relevant spiders are rare, and of those treatment with antivenom is rarer. Despite extensive publicity and rumor, there is no conclusive evidence that the venom of any Australian spiders is responsible for necrotic arachnidism. The complexity and diversity of spider venoms, combined with potent activity on a range of targets in mammalian and insect systems, have attracted interest in the potential of spider venoms as a source of insecticidal and therapeutic leads. The venom of species of Australian funnel-web spider has received the most attention for study, with more than 75 venom peptides identified from nine toxin families. Recent work has identified venom peptides from the venom of Australian tarantulas with potential as insecticidal and therapeutic leads. This chapter provides an overview of spiders in Australia and their medical and clinical importance and provides a current comprehensive review of the published toxins from Australian spider venoms.

A quantitative proteomic analysis of the tegumental proteins from Schistosoma mansoni schistosomula reveals novel potential therapeutic targets - Javier Sotillo, Mark Pearson, Luke Becker, Jason Mulvenna, Alex Loukas, 2015

The tegument of Schistosoma mansoni plays an integral role in host–parasite interactions, particularly during the transition from the free-living cercariae to the intra-mammalian schistosomula stages. This developmental period is characterised by the transition from a trilaminate surface to a heptalaminate tegument that plays key roles in immune evasion, nutrition and excretion. Proteins exposed at the surface membranes of newly transformed schistosomula are therefore thought to be prime targets for the development of new vaccines and drugs for schistosomiasis. Using a combination of tegumental labelling and high-throughput quantitative proteomics, more than 450 proteins were identified on the apical membrane of S. mansoni schistosomula, of which 200 had significantly regulated expression profiles at different stages of schistosomula development in vitro, including glucose transporters, sterols, heat shock proteins, antioxidant enzymes and peptidases. Current vaccine antigens were identified on the apical membrane (Sm-TSP-1, calpain) or sub-tegumental (Sm-TSP-2, Sm29) fractions of the schistosomula, displaying localisation patterns that, in some cases, differ from that in the adult stage fluke. This work provides the first known in-depth proteomic analysis of the surface-exposed proteins in the schistosomula tegument, and some of the proteins identified are clear targets for the generation of new vaccines and drugs against schistosomiasis.

Ketogenic diet reverses behavioral abnormalities in an acute NMDA receptor hypofunction model of schizophrenia - Ann Katrin Kraeuter, Heather Loxton, Bruna Costa Lima, Donna Rudd, Zoltán Sarnyai, 2015

Schizophrenia is a devastating, chronic mental illness that affects close to one percent of the population worldwide. It is characterised by hallucinations (hearing voices), delusions (unshakable beliefs in something untrue), social withdrawal and problems with thinking, causing suffering for the patient and major challenges to their families. At present there is no cure for schizophrenia. The medications used only target some of the symptoms and they produce side effects such as movement disorder, weight gain and cardiovascular disease in the long-term, prompting the need to develop more effective treatment with fewer side effects. Feeding mice with ketogenic diet, which is high on fat but very low on carbohydrates (sugars), leads to the normalisation of behaviours that resembles to schizophrenia in a well-established animal model of the disorder. 

Prolonged Subcutaneous Administration of Oxytocin Accelerates Angiotensin II-Induced Hypertension and Renal Damage in Male Rats James Phie, Nagaraja Haleagrahara, Patricia Newton, Constantin Constantinoiu, Zoltan Sarnyai, Lisa Chilton, Robert Kinobe, 2015

Oxytocin and its receptor are synthesised in the heart and blood vessels but effects of chronic activation of this peripheral oxytocinergic system on cardiovascular function are not known. In acute studies, systemic administration of low dose oxytocin exerted a protective, preconditioning effect in experimental models of myocardial ischemia and infarction. In this study, we investigated the effects of chronic administration of low dose oxytocin following angiotensin II-induced hypertension, cardiac hypertrophy and renal damage. Angiotensin II (40 μg/Kg/h) only, oxytocin only (20 or 100 ng/Kg/h), or angiotensin II combined with oxytocin (20 or 100 ng/Kg/h) were infused subcutaneously in adult male Sprague-Dawley rats for 28 days. At day 7, oxytocin or angiotensin-II only did not change hemodynamic parameters, but animals that received a combination of oxytocin and angiotensin-II had significantly elevated systolic, diastolic and mean arterial pressure compared to controls (P < 0.01). Hemodynamic changes were accompanied by significant left ventricular cardiac hypertrophy and renal damage at day 28 in animals treated with angiotensin II (P < 0.05) or both oxytocin and angiotensin II, compared to controls (P < 0.01). Prolonged oxytocin administration did not affect plasma concentrations of renin and atrial natriuretic peptide, but was associated with the activation of calcium-dependent protein phosphatase calcineurin, a canonical signaling mechanism in pressure overload-induced cardiovascular disease. These data demonstrate that oxytocin accelerated angiotensin-II induced hypertension and end-organ renal damage, suggesting caution should be exercised in the chronic use of oxytocin in individuals with hypertension.

