Drosophila melanogaster as a function-based high-throughput screening model for antinephrolithiasis agents in kidney stone patients.

Kidney stone disease involves the aggregation of stone-forming salts consequent to solute supersaturation in urine. The development of novel therapeutic agents for this predominantly metabolic and biochemical disorder have been hampered by the lack of a practical pre-clinical model amenable to drug screening. Here, Drosophila melanogaster, an emerging model for kidney stone disease research, was adapted as a high-throughput functional drug screening platform independent of the multifactorial nature of mammalian nephrolithiasis. Through functional screening, the therapeutic potential of a novel compound commonly known as arbutin that specifically binds to oxalate, a key component of kidney calculi, was identified. Through isothermal titration calorimetry, high-performance liquid chromatography and atomic force microscopy, arbutin was determined to interact with calcium and oxalate in both free and bound states, disrupting crystal lattice structure, growth and crystallization. When used to treat patient urine samples, arbutin significantly abrogated calculus formation in vivo and outperformed potassium citrate in low pH urine conditions, owing to its oxalate-centric mode of action. The discovery of this novel antilithogenic compound via D. melanogaster, independent of a mammalian model, brings greater recognition to this platform, for which metabolic features are primary outcomes, underscoring the power of D. melanogaster as a high-throughput drug screening platform in similar disorders. This is the first description of the use of D. melanogaster as the model system for a high-throughput chemical library screen. This article has an associated First Person interview with the first authors of the paper.

Identification of Dermcidin as a novel binding protein of Nck1 and characterization of its role in promoting cell migration.

A distinct feature of hepatocellular carcinoma (HCC) is the tendency of tumor cells to disperse throughout the liver. Nck family adaptor proteins function to couple tyrosine phosphorylation signals to regulate actin cytoskeletal reorganization that leads to cell motility. In order to explore the role of Nck in HCC development, we performed GST pull-down assay using the SH2 domain of Nck1 as bait. The resulting precipitates were separated by 2-DE. Mass spectrometry analysis revealed a group of Nck1 SH2 domain-binding proteins that were differentially expressed in HCC. One of these proteins, dermcidin (DCD), and its interaction with Nck1, was further validated in vitro. GST pull-down assay revealed that Nck1 SH2 domain binds to the phosphotyrosine residue at position 20 (Y20) of the DCD. Pervandate treatment significantly enhanced the interaction between DCD and Nck1. Moreover, we demonstrated that forced expression of DCD could activate Rac1 and Cdc42 and promoted cell migration. Taken together, these data suggest a role of DCD in tumor metastasis.

Bisdesmosidic saponins from Securidaca longepedunculata roots: evaluation of deterrency and toxicity to Coleopteran storage pests.

Powdered dry root bark of Securidaca longepedunculata was mixed with maize and cowpea and effectively reduced the numbers of Sitophilus zeamais and Callosobruchus maculatus emerging from these commodities, respectively, more than 9 months after treatment. This effect was reciprocated in grain treated with a methanol extract of the root bark, indicating that compounds were present that were oviposition deterrents or directly toxic to the adults or larvae. Two new bisdesmosidic saponins, 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(4-methoxycinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside A) and 3-O-beta-D-glucopyranosyl-28-O-(alpha-L-arabinopyranosyl-(1 --> 3)-beta-D-xylopyranosyl-(1 --> 4)[beta-D-apiofuranosyl-(1 --> 3)]-alpha-L-rhamnopyranosyl-(1 --> 2)-[4-O-(3,4,5-trimethoxy-(E)-cinnamoyl-beta-D-fucopyranosyl)])-medicagenic acid (securidacaside B), were isolated from the methanol extract of the roots of S. longepedunculata and characterized by spectroscopic methods. Securidacaside A, which occurred as (E)- and (Z)-regioisomers, showed deterrency and toxicity toward C. maculatus and S. zeamais and could contribute to the biological activity of the methanol extract. The potential to optimize the use of this plant for stored product protection using water extracts, which would be appropriate technology for target farmers, is discussed.

Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization.

We have analyzed the means by which the Nck family of adaptor proteins couples adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. The clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its Src homology 2 (SH2) domain and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit the high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic Escherichia coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation.