Options for active case detection of visceral leishmaniasis in endemic districts of India, Nepal and Bangladesh, comparing yield, feasibility and costs

Background

The VL elimination strategy requires cost-effective tools for case detection and management. This intervention study tests the yield, feasibility and cost of 4 different active case detection (ACD) strategies (camp, index case, incentive and blanket approach) in VL endemic districts of India, Nepal and Bangladesh.

Methodology/Principal Findings

First, VL screening (fever more than 14 days, splenomegaly, rK39 test) was performed in camps. This was followed by house to house screening (blanket approach). An analysis of secondary VL cases in the neighborhood of index cases was simulated (index case approach). A second screening round was repeated 4–6 months later. In another sub-district in India and Nepal, health workers received incentives for detecting new VL cases over a 4 month period (incentive approach). This was followed by house screening for undetected cases. A total of 28 new VL cases were identified by blanket approach in the 1^st screening round, and used as ACD gold standard. Of these, the camp approach identified 22 (sensitivity 78.6%), index case approach identified 12 (sensitivity – 42.9%), and incentive approach identified 23 new VL cases out of 29 cases detected by the house screening (sensitivity – 79.3%). The effort required to detect a new VL case varied (blanket approach – 1092 households, incentive approach – 978 households; index case approach – 788 households had to be screened). The cost per new case detected varied (camp approach $21 – $661; index case approach $149 – $200; incentive based approach $50 – $543; blanket screening $112 – $629). The 2^nd screening round yielded 20 new VL cases. Sixty and nine new PKDL cases were detected in the first and second round respectively.

Conclusions/Significance

ACD in the VL elimination campaign has a high yield of new cases at programme costs which vary according to the screening method chosen. Countries need the right mix of approaches according to the epidemiological profile, affordability and organizational feasibility.     

A Suppressor Screen of the Chimeric AtCNGC11/12 Reveals Residues Important for Intersubunit Interactions of Cyclic Nucleotide-Gated Ion Channels

