Dengue virus (DENV) infection in neuronal cells was speculated to trigger neuropathy. Herein, we determined the blockade of DENV infection by targeting endocytic pathways in vitro and in vivo. In DENV-infected mouse brains, we previously showed that viral proteins are expressed in neuronal cells around the hippocampus with accompanying neurotoxicity. DENV caused infection, including entry, double-stranded (ds)RNA replication, protein expression, and virus release, followed by cytotoxicity in the mouse neuronal Neuro-2a cell line. Pharmacologically blocking clathrin-mediated endocytosis of the DENV retarded viral replication. Targeting vacuolar-type H+-ATPase (V-ATPase)-based endosomal acidification effectively blocked the DENV replication process, but had no direct effect on viral translation. Blockade of the clathrin- and V-ATPase-based endocytic pathways also attenuated DENV-induced neurotoxicity. Inhibiting endosomal acidification effectively retarded DENV infection, acute viral encephalitis, and mortality. These results demonstrate that clathrin mediated endocytosis of DENV followed by endosomal acidification-dependent viral replication in neuronal cells, which can lead to neurotoxicity.

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MPT0L145 has been developed as a FGFR inhibitor exhibiting significant anti-bladder cancer activity and via promoting autophagy-dependent cell death. Here, we aim to elucidate the underlying mechanisms. Autophagy flux, morphology, and intracellular organelles were evaluated by Western blotting, transmission electron microscope, and fluorescence microscope. Molecular docking and surface plasmon resonance assay were performed to identify drug-protein interaction. Lentiviral delivery of cDNA or shRNA and CRISPR/Cas9-mediated genome editing was used to modulate gene expression. Mitochondrial oxygen consumption rate was measured by a Seahorse XFe24 extracellular flux analyzer, and ROS level was measured by flow cytometry. MPT0L145 