Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

A herbivore tag-and-trace system reveals contact- and density-dependent repellence of a root toxin

Foraging behavior of root feeding organisms strongly affects plant-environment-interactions and ecosystem processes. However, the impact of plant chemistry on root herbivore movement in the soil is poorly understood. Here, we apply a simple technique to trace the movement of soil-dwelling insects in their habitats without disturbing or restricting their interactions with host plants. We tagged the root feeding larvae of Melolontha melolontha with a copper ring and repeatedly located their position in relation to their preferred host plant, Taraxacum officinale, using a commercial metal detector. This method was validated and used to study the influence of the sesquiterpene lactone taraxinic acid β-D-glucopyranosyl ester (TA-G) on the foraging of M. melolontha. TA-G is stored in the latex of T. officinale and protects the roots from herbivory. Using behavioral arenas with TA-G deficient and control plants, we tested the impact of physical root access and plant distance on the effect of TA-G on M. melolontha. The larvae preferred TA-G deficient plants to control plants, but only when physical root contact was possible and the plants were separated by 5 cm. Melolontha melolontha showed no preference for TA-G deficient plants when the plants were grown 15 cm apart, which may indicate a trade-off between the cost of movement and the benefit of consuming less toxic food. We demonstrate that M. melolontha integrates host plant quality and distance into its foraging patterns and suggest that plant chemistry affects root herbivore behavior in a plant-density dependent manner. © 2017, Springer Science+Business Media New York

Interaction of 8-Hydroxyquinoline with Soil Environment Mediates Its Ecological Function

Background: Allelopathic functions of plant-released chemicals are often studied through growth bioassays assuming that these chemicals will directly impact plant growth. This overlooks the role of soil factors in mediating allelopathic activities of chemicals, particularly non-volatiles. Here we examined the allelopathic potential of 8-hydroxyquinoline (HQ), a chemical reported to be exuded from the roots of Centaurea diffusa. Methodology/Principal Findings: Growth bioassays and HQ recovery experiments were performed in HQ-treated soils (non-sterile, sterile, organic matter-enriched and glucose-amended) and untreated control soil. Root growth of either Brassica campestris or Phalaris minor was not affected in HQ-treated non-sterile soil. Soil modifications (organic matter and glucose amendments) could not enhance the recovery of HQ in soil, which further supports the observation that HQ is not likely to be an allelopathic compound. Hydroxyquinoline-treated soil had lower values for the CO2 release compared to untreated non-sterile soil. Soil sterilization significantly influenced the organic matter content, PO 4-P and total organic nitrogen levels. Conclusion/Significance: Here, we concluded that evaluation of the effect of a chemical on plant growth is not enough in evaluating the ecological role of a chemical in plant-plant interactions. Interaction of the chemical with soil factors largel

Evaluation of groundwater quality and its suitability for drinking and agricultural use in Thanjavur city, Tamil Nadu, India

As groundwater is a vital source of water for domestic and agricultural activities in Thanjavur city due to lack of surface water resources, groundwater quality and its suitability for drinking and agricultural usage were evaluated. In this study, 102 groundwater samples were collected from dug wells and bore wells during March 2008 and analyzed for pH, electrical conductivity, temperature, major ions, and nitrate. Results suggest that, in 90% of groundwater samples, sodium and chloride are predominant cation and anion, respectively, and NaCl and CaMgCl are major water types in the study area. The groundwater quality in the study site is impaired by surface contamination sources, mineral dissolution, ion exchange, and evaporation. Nitrate, chloride, and sulfate concentrations strongly express the impact of surface contamination sources such as agricultural and domestic activities, on groundwater quality, and 13% of samples have elevated nitrate content (>45 mg/l as NO3). PHREEQC code and Gibbs plots were employed to evaluate the contribution of mineral dissolution and suggest that mineral dissolution, especially carbonate minerals, regulates water chemistry.Groundwater suitability for drinking usage was evaluated by the World Health Organization and Indian standards and suggests that 34% of samples are not suitable for drinking. Integrated groundwater suitability map for drinking purposes was created using drinking water standards based on a concept that if the groundwater sample exceeds any one of the standards, it is not suitable for drinking. This map illustrates that wells in zones 1, 2, 3, and 4 are not fit for drinking purpose. Likewise, irrigational suitability of groundwater in the study region was evaluated, and results suggest that 20% samples are not fit for irrigation. Groundwater suitability map for irrigation was also produced based on salinity and sodium hazards and denotes that wells mostly situated in zones 2 and 3 are not suitable for irrigation. Both integrated suitability maps for drinking and irrigation usage provide overall scenario about the groundwater quality in the study area. Finally, the study concluded that groundwater quality is impaired by man-made activities, and proper management plan is necessary to protect valuable groundwater resources inThanjavur city

Local Translation in Primary Afferent Fibers Regulates Nociception

Recent studies have demonstrated the importance of local protein synthesis for neuronal plasticity. In particular, local mRNA translation through the mammalian target of rapamycin (mTOR) has been shown to play a key role in regulating dendrite excitability and modulating long-term synaptic plasticity associated with learning and memory. There is also increased evidence to suggest that intact adult mammalian axons have a functional requirement for local protein synthesis in vivo. Here we show that the translational machinery is present in some myelinated sensory fibers and that active mTOR-dependent pathways participate in maintaining the sensitivity of a subpopulation of fast-conducting nociceptors in vivo. Phosphorylated mTOR together with other downstream components of the translational machinery were localized to a subset of myelinated sensory fibers in rat cutaneous tissue. We then showed with electromyographic studies that the mTOR inhibitor rapamycin reduced the sensitivity of a population of myelinated nociceptors known to be important for the increased mechanical sensitivity that follows injury. Behavioural studies confirmed that local treatment with rapamycin significantly attenuated persistent pain that follows tissue injury, but not acute pain. Specifically, we found that rapamycin blunted the heightened response to mechanical stimulation that develops around a site of injury and reduced the long-term mechanical hypersensitivity that follows partial peripheral nerve damage - a widely used model of chronic pain. Our results show that the sensitivity of a subset of sensory fibers is maintained by ongoing mTOR-mediated local protein synthesis and uncover a novel target for the control of long-term pain states

PAX4 Enhances Beta-Cell Differentiation of Human Embryonic Stem Cells

Background Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of β-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. Methods and Findings Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a β-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca2+ homeostasis of β-cells developing in embryoid bodies produced from such HESC. Cells expressing key β-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. Conclusion Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative β-cells. Our findings provide a novel foundation to study the mechanism of pancreatic β-cells differentiation during early human development and to help evaluate strategies for the generation of purified β-cells for future clinical applications

Effect of Supplementation with Zinc and Other Micronutrients on Malaria in Tanzanian Children: A Randomised Trial

Hans Verhoef and colleagues report findings from a randomized trial conducted among Tanzanian children at high risk for malaria. Children in the trial received either daily oral supplementation with either zinc alone, multi-nutrients without zinc, multi-nutrients with zinc, or placebo. The investigators did not find evidence from this study that zinc or multi-nutrients protected against malaria episodes