Viability of developmental stages of Schistosoma mansoni quantified with xCELLigence worm real-time motility assay (xWORM) Gabriel Rinaldi, Alex Loukas, Paul J. Brindley, Jeff T. Irelan, Michael J. Smout, 2015

Infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide. Where anthelmintic drugs are available, drug resistance is a major problem in livestock parasites, and a looming threat to public health. Monitoring the efficacy of these medicines and screening for new drugs has been hindered by the lack of objective, high-throughput approaches. Several cell monitoring technologies have been adapted for parasitic worms, including video-, fluorescence-, metabolism enzyme- and impedance-based tools that minimize the screening bottleneck. Using the xCELLigence impedance-based system we previously developed a motility-viability assay that is applicable for a range of helminth parasites. Here we have improved substantially the assay by using diverse frequency settings, and have named it the xCELLigence wormreal-time motility assay (xWORM). By utilizing strictly standardized mean difference analysis we compared the xWORM output measured with 10, 25 and 50 kHz frequencies to quantify the motility of schistosome adults (human blood flukes) and hatching of schistosome eggs. Furthermore, we have described a novel application of xWORM to monitor movement of schistosome cercariae, the developmental stage that is infectious to humans. For all three stages, 25 kHz was either optimal or near-optimal for monitoring and quantifying schistosome motility. These improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large.,

Mononuclear polypyridylruthenium(II) complexes with high membrane permeability in gram-negative bacteria—in particular Pseudomonas aeruginosa. Gorle et al, 2015

Ruthenium(II) complexes containing the tetradentate ligand bis[4(4'-methyl 2,2'-bipyridyl)]-1,n-alkane ("bb(n)"; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bb(n))]²⁺ (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-a-[Ru(phen)(bb₁₂)]²⁺ species showed good activity against Gram-negative species. In particular, the cis-a-[Ru(phen)(bb₁₂)]²⁺ complex was two to four times more active than the cis-b [Ru(phen)(bb₁₂)]²⁺complex against the Gram-negative strains. The cis-a- and cis-b-[Ru(phen)(bb₁₂)]²⁺ complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-a- and cis-b-[Ru(phen)(bb₁₂)]²⁺ complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-a-[Ru(phen)(bb₁₂)]²⁺ complex into live P. aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-a- and cis-b-[Ru(phen)(bb(n))]²⁺ complexes exhibited relatively strong binding to DNA (Kb≈10⁶M⁻¹ ), but no significant difference between the geometric isomers was observed.

Ruthenium complexes as antimicrobial agents. Li et al, 2015

One of the major advances in medical science has been the development of antimicrobials; however, a consequence of their widespread use has been the emergence of drug-resistant populations of microorganisms.  There is clearly a need for the development of new antimicrobials - but more importantly, there is the need for the development of new classes of antimicrobials, rather than drugs based upon analogues of known scaffolds.  Due to the success of the platinum anticancer agents, there has been considerable interest in the development of therapeutic agents based upon other transition metals – and in particular ruthenium(II/III) complexes, due to their well known interaction with DNA.  There have been many studies of the anticancer properties and cellular localisation of a range of ruthenium complexes in eukaryotic cells over the last decade.  However, only very recently has there been significant interest in their antimicrobial properties.   This review highlights the types of ruthenium complexes that have exhibited significant antimicrobial activity and discusses the relationship between chemical structure and biological processing - including site(s) of intracellular accumulation - of the ruthenium complexes in both bacterial and eukaryotic cells.

RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells.  Li et al, 2015

Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2).  Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand (“bb12”) showed a high degree of selectivity for the nucleus of the eukaryotic cells.  Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA.  No significant differences were observed in the intracellular localisation between the delta,delta and lambda,lambda enantiomers of the dinuclear complex.  Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells.  However, when the activity of the least cytotoxic compound (the delta,delta enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria.  Circular dichroism (CD) spectroscopy was used to examine the effect of the delta,delta and lambda,lambda enantiomers of the dinuclear complex on the solution conformations of RNA and DNA.  The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.

