Soil-transmitted helminth infections among plantation sector schoolchildren in Sri Lanka: prevalence after ten years of preventive chemotherapy

Background

The plantation sector in Sri Lanka lags behind the rest of the country in terms of living conditions and health. In 1992, a sector-wide survey of children aged 3–12 years and women of reproductive age showed >90% prevalence of soil-transmitted helminth infections. Biannual mass de-worming targeting children aged 3–18 years started in 1994 and was continued until 2005. The present study was carried out to assess the status of infection four years after cessation of mass de-worming.

Methods/Findings

A school-based cross-sectional survey was carried out. Faecal samples from approximately 20 children from each of 114 schools in five districts were examined using the modified Kato-Katz technique. Data regarding the school, the child''s family and household sanitation were recorded after inspection of schools and households. Multivariate analysis was carried out using logistic regression, to identify risk factors for infection. Faecal samples were obtained from 1890 children. In 4/5 districts, >20% were infected with one or more helminth species. Overall combined prevalence was 29.0%; 11.6% had infections of moderate-heavy intensity. The commonest infection was Ascaris lumbricoides, present in all five districts, as was Trichuris trichiura. Hookworm was not detected in two districts. Multivariate analysis identified low altitude and maternal under-education as risk factors for all three infections. Poor household sanitation was identified as a risk factor for A. lumbricoides and hookworm, but not T. trichiura infections.

Conclusions/Significance

The results indicate that regular mass de-worming of plantation sector children should be resumed along with more emphasis on better sanitation and health education. They show that even after 10 years of mass chemotherapy, prevalence can bounce back after cessation of preventive chemotherapy, if the initial force of transmission is strong and other long-term control measures are not concomitantly implemented.     

RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice

Mice with a malignant hyperthermia mutation (Y522S) in the ryanodine receptor (RyR1) display muscle contractures, rhabdomyolysis, and death in response to elevated environmental temperatures. We demonstrate that this mutation in RyR1 causes Ca(2+) leak, which drives increased generation of reactive nitrogen species (RNS). Subsequent S-nitrosylation of the mutant RyR1 increases its temperature sensitivity for activation, producing muscle contractures upon exposure to elevated temperatures. The Y522S mutation in humans is associated with central core disease. Many mitochondria in the muscle of heterozygous Y522S mice are swollen and misshapen. The mutant muscle displays decreased force production and increased mitochondrial lipid peroxidation with aging. Chronic treatment with N-acetylcysteine protects against mitochondrial oxidative damage and the decline in force generation. We propose a feed-forward cyclic mechanism that increases the temperature sensitivity of RyR1 activation and underlies heat stroke and sudden death. The cycle eventually produces a myopathy with damaged mitochondria.     

3'-Azido-3'-deoxythymidine-(5')-tetraphospho-(5')-adenosine, the product of ATP-mediated excision of chain-terminating AZTMP, is a potent chain-terminating substrate for HIV-1 reverse transcriptase

The resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) involves phosphorolytic excision of chain-terminating AZT-5'-monophosphate (AZTMP). Both pyrophosphate (PPi) and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. PPi-mediated excision produces AZT-5'-triphosphate (AZTTP), which could be immediately re-used as a substrate for viral DNA chain termination. In contrast, ATP-mediated excision produces the novel compound AZT-(5')-tetraphospho-(5')-adenosine (AZTp4A). Since little is known of the interaction of AZTp4A with HIV-1 RT, we carried out kinetic and molecular modeling studies to probe this. AZTp4A was found to be a potent inhibitor of HIV-1 RT-catalyzed DNA synthesis and of both ATP- and PPi-mediated AZTMP excision. AZTp4A is in fact an excellent chain-terminating substrate for AZT-resistant RT-catalyzed DNA synthesis, better than AZTTP (k(pol)/Kd = 6.2 and 11.9 for AZTTP and AZTp4A, respectively). The affinity of AZT-resistant HIV-1 RT for AZTp4A is at least 30,000-fold greater than that for the excision substrate ATP and approximately 10-fold greater than that for AZTTP. Dissociation of newly formed AZTp4A from RT may therefore provide a significant rate-limiting step for continued HIV-1 DNA synthesis. Our studies show that the products of PPi- and ATP-mediated excision of chain-terminating AZTMP (AZTTP and AZTp4A, respectively) are both potent chain-terminating substrates for HIV-1 RT, suggesting that there is no obvious benefit to HIV using ATP instead of PPi as the excision substrate.     

