The Sigma Class glutathione transferase from the liver fluke Fasciola hepatica

BACKGROUND: Liver fluke infection of livestock causes economic losses of over US$ 3 billion worldwide per annum. The disease is increasing in livestock worldwide and is a re-emerging human disease. There are currently no commercial vaccines, and only one drug with significant efficacy against adult worms and juveniles. A liver fluke vaccine is deemed essential as short-lived chemotherapy, which is prone to resistance, is an unsustainable option in both developed and developing countries. Protein superfamilies have provided a number of leading liver fluke vaccine candidates. A new form of glutathione transferase (GST) family, Sigma class GST, closely related to a leading Schistosome vaccine candidate (Sm28), has previously been revealed by proteomics in the liver fluke but not functionally characterised. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript we show that a purified recombinant form of the F. hepatica Sigma class GST possesses prostaglandin synthase activity and influences activity of host immune cells. Immunocytochemistry and western blotting have shown the protein is present near the surface of the fluke and expressed in eggs and newly excysted juveniles, and present in the excretory/secretory fraction of adults. We have assessed the potential to use F. hepatica Sigma class GST as a vaccine in a goat-based vaccine trial. No significant reduction of worm burden was found but we show significant reduction in the pathology normally associated with liver fluke infection. CONCLUSIONS/SIGNIFICANCE: We have shown that F. hepatica Sigma class GST has likely multi-functional roles in the host-parasite interaction from general detoxification and bile acid sequestration to PGD synthase activity

Quantitative Analysis of Immune Response and Erythropoiesis during Rodent Malarial Infection

Malarial infection is associated with complex immune and erythropoietic responses in the host. A quantitative understanding of these processes is essential to help inform malaria therapy and for the design of effective vaccines. In this study, we use a statistical model-fitting approach to investigate the immune and erythropoietic responses in Plasmodium chabaudi infections of mice. Three mouse phenotypes (wildtype, T-cell-deficient nude mice, and nude mice reconstituted with T-cells taken from wildtype mice) were infected with one of two parasite clones (AS or AJ). Under a Bayesian framework, we use an adaptive population-based Markov chain Monte Carlo method and fit a set of dynamical models to observed data on parasite and red blood cell (RBC) densities. Model fits are compared using Bayes' factors and parameter estimates obtained. We consider three independent immune mechanisms: clearance of parasitised RBCs (pRBC), clearance of unparasitised RBCs (uRBC), and clearance of parasites that burst from RBCs (merozoites). Our results suggest that the immune response of wildtype mice is associated with less destruction of uRBCs, compared to the immune response of nude mice. There is a greater degree of synchronisation between pRBC and uRBC clearance than between either mechanism and merozoite clearance. In all three mouse phenotypes, control of the peak of parasite density is associated with pRBC clearance. In wildtype mice and AS-infected nude mice, control of the peak is also associated with uRBC clearance. Our results suggest that uRBC clearance, rather than RBC infection, is the major determinant of RBC dynamics from approximately day 12 post-innoculation. During the first 2–3 weeks of blood-stage infection, immune-mediated clearance of pRBCs and uRBCs appears to have a much stronger effect than immune-mediated merozoite clearance. Upregulation of erythropoiesis is dependent on mouse phenotype and is greater in wildtype and reconstitited mice. Our study highlights the informative power of statistically rigorous model-fitting techniques in elucidating biological systems

The Ruegeria pomeroyi acuI Gene Has a Role in DMSP Catabolism and Resembles yhdH of E. coli and Other Bacteria in Conferring Resistance to Acrylate

The Escherichia coli YhdH polypeptide is in the MDR012 sub-group of medium chain reductase/dehydrogenases, but its biological function was unknown and no phenotypes of YhdH− mutants had been described. We found that an E. coli strain with an insertional mutation in yhdH was hyper-sensitive to inhibitory effects of acrylate, and, to a lesser extent, to those of 3-hydroxypropionate. Close homologues of YhdH occur in many Bacterial taxa and at least two animals. The acrylate sensitivity of YhdH− mutants was corrected by the corresponding, cloned homologues from several bacteria. One such homologue is acuI, which has a role in acrylate degradation in marine bacteria that catabolise dimethylsulfoniopropionate (DMSP) an abundant anti-stress compound made by marine phytoplankton. The acuI genes of such bacteria are often linked to ddd genes that encode enzymes that cleave DMSP into acrylate plus dimethyl sulfide (DMS), even though these are in different polypeptide families, in unrelated bacteria. Furthermore, most strains of Roseobacters, a clade of abundant marine bacteria, cleave DMSP into acrylate plus DMS, and can also demethylate it, using DMSP demethylase. In most Roseobacters, the corresponding gene, dmdA, lies immediately upstream of acuI and in the model Roseobacter strain Ruegeria pomeroyi DSS-3, dmdA-acuI were co-regulated in response to the co-inducer, acrylate. These observations, together with findings by others that AcuI has acryloyl-CoA reductase activity, lead us to suggest that YdhH/AcuI enzymes protect cells against damaging effects of intracellular acryloyl-CoA, formed endogenously, and/or via catabolising exogenous acrylate. To provide “added protection” for bacteria that form acrylate from DMSP, acuI was recruited into clusters of genes involved in this conversion and, in the case of acuI and dmdA in the Roseobacters, their co-expression may underpin an interaction between the two routes of DMSP catabolism, whereby the acrylate product of DMSP lyases is a co-inducer for the demethylation pathway