Structural diversity of lanthanum saccharinates induced by 1,10-phenanthroline: A synthetic and X-ray crystallographic study. Vasilescu, Tas, Junk 2015

Four new lanthanum(III) complexes of formulae [La(sac)3(phen)2(H2O)2]·(phen)(H2O)2 (1), [La(sac)3(phen)2(H2O)2]·(phen) (2), [La(sac)2(phen)(H2O)4]·(sac) (3) and [La(sac)2(phen)2(H2O)3]·(sac)(phen)(H2O) (4) (phen = 1,10 phenanthroline, sac = saccharinate) have been synthesised and characterised by single crystal X-ray diffraction, IR, elemental analysis and thermogravimetric analysis. The binding mode of the sac anion was manipulated through the use of phen as structure inducer. In all four complexes the La(III) ion is in a nine-coordinate environment. The structures of 1 and 2 are similar, comprising of three sac anions coordinating in a monodentate fashion through the carbonyl O-atom, two bidentate phen and two water molecules coordinating directly to the same La(III) ion; a third, uncoordinated phen moiety also forms part of the unit cell. In addition, 1 is characterized by the presence of two crystallization water molecules which are absent in 2. In compound 3 the coordination sphere of La(III) is completed by a bidentate chelating sac through the N- and carbonyl O-atoms, a monodentate sac, a bidentate phen and four water ligands; a third, uncoordinated sac is also present in the crystal lattice. Compound 4 contains two bidentate phen, three water ligands and two monodentate O(carbonyl) sac ligands. The presence of three sac anions coordinating to the same lanthanide ion as well as a bidentate N–Ln–O chelating sac, confirmed by single crystal X-ray crystallography, is unprecedented for rare-earth saccharinates.

Tri- and tetra-nuclear polypyridyl ruthenium(II) complexes as antimicrobial agents. Gorle et al, 2014

A series of inert tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'- methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bbn" for n = 10, 12 and 16) have been synthesised and their potential as antimicrobial agents examined. Due to the modular nature of the synthesis of the oligonuclear complexes, it was possible to make both linear and non-linear tetra nuclear ruthenium species. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria − Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In order to gain an understanding of the relative antimicrobial activities, the cellular uptake and water–octanol partition coefficients (log P) were determined for a selection of the ruthenium complexes. Although the trinuclear complexes were the most lipophilic based upon log P values and showed the greatest cellular uptake, the linear tetranuclear complexes were generally more active, with MIC values <1 μM against the Gram positive bacteria. Similarly, although the non-linear tetranuclear complexes were slightly more lipophilic and were taken up to a greater extent by the bacteria, they were consistently less active than their linear counterparts. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive S. aureus and MRSA. The results demonstrate that the lower antimicrobial activity of polypyridylruthenium(II) complexes towards Gram negative bacteria, particularly P. aeruginosa, is not strongly correlated to the cellular accumulation but rather to a lower intrinsic ability to kill the Gram negative cells.

Multinuclear ruthenium(II) complexes as anticancer agents, Gorle et al, 2014

A series of dinuclear ruthenium(II) complexes that contain labile chlorido ligands, [{Ru(tpy)Cl}2{μ-bbn}]2+ {designated Cl-Rubbn; tpy = 2,2':6',2"-terpyridine, bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 10, 12, 14 or 16)} and derivatives containing nitro substituents on the tpy ligand and/or secondary amines within the bbn linking chain have been synthesised and their potential as anticancer agents examined.  Some of the Cl-Rubbn species showed good anticancer activity against MCF-7 and MDA-MB-231 breast cancer cell lines, with the Cl-Rubb12 complex being four-times more active than cisplatin.  Inclusion of nitro substituents on the tpy ligands of Cl-Rubb12 resulted in significantly decreased anticancer activity.  The incorporation of amine groups into the linking ligand did not increase the anticancer activity of the Cl-Rubbn complexes. The Cl-Rubbn complexes and those containing amine groups in the linking chain aquated at approximately the same rate, with 50% aquation within 120 minutes.  By comparison, the complexes containing nitro substituents on the tpy ligand aquated extremely slowly, with 60% of the chlorido complex remaining 24 hours after they were dissolved in water.  Cyclic voltammetry with the model mononuclear complex [Ru{(NO2)3tpy}(Me2bpy)Cl]+ {(NO2)3tpy = 4,4',4"-trinitro-2,2':6',2"-terpyridine} showed that the nitro substituents exerted a strong effect on the ruthenium centre, with the anodic peak corresponding to the Ru(III/II) couple shifted positively by 300 mV compared to that from the non-nitrated parent complex [Ru(tpy)(Me2bpy)Cl]+1H NMR studies of the reaction of the Cl-Rubbn complexes with GMP indicated that the ruthenium complexes covalently bound the nucleotide slowly, with 33% bound in 24 hours.  However, the results of this study suggest that the cytotoxicity of the dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA.