To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.Cyclic nucleotide-gated ion channels (CNGCs) were first discovered in retinal photoreceptors and olfactory sensory neurons (Zagotta and Siegelbaum, 1996; Kaupp and Seifert, 2002). CNGCs play crucial roles for the signal transduction in these neurons that are excited by photons and odorants, respectively. In mammalian genomes, six CNGC genes have been found and named CNGA1 to CNGA4, CNGB1, and CNGB3 (Kaupp and Seifert, 2002). It has been reported that in mammalian cells, CNGCs function as heterotetramers that are composed of A and B subunits with cell-specific stoichiometry (Kaupp and Seifert, 2002; Cukkemane et al., 2011). For example, CNGCs in rod photoreceptors are composed of three A1 subunits and one B1a subunit, whereas in cone photoreceptors, they are believed to be composed of two A3 and two B3 subunits (Zhong et al., 2002; Peng et al., 2004). The structure of each subunit is similar to that of the voltage-gated K⁺-selective ion channel (Shaker) proteins, including a cytoplasmic N terminus, six membrane-spanning regions (S1–S6), a pore domain located between S5 and S6, and a cytoplasmic C terminus (Zagotta and Siegelbaum, 1996). However, CNGCs are only weakly voltage dependent and are opened by the direct binding of cyclic nucleotides (cAMP and cGMP), which are universally important secondary messengers that control diverse cellular responses (Fesenko et al., 1985). The cytoplasmic C terminus contains a cyclic nucleotide-binding domain (CNBD) and a C-linker region that connects the CNBD to the S6 domain. CNGC activity is also regulated by feedback inhibitory mechanisms involving the Ca²⁺ sensor protein, calmodulin (CaM). CaM-binding sites in animal CNGCs have been found in various regions of both the C- and N-terminal domains (Ungerer et al., 2011). It has been reported that the subunit composition has significant influence on the mode of CaM-mediated regulation (Kramer and Siegelbaum, 1992; Bradley et al., 2004; Song et al., 2008).On the other hand, plant CNGCs have only been investigated much more recently. The first plant CNGC, HvCBT1, was identified as a CaM-binding transporter protein in barley (Hordeum vulgare; Schuurink et al., 1998). Subsequently, several CNGCs were identified from Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum; Arazi et al., 1999; Köhler and Neuhaus, 1998; Köhler et al., 1999). Interestingly, the Arabidopsis genome sequencing project identified a large family comprising 20 members (AtCNGC1–AtCNGC20), indicating a significant expansion of Arabidopsis CNGCs that suggests a higher level of diversity and functional importance in plants (Mäser et al., 2001). To date, possible biological functions of Arabidopsis CNGCs in development, ion homeostasis, thermal sensing, as well as pathogen resistance have been reported (Kaplan et al., 2007; Chin et al., 2009; Dietrich et al., 2010; Moeder et al., 2011; Finka et al., 2012). With respect to structure, plant CNGCs are believed to have a similar architecture to their animal counterparts (Chin et al., 2009). However, only a handful of studies on the structure-function analysis of plant CNGCs have been published so far, and this field is still very much in its infancy (Hua et al., 2003; Bridges et al., 2005; Kaplan et al., 2007; Baxter et al., 2008; Chin et al., 2010).Previously, we have reported two functionally important residues in plant CNGCs (Baxter et al., 2008; Chin et al., 2010). These residues were discovered using a suppressor screen of the rare gain-of-function Arabidopsis mutant constitutive expresser of PR genes22 (cpr22; Yoshioka et al., 2006). The cpr22 mutant, which has a deletion between AtCNGC11 and AtCNGC12 resulting in a novel but functional chimeric CNGC (AtCNGC11/12), exhibits multiple resistance responses without pathogen infection in the hemizygous state and conditional lethality in the homozygous state (Yoshioka et al., 2001, 2006; Moeder et al., 2011). It has been reported that the cpr22 phenotype is attributable to the expression of AtCNGC11/12 and its channel activity (Yoshioka et al., 2006; Baxter et al., 2008), thereby making the suppressor screen an invaluable tool for identifying intragenic mutants to further elucidate the structure-function relationship of plant CNGCs (Baxter et al., 2008; Chin et al., 2010).In this study, we describe a total of 29 mutant alleles of AtCNGC11/12, including the three previously published alleles (Baxter et al., 2008; Chin at al., 2010), and compare their predicted three-dimensional structural positions with equivalent mutations of a human CNGC, CNGA3. In this analysis, two AtCNGC11/12 mutations emerged as counterparts of human mutations (Wissinger et al., 2001). Both the AtCNGC11/12 as well as the human CNGA3 mutations were computationally predicted to affect intersubunit interactions. This prediction was experimentally validated by size-exclusion chromatography (FPLC) as well as bimolecular fluorescence complementation (BiFC) in combination with site-direct mutagenesis using recombinant C-terminal peptides.     

Association of Single Nucleotide Polymorphisms in Wnt Signaling Pathway Genes with Breast Cancer in Saudi Patients

Breast cancer is a complex heterogeneous disease involving genetic and epigenetic alterations in genes encoding proteins that are components of various signaling pathways. Candidate gene approach have identified association of genetic variants in the Wnt signaling pathway genes and increased susceptibility to several diseases including breast cancer. Due to the rarity of somatic mutations in key genes of Wnt pathway, we investigated the association of genetic variants in these genes with predisposition to breast cancers. We performed a case-control study to identify risk variants by examining 15 SNPs located in 8 genes associated with Wnt signaling. Genotypic analysis of individual locus showed statistically significant association of five SNPs located in β-catenin, AXIN2, DKK3, SFRP3 and TCF7L2 with breast cancers. Increased risk was observed only with the SNP in β-catenin while the other four SNPs conferred protection against breast cancers. Majority of these associations persisted after stratification of the cases based on estrogen receptor status and age of on-set of breast cancer. The rs7775 SNP in exon 6 of SFRP3 gene that codes for either arginine or glycine exhibited very strong association with breast cancer, even after Bonferroni''s correction. Apart from these five variants, rs3923086 in AXIN2 and rs3763511 in DKK4 that did not show any association in the overall population were significantly associated with early on-set and estrogen receptor negative breast cancers, respectively. This is the first study to utilize pathway based approach to identify association of risk variants in the Wnt signaling pathway genes with breast cancers. Confirmation of our findings in larger populations of different ethnicities would provide evidence for the role of Wnt pathway as well as screening markers for early detection of breast carcinomas.     