Area-level socioeconomic characteristics and incidence of metabolic syndrome: a prospective cohort study

BACKGROUND The evidence linking socioeconomic environments and metabolic syndrome (MetS) has primarily been based on cross-sectional studies. This study prospectively examined the relationships between area-level socioeconomic position (SEP) and the incidence of MetS. METHODS A prospective cohort study design was employed involving 1,877 men and women aged 18+ living in metropolitan Adelaide, Australia, all free of MetS at baseline. Area-level SEP measures, derived from Census data, included proportion of residents completing a university education, and median household weekly income. MetS, defined according to International Diabetes Federation, was ascertained after an average of 3.6 years follow up. Associations between each area-level SEP measure and incident MetS were examined by Poisson regression Generalised Estimating Equations models. Interaction between area- and individual-level SEP variables was also tested. RESULTS A total of 156 men (18.7%) and 153 women (13.1%) developed MetS. Each percentage increase in the proportion of residents with a university education corresponded to a 2% lower risk of developing MetS (age and sex-adjusted incidence risk ratio (RR) = 0.98; 95% confidence interval (CI) =0.97-0.99). This association persisted after adjustment for individual-level income, education, and health behaviours. There was no significant association between area-level income and incident MetS overall. For the high income participants, however, a one standard deviation increase in median household weekly income was associated with a 29% higher risk of developing MetS (Adjusted RR = 1.29; 95%CI = 1.04-1.60). CONCLUSIONS While area-level education was independently and inversely associated with the risk of developing MetS, the association between area-level income and the MetS incidence was modified by individual-level income.Anh D Ngo, Catherine Paquet, Natasha J Howard, Neil T Coffee, Robert Adams, Anne Taylor and Mark Danie

Pre-referral rectal artesunate is no “magic bullet” in weak health systems

Abstract Severe malaria is a potentially fatal condition that requires urgent treatment. In a clinical trial, a sub-group of children treated with rectal artesunate (RAS) before being referred to a health facility had an increased chance of survival. We recently published in BMC Medicine results of the CARAMAL Project that did not find the same protective effect of pre-referral RAS implemented at scale under real-world conditions in three African countries. Instead, CARAMAL identified serious health system shortfalls that impacted the entire continuum of care, constraining the effectiveness of RAS. Correspondence to the article criticized the observational study design and the alleged interpretation and consequences of our findings. Here, we clarify that we do not dispute the life-saving potential of RAS, and discuss the methodological criticism. We acknowledge the potential for confounding in observational studies. Nevertheless, the totality of CARAMAL evidence is in full support of our conclusion that the conditions under which RAS can be beneficial were not met in our settings, as children often failed to complete referral and post-referral treatment was inadequate. The criticism did not appear to acknowledge the realities of highly malarious settings documented in detail in the CARAMAL project. Suggesting that trial-demonstrated efficacy is sufficient to warrant large-scale deployment of pre-referral RAS ignores the paramount importance of functioning health systems for its delivery, for completing post-referral treatment, and for achieving complete cure. Presenting RAS as a “magic bullet” distracts from the most urgent priority: fixing health systems so they can provide a functioning continuum of care and save the lives of sick children. The data underlying our publication is freely accessible on Zenodo

Expression and purification of recombinant FhGST-S1.

<p>A) ESI mass spectrum of the GSH-affinity purified rFhGST-S1 showing the MW of rFhGST-S1 at 24536.39±0.77 Da. B) SDS-PAGE gel of the expression and purification of rFhGST-S1. Lane 1. <i>E. coli</i> total cytosolic protein. Lane 2. GSH-affinity purified recombinant rFhGST-S1 protein. Ran on 12.5% SDS PAGE and coomassie blue stained.</p

Western Blotting localising FhGST-S1 in embryonating eggs, NEJs, adults and adult ES products.

<p>10 µg of each protein sample was resolved through 14% SDS PAGE and electrophoretically transferred to Hybond C nitrocellulose membrane. Membranes were amido black stained membrane to assess protein transfer (A) Membranes were incubated with anti-FhGST-S1 antibody diluted 1∶30,000 and developed using the BCIP/NBT liquid substrate system according to manufacturer's instructions (B). 1: Low Molecular Weight Marker (GE Biosciences); 2: Day 0 Egg; 3: Day 2 Egg; 4: Day 7 Egg; 5: Day 9 Egg; 6: NEJ Somatic Sample; 7: Adult Somatic Sample; 8: NEJ ES Products; 9: Adult ES Products; 10: Uninfected <i>Galba truncatula</i> –ve Control.</p