Dinuclear ruthenium(II) antimicrobial agents that selectively target polysomes in vivo. Li et al, 2014

Wide-field fluorescence microscopy at high magnification was used to study the intracellular binding site of Rubb16 in Escherichia coli.  Upon incubation of E. coli cells at the minimum inhibitory concentration, Rubb16 localised at ribosomes with no significant DNA binding observed.  Furthermore, Rubb16 condensed the ribosomes when they existed as polysomes.  It is postulated that the condensation of polysomes would halt protein production, and thereby inhibit bacterial growth.  The results of this study indicate that the family of inert dinuclear ruthenium complexes Rubbn selectively target RNA over DNA in vivo. Selective RNA targeting could be advantageous for the development of therapeutic agents, and because of differences in ribosome structure between bacteria and eukaryotic cells, the Rubbn complexes could be selectively toxic to bacteria.  In support of this hypothesis, the toxicity of Rubb16 was found to be significantly less to liver and kidney cell lines than against a range of bacteria.

Protein binding by dinuclear polypyridyl ruthenium(II) complexes and the effect of cucurbit[10]uril encapsulation.  Li et al, 2013.

The effect of human serum on the minimum inhibitory/bactericidal concentrations of the potential antimicrobial agents ΔΔ-[{Ru(phen)2}2(μ-bbn)]4+ {ΔΔ-Rubbn; where phen = 1,10-phenanthroline, bbn = 1,n-bis[4(4’-methyl-2,2’-bipyridyl)]-alkane for n = 12 and 16} against four strains of bacteria – Gram positive Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli and Pseudomonas aeruginosa – has been determined.  The results demonstrated that the ruthenium(II) complexes have significantly decreased in vitro activity in serum.  Fluorescence spectroscopy was used to confirm that the decrease in antimicrobial activity was due to the strong binding of the ruthenium complexes with the serum proteins human serum albumin (HSA) and transferrin.  A series of ruthenium complexes showed stronger binding to HSA than apo-transferrin but comparable or less than with holotransferrin, with the binding affinity to all three proteins decreasing in the order trinuclear > dinuclear > mononuclear.  The dinuclear complex ΔΔ-Rubb12 displaced warfarin from HSA, tentatively suggesting that the ruthenium complexes bind at or near the warfarin-binding site, Sudlow’s site 1.  The binding of ΔΔ-Rubb12 and ΔΔ-Rubb16 to the macrocyclic host molecule cucurbit[10]uril (Q[10]) was examined by NMR spectroscopy.  The large upfield 1H NMR chemical shift changes observed for the methylene protons in the bridging ligands upon addition of Q[10], coupled with the observation of a range of intermolecular ROEs in ROESY spectra, indicated that the dinuclear complexes bound Q[10] with the bridging ligand within the cavity and the metal centres positioned outside the portals. NMR and fluorescence spectroscopy demonstrated that the Q[10]-encapsulated ruthenium complexes directly bound HSA, and with similar affinity to the corresponding free metal complexes.

Chlorido-containing ruthenium(II) and iridium(III) complexes as antimicrobial agents.  Pandrala et al, 2013

A series of polypyridyl-ruthenium(II) and -iridium(III) complexes that contain labile chlorido ligands, [{M(tpy)Cl}2{μ-bbn}]2/4+ {Cl-Mbbn; where M = Ru or Ir; tpy = 2,2':6',2"-terpyridine; and bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 7, 12 or 16)}  have been synthesised and their potential as antimicrobial agents examined.  The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the series of metal complexes against four strains of bacteria - Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) – have been determined.  All the ruthenium complexes were highly active and bactericidal.  In particular, the Cl-Rubb12 complex showed excellent activity against all bacterial cell lines with MIC values of 1 mg/ml against the Gram positive bacteria and 2 and 8 mg/ml against E. coli and P. aeruginosa, respectively.  The corresponding iridium(III) complexes also showed significant antimicrobial activity in terms of MIC values; however and surprisingly, the iridium complexes were bacteriostatic rather than bactericidal.  The inert iridium(III) complex, [{Ir(phen)2}2{μ-bb12}]6+ {where phen = 1,10-phenanthroline) exhibited no antimicrobial activity, suggesting that it could not cross the bacterial membrane.  The mononuclear model complex, [Ir(tpy)(Me2bpy)Cl]Cl2 (where Me2bpy = 4,4'-dimethyl-2,2'-bipyridine), was found to aquate very rapidly, with the pKa of the iridium-bound water in the corresponding aqua complex determined to be 6.0.  This suggests the dinuclear complexes [Ir(tpy)Cl}2{μ-bbn}]4+ aquate and deprotonate rapidly and enter the bacterial cells as 4+ charged hydroxo species.