A slow-tight binding inhibitor of dopamine beta-monooxygenase: a transition state analogue for the product release step

The steady-state kinetic data show that 3-hydroxy-4-phenylthiazole-2(3H)-thione (3H4PTT) is a potent tight-binding inhibitor for dopamine beta-monooxygenase (DbetaM) with a dissociation constant of 0.9 nM. Ackermann-Potter plots of the enzyme dependence of the inhibition revealed that the stoichiometry of the enzyme inhibition by 3H4PTT is 1:1. Pre-steady-state progress curves at varying inhibitor with fixed reductant and enzyme concentrations clearly show the slow binding behavior of the inhibitor. The observed kinetic behavior is consistent with the apparent direct formation of the tightly bound E x I* complex. The k(on) and k(off) for 3H4PTT which were determined under pre-steady-state conditions at variable inhibitor concentrations were found to be (1.85 +/- 0.07) x 10(6) M(-1) s(-1) and (1.9 +/- 0.6) x 10(-3) s(-1), respectively. The dissociation constant calculated from these rates was similar to that determined under steady-state conditions, confirming that 3H4PTT is a kinetically well-behaved inhibitor. The steady-state as well as pre-steady-state kinetic studies at variable DMPD concentrations show that the inhibition is competitive with respect to the reductant, demonstrating the exclusive interaction of 3H4PTT with the oxidized form of the enzyme. The kinetic behavior and the structural properties of 3H4PTT are consistent with the proposal that the E x 3H4PTT complex may mimic the transition state for the product (protonated) release step of the enzyme. Therefore, 3H4PTT could be used as a convenient probe to examine the properties of the E x P complex of the DbetaM reaction and also as an active site titrant for the oxidized enzyme.     

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.     

Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram‐positive pathogens. Teicoplanin is distinguished from the vancomycin‐type glycopeptide antibiotics, by the presence of an additional cross‐link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol‐coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2‐Å resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron‐bound water molecule. Sequence comparisons with other phenol‐coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross‐linking mechanisms that occur during glycopeptide antibiotics biosynthesis. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Differences in Breast Cancer Survival between Public and Private Care in New Zealand: Which Factors Contribute?

BackgroundPatients who received private health care appear to have better survival from breast cancer compared to those who received public care. This study investigated if this applied to New Zealand women and identified factors that could explain such disparities.MethodsThis study involved all women who were diagnosed with primary breast cancer in two health regions in New Zealand, covering about 40% of the national population, between June 2000 and May 2013. Patients who received public care for primary treatment, mostly surgical treatment, were compared with those who received private care in terms of demographics, mode of presentation, disease factors, comorbidity index and treatment factors. Cox regression modelling was performed with stepwise adjustments, and hazards of breast cancer specific mortality associated with the type of health care received was assessed.ResultsOf the 14,468 patients, 8,916 (61.6%) received public care. Compared to patients treated in private care facilities, they were older, more likely to be Māori, Pacifika or Asian and to reside in deprived neighbourhoods and rural areas, and less likely to be diagnosed with early staged cancer and to receive timely cancer treatments. They had a higher risk of mortality from breast cancer (hazard ratio: 1.95; 95% CI: 1.75, 2.17), of which 80% (95% CI: 63%, 100%) was explained by baseline differences, particularly related to ethnicity, stage at diagnosis and type of loco-regional therapy. After controlling for these demographic, disease and treatment factors, the risk of mortality was still 14% higher in the public sector patients.ConclusionsEthnicity, stage at diagnosis and type of loco-regional therapy were the three key contributors to survival disparities between patients treated in public and private health care facilities in New Zealand. The findings underscore the need for more efforts to improve the quality, timeliness and equitability of public cancer care services.     

Allelic variation in the Depressaria pastinacella CYP6AB3 protein enhances metabolism of plant allelochemicals by altering a proximal surface residue and potential interactions with cytochrome P450 reductase

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V(max) of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V(max) of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val(92) on the proximal face of CYP6AB3v1 with Ala(92) in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants.     

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

Unregulated Ca²⁺ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na⁺-Ca²⁺ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd^−/−), Dysf^−/−, and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd^−/− mice. Measured increases in baseline Na⁺ and Ca²⁺ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca²⁺ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca²⁺ levels. Indeed, Atp1a2^+/− (encoding Na⁺-K⁺ ATPase α2) mice, which have reduced Na⁺ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na⁺-K⁺ ATPase inhibitor digoxin. Treatment of Sgcd^−/− mice with ranolazine, a broadly acting Na⁺ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.