Resolvin D1 Prevents the Impairment in the Retention Memory and Hippocampal Damage in Rats Fed a Corn Oil-Based High Fat Diet by Upregulation of Nrf2 and Downregulation and Inactivation of p66Shc

This study investigated the effect of a high-fat diet rich in corn oil (CO-HFD) on the memory retention and hippocampal oxidative stress, inflammation, and apoptosis in rats, and examined if the underlying mechanisms involve modulating Resolvin D1 (RvD1) levels and activation of p⁶⁶Shc. Also, we tested if co-administration of RvD1 could prevent these neural adverse effects induced by CO-HFD. Adult male Wistar rats were divided into 4 groups (n?=?18/each) as control fed standard diet (STD) (3.82 kcal/g), STD?+?RvD1 (0.2 µg/Kg, i.p/twice/week), CO-HFD (5.4 kcal/g), and CO-HFD?+?RvD1. All treatments were conducted for 8 weeks. With normal fasting glucose levels, CO-HFD induced hyperlipidemia, hyperinsulinemia, increased HOMA-IRI and reduced the rats’ memory retention. In parallel, CO-HFD increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), cytoplasmic cytochrome-c, and cleaved caspase-3 and significantly decreased levels of glutathione (GSH), Bcl-2, and manganese superoxide dismutase (MnSOD) in rats’ hippocampi. Besides, CO-HFD significantly reduced hippocampal levels of docosahexaenoic acid (DHA) and RvD1, as well as total protein levels of Nrf2 and significantly increased nuclear protein levels of p-NF-κB. Concomitantly, CO-HFD increased hippocampal protein levels of p-JNK, p53, p⁶⁶Shc, p-p⁶⁶Shc, and NADPH oxidase. However, without altering plasma and serum levels of glucose, insulin, and lipids, co-administration of RvD1 to CO-HFD completely reversed all these events. It also resulted in similar effects in the STD fed-rats. In conclusion, CO-HFD impairs memory function and induces hippocampal damage by reducing levels of RvD1 and activation of JNK/p53/p⁶⁶Shc/NADPH oxidase, effects that are prevented by co-administration of RvD1.

     

The identification of 4,7-disubstituted naphthoic acid derivatives as UDP-competitive antagonists of P2Y14

A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY₁₄ with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y₁₄ with a good pharmacokinetic profile.     

14-3-3 Binding to ataxin-1(ATXN1) regulates its dephosphorylation at Ser-776 and transport to the nucleus

Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65.     

Optical study of stress hormone‐induced nanoscale structural alteration in brain using partial wave spectroscopic microscopy

Chronic stress affects nano to microscale structures of the brain cells/tissues due to suppression of neural growths and reconnections, hence the neuronal activities. This results in depression, memory loss and even death of the brain cells. Our recently developed novel optical technique, partial wave spectroscopic microscopy has nanoscale sensitivity, and hence, can detect nanoscale changes in brain tissues due to stress. In this study, we applied this technique to quantify the stress related structural changes in the corticosterone‐treated mouse model of stress. Our results show that brains from corticosterone‐treated mice showed higher nanoscale structural disorder in the hippocampal region as compared to the brain from normal (vehicle) mice. The increase in structural alteration correlates with the duration of the stress. We further quantified the relative changes and the spatial localization of these changes in this mouse model and found out that the maximum changes occurred nearly symmetrically in both regions of the hippocampus. The mRNA for stress‐related genes, brain‐derived neurotrophic factor and tyrosine kinase‐coupled receptor were also significantly reduced in the hippocampus of corticosterone‐treated mice compared to that in control mice. These results indicate that chronic corticosterone treatment induces nanoscale structural alterations in mouse brain that corresponds to changes in stress‐related gene expression.     

Biophysical Studies of Bacterial Topoisomerases Substantiate Their Binding Modes to an Inhibitor

Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G⁺) and Gram-negative (G⁻) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G⁺ strain (Streptococcus pneumoniae (sParE)) and a G⁻ strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between β2 and α3, and the β2 and β6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher K_D than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.