Dinuclear polypyridylruthenium(II) complexes:  flow cytometry studies of their accumulation in bacteria and the effect on the bacterial membrane. Li et al, 2013.

Objectives: To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)2}2{m-bbn}]4+ (Rubbn) and the mononuclear complexes [Ru(Me4phen)3]2+ and [Ru(phen)2(bb7)]2+ in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane.

Methods: The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures.  The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability.

Results: The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb12 and Rubb16.  No decrease in accumulation was observed for Rubb12 in ATP-inhibited cells.  While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb12 was observed; however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP.  Except for the mononuclear complex [Ru(Me4phen)3]2+, incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus.

Conclusions: The results indicate that the potential new antimicrobial Rubbn complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane.

Iridium(III) complexes containing 1,10-phenanthroline and derivatives:  synthetic, stereochemical, and structural studies, and their antimicrobial activity.  Pandrala et al, 2013.

A convenient synthetic strategy is reported for the series of complexes [Ir(pp)3]3+ (where pp = phen, Me2phen and Me4phen) through the intermediacy of the appropriate [Ir(pp)2(CF3SO3)2]+ species.  In the case of [Ir(phen)3]3+, the cation was resolved into its enantiomeric forms, for which the absolute configurations were determined by X-ray diffraction.  The availability for the first time of the CD spectra allowed comparison with computed CD spectra.  Measurement of the antimicrobial activity of the [Ir(pp)3]3+ species {and the [Ir(pp)2X2]+ (X = Cl-, CF3SO3-) precursors involved in their synthesis}, as well as cell uptake studies with the four bacterial strains S. aureus, methicillin-resistant S. aureus (MRSA), E. coli, and P. aeruginosa, indicated that they showed little activity compared with their Ru(II) analogues. The results suggest that it is unfavourable for an individual metal centre with a 3+ charge to pass across the bacterial cell membrane.

Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection. Gaze et al, 2012

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-β) response, with some evidence of a Th1 (IFN-γ and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation ofIL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.

In vitro susceptibility and cellular uptake for a new class of antimicrobial agents: dinuclear ruthenium(II) complexes. Li at al, 2012

This study determined the in vitro susceptibility and cellular uptake for a series of dinuclear ruthenium(II) complexes [{Ru(phen)2}2{μ-bbn}]4+ (Rubbn), and the mononuclear complexes [Ru(Me4phen)3]2+ and [Ru(phen)2(bb7)]2+ against Staphylococcus aureus, methicillin-resistant S. aureus, Escherichia coli and Pseudomonas aeruginosa by MIC and MBC assays, and time–kill curve experiments, while the cellular uptake was evaluated by monitoring the fluorescence of the complexes remaining in the supernatant of the cultures after incubation for various periods of time, flow cytometry and confocal microscopy.  It was observed that Rubb12 and Rubb16 were highly active, with MIC and MBC values of 1–2 mg/L (0.5–1 mM) for the two Gram-positive strains and 2–4 mg/L for E. coli and 16–32 mg/L for P. aeruginosa. Rubb16 showed equal or better activity (on a molar basis) to gentamicin and ampicillin for all strains apart from P. aeruginosa. The relative MBC to MIC values indicated that Rubb12 and Rubb16 are bactericidal, and from the time–kill curve experiments, the ruthenium complexes can kill the bacteria within 2–6 h. The cellular uptake studies demonstrated that the observed antimicrobial activity is correlated with the level of uptake of the ruthenium complexes. Confocal microscopy confirmed the cellular uptake of Rubb16, and tentatively suggested that the ruthenium complex is localised in the bacteria. These results indicate that the inert dinuclear ruthenium(II) complexes Rubb12 and Rubb16 have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complexes can be readily further modified in order to increase their selectivity for bacteria over human cells.

