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.

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helixloop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

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.

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop...

Our results show that KIM-1 is a promising biomarker of subclinical AKI associated with hydronephrosis in urological patients. NGAL values were influenced by the presence of leukocyturia, limiting its usefulness in this population.

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. .

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The release of extracellular vesicles (EV) from human multipotent stromal cells (MSC) has been proposed as a mechanism by which MSC mediate regenerative functions in vivo. Our recent work has characterized MSC derived from human pancreatic tissues (Panc-MSC) that generated a tissue regenerative secretome which could be used as an injectable biotherapeutic agent in vivo. Despite these advancements, it remains unknown whether tissue regenerative stimuli released by Panc-MSC are released independent or within extracellular vesicles (EVs).Herein, this study demonstrates ultrafiltration is a simple method to enrich for EVs which can be injected in murine models of blood vessel or pancreatic islet regeneration. The enrichment of EVs from Panc-MSC conditioned media (CM) was validated using nanoscale flow cytometry and atomic force microscopy; in addition to the exclusive detection of classical EV-markers CD9, CD81, CD63 using label-free mass spectrometry. Additionally, we identified several proregenerative stimuli, such as WNT5A or ANGPT1, exclusively detected in Panc-MSC EVenriched versus EV-depleted conditioned media. Human microvascular endothelial cell tubule formation was enhanced in response to both Panc-MSC CM fractions in vitro yet only intramuscular injection of EV-enriched CM demonstrated vascular regenerative functions in NOD/SCID mice with unilateral hind-limb ischemia (*