An approach to therapeutic agents through selective targeting of destabilised nucleic acid duplex sequences. Li et al, 2012

The binding of ΔΔ/ΛΛ-[{Ru(phen)2}2(μ-bbn)]4+ {where phen = 1,10-phenanthroline, bbn = 1,n-bis[4(4'-methyl-2,2'-bipyridyl)]-alkane (ΔΔ/ΛΛ-Rubbn} to the non-self complementary oligonucleotide 5′-d(CGCGATAAGCCGC·5'-GCGGCATTACGCG) (3-DB) has been examined using a 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) displacement assay. The 3-DB oligonucleotide contains two single adenine bulge nucleotides that are separated by three base pairs. 1H NMR spectroscopy data demonstrated that the adenine bases are intra-helical and that the segment containing the two bulge nucleotides and the three A·T base pairs between the bulges forms a destabilised segment within the stable duplex oligonucleotide. The DAPI displacement assay demonstrated that ΔΔ-Rubb7-bound 3-DB with higher affinity than the other members of the ΔΔ/ΛΛ-Rubbn series. Molecular models suggested that the seven-carbon chain length in ΔΔ-Rubb7 was ideal to span the distance between the two bulge sites. The binding of ΔΔ-Rubb₇ to 3-DB was also studied by 1H NMR spectroscopy and molecular modelling. The selective changes in chemical shifts for the resonances from 3-DB upon addition of ΔΔ-Rubb7 suggested that the metal complex specifically bound at the destabilised segment between A5 and A19. Observation in NOESY spectra of NOE cross peaks between 3-DB and ΔΔ-Rubb7 confirmed that one of the ruthenium centres bound at the A5 bulge site, with the other metal centre positioned at the A19 bulge. In addition, ΔΔ-Rubb7 was found to bind chromosomal DNA extracted from a suspension of Staphylococcus aureus that had been incubated with the ruthenium(II) complex. As inert dinuclear ruthenium(II) complexes are capable of being transported into a bacterial cell and bind chromosomal DNA, it is possible that they could be developed into anti-microbial agents that specifically target destabilised segments of DNA that are recognised by essential DNA-binding proteins.

Characterising the Mucosal and Systemic Immune Responses to Experimental Human Hookworm Infection. Gaze et al, 2012

The mucosal cytokine response of healthy humans to parasitic helminths has never been reported. We investigated the systemic and mucosal cytokine responses to hookworm infection in experimentally infected, previously hookworm naive individuals from non-endemic areas. We collected both peripheral blood and duodenal biopsies to assess the systemic immune response, as well as the response at the site of adult worm establishment. Our results show that experimental hookworm infection leads to a strong systemic and mucosal Th2 (IL-4, IL-5, IL-9 and IL-13) and regulatory (IL-10 and TGF-b) response, with some evidence of a Th1 (IFN-c and IL-2) response. Despite upregulation after patency of both IL-15 and ALDH1A2, a known Th17-inducing combination in inflammatory diseases, we saw no evidence of a Th17 (IL-17) response. Moreover, we observed strong suppression of mucosal IL-23 and upregulation of IL-22 during established hookworm infection, suggesting a potential mechanism by which Th17 responses are suppressed, and highlighting the potential that hookworms and their secreted proteins offer as therapeutics for human inflammatory diseases.

The antimicrobial activity of inert oligonuclear polypyridylruthenium(II) complexes against pathogenic bacteria, including MRSA. Li et al 2011

The minimum inhibitory concentrations (MIC) of a series of synthetic inert polypyridylruthenium(II) complexes against four strains of bacteria – Gram positive Staphylococcus aureus (S.aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E.coli) and Pseudomonas aeruginosa (P.aeruginosa) – have been determined.  The results demonstrate that for the dinuclear ruthenium(II) complexes DD/LL-[{Ru(phen)2}2{m-bbn}]4+ {where phen = 1,10-phenanthroline; bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16)} the complexes linked by the bb12, bb14 and bb16 ligands are highly active, with MIC values of 1 µg/mL against both S.aureus and MRSA, and 2-4 and 8-16 µg/mL against E.coli and P.aeruginosa, respectively.  The mononuclear complex [Ru(Me4phen)3]2+ showed equal activity (on a mole basis) against S. aureus compared with the Rubb12, Rubb14 and Rubb16, but was considerably less active against MRSA and the two Gram negative bacteria.  For the dinuclear Rubbn family of complexes, the antimicrobial activity was related to the octanol-water partition coefficient (log P).  However, the highly lipophilic mononuclear complex ∆-[Ru(phen)2(bb16)]2+ was significantly less active than Rubb16, highlighting the importance of the dinuclear structure.  Preliminary toxicity assays were also carried out for the DD isomers of Rubb7, Rubb10, Rubb12 and Rubb16 against two human cells lines, fresh red blood cells and THP-1 cells.  The results showed that the dinuclear ruthenium complexes are significantly less toxic to human cells compared to bacterial cells, with the HC50 and IC50 values 100-fold higher than the MIC for the complex that showed the best potential - DD-Rubb12.

Suppression of Inflammatory Immune Responses in Celiac Disease by Experimental Hookworm Infection McSorley et al, 2011

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)c and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-c and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.

Mechanism of Cytotoxicity and Cellular Uptake of Lipophilic Inert Dinuclear Polypyridylruthenium(II) Complexes. Pisani et al, 2011

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of — and mode of cell death induced by — dinuclear ruthenium(II) complexes ΔΔ/ΛΛ-[{Ru(phen)2}2{μ-bbn}]4+ (Rubbn), where phen is 1,10-phenanthroline, bbn is bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane (n = 2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bbn ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ-Rubb16 complex displayed the highest cytotoxicity of the series, with an IC50 value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bbn bridge of the metal complex. ΔΔ-Rubb16 entered the L1210 cells by passive diffusion (with a minor contribution from protein-mediated active transport), inducing cell death via apoptosis. Additionally, metal-complex uptake in leukaemia cells was approximately 16-times that observed in healthy B cells highlighting that the bbn series of complexes may have potential as selective anticancer drugs.

Seafood plays an important role in human nutrition and health. The growing international trade in seafood species and products has added to the popularity and frequency of consumption of a variety of seafood products across many countries. This increased production and consumption of seafood has been accompanied by more frequent reports of adverse health problems among consumers as well as processors of seafood. Adverse reactions to seafood are often generated by contaminants but can also be mediated by the immune system and cause allergies. These reactions can result from exposure to the seafood itself or various non-seafood components in the product. Non-immunological reactions to seafood can be triggered by contaminants such as parasites, bacteria, viruses, marine toxins and biogenic amines. Ingredients added during processing and canning of seafood can also cause adverse reactions. Importantly all these substances are able to trigger symptoms which are similar to true allergic reactions, which are mediated by antibodies produced by the immune system against specific allergens. Allergic reactions to ‘shellfish’, which comprises the groups of crustaceans and molluscs, can generate clinical symptoms ranging from mild urticaria and oral allergy syndrome to life-threatening anaphylactic reactions. The prevalence of crustacean allergy seems to vary largely between geographical locations, most probably as a result of the availability of seafood. The major shellfish allergen is tropomyosin, although other allergens may play an important part in allergenicity such as arginine kinase and myosin light chain. Current observations regard tropomyosin to be the major allergen responsible for molecular and clinical cross-reactivity between crustaceans and molluscs, but also to other inhaled invertebrates such as house dust mites and insects. Future research on the molecular structure of tropomyosins with a focus on the immunological and particularly clinical cross-reactivity will improve diagnosis and management of this potentially lifethreatening allergy and is essential for future immunotherapy.

Enhanced Protective Efficacy of a Chimeric Form of the Schistosomiasis Vaccine Antigen Sm-TSP-2

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund’s adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-c from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.

Kalata B8, a novel antiviral circular protein, exhibits conformational flexibility in the cystine knot motif

The cyclotides are a family of circular proteins with a range of biological activities and potential pharmaceutical and agricultural applications. The biosynthetic mechanism of cyclization is un- known and the discovery of novel sequences may assist in achiev- ing this goal. In the present study, we have isolated a new cyclo- tide from Oldenlandia affinis, kalata B8, which appears to be a hybrid of the two major subfamilies (Mo ̈bius and bracelet) of currently known cyclotides. We have determined the three-di- mensional structure of kalata B8 and observed broadening of resonances directly involved in the cystine knot motif, suggesting flexibility in this region despite it being the core structural element of the cyclotides. The cystine knot motif is widespread throughout Nature and inherently stable, making this apparent flexibility a surprising result. Furthermore, there appears to be isomerization of the peptide backbone at an Asp-Gly sequence in the region involved in the cyclization process. Interestingly, such isomerization has been previously characterized in related cyclic knottins from Momordica cochinchinensis that have no sequence similarity to kalata B8 apart from the six conserved cysteine residues and may result from a common mechanism of cyclization. Kalata B8 also provides insight into the structure– activity relationships of cyclotides as it displays anti-HIV activity but lacks haemolytic activity. The ‘uncoupling’ of these two activities has not previously been observed for the cyclotides and may be related to the unusual hydrophilic nature of the peptide.

Bioconjugates composed of chlorotoxin and near infrared fluorescent (NIRF) moieties are being advanced toward human clinical trials as intra-operative imaging agents that will enable surgeons to visualize small foci of cancer. In previous studies, the NIRF molecules were conjugated to chlorotoxin, which results in a mixture of mono-, di-, and tri-labeled peptide. Here we report a new chemical entity that bound only a single NIRF molecule. The lysines at positions 15 and 23 were substituted with either Ala or Arg, which resulted in only mono-labeled peptide that was functionally equivalent to native chlorotoxin:Cy5.5. We also analyzed the serum stability and serum half life of cyclized chlorotoxin, which showed an 11 hour serum half life and resulted in a mono-labeled product. Based on these data, we propose to advance a mono-labeled chlorotoxin to human clinical trials.

Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharma- ceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as tem- plates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge signifi- cance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applica- tions, including plant-produced designer peptide drugs.

Evidence for associations between the purinergic receptor P2X7 (P2RX7) and toxoplasmosis

Congenital Toxoplasma gondii infection can result in intracranial calcification, hydrocephalus, and retinochoroiditis. Acquired infection is commonly associated with ocular disease. Pathology is characterized by strong pro-inflammatory responses. Ligation of ATP by purinergic receptor P2X7, encoded by P2RX7, stimulates pro-inflammatory cytokines and can lead directly to killing

of intracellular pathogens. To determine whether P2X7 plays a role in susceptibility to congenital toxoplasmosis, we examined polymorphisms at P2RX7 in 149 child/parent trios from North America. We found association (FBAT Z scores ±2.429; P= 0.015) between the derived C(+)G() allele (f= 0.68; OR= 2.06; 95% CI: 1.14–3.75) at SNP rs1718119 (1068T>C; Thr-348-Ala), and a second synonymous variant rs1621388 in linkage disequilibrium with it, and clinical signs of disease per se. Analysis of clinical sub-groups showed no association with hydrocephalus, with effect sizes for associations with retinal disease and brain calcifications enhanced (OR=3.0 to 4.25; 0.004<P<0.009) when hydrocephalus was removed from the analysis. Association with toxoplasmic retinochoroiditis was replicated (FBAT Z scores ±3.089; P= 0.002) in a small family- based study (60 families; 68 affected offspring) of acquired infection in Brazil, where the ancestral T(+) allele (f= 0.296) at SNP rs1718119 was strongly protective (OR= 0.27; 95% CI: 0.09–0.80).

Sexual-stage glycoproteins of Eimeria are important components of the oocyst wall, a structure that ensures the efficient transmission of these and related parasites. In this study, the primary enzyme in the glycosylation pathway of Eimeria tenella, glucosamine:fructose-6-phosphate aminotransferase (EtGFAT), has been charac- terized as a macrogamete-specific protein. Although the transcription of EtGFAT was observed early in macrogamete development, protein expression was restricted to mature macrogametes, prior to their conver- sion into unsporulated oocysts. Genes coding for three other enzymes required for N-acetylgalactosamine (GalNAc) synthesis were also transcribed during E. tenella macrogamete development. Gene transcription of the enzyme responsible for the O-linked transfer of GalNAc to proteins, EtGalNAc-T, was upregulated primarily in unsporulated oocyst stages, and accordingly, a significant increase in GalNAc levels was observed in E. tenella gametocytes and oocysts. Gam56 and Gam82, two well-characterized glycoproteins of Eimeria macrogametes and the oocyst wall, contain high levels of GalNAc and represent probable targets of GalNAc O linkage. It appears that the glycosylation pathway, specifically relating to the formation of GalNAc O links, is dramatically upregulated in E. tenella sexual stages and may play a role in directing a number of macrogamete proteins to the developing oocyst wall.

The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-c, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other. Regardless, of whether F. hepatica infection preceded or succeeded T. gondii infection, there was little impact on the production of the Th1 cytokines IL-12, IFN-c or on the development of classically-activated macrophages induced by T. gondii. By contrast, the production of helminth-specific Th2 cytokines, such as IL-4 and IL-5, was suppressed by infection with T. gondii. Additionally, the recruitment and alternative activation of macrophages by F. hepatica was blocked or reversed by subsequent infection with T. gondii. The clinical symptoms of toxoplasmosis and the survival rate of infected mice were not significantly altered by the helminth. Despite previous studies showing that F. hepatica suppressed the classical activation of macrophages and the Th1-driven responses of mice to bystander microbial infection, as well as reduced their ability to reject these, here we found that the potent immune responses to T. gondii were capable of suppressing the responses to helminth infection. Clearly, the outcome of particular infections in polyparasitoses depends on the means and potency by which each pathogen controls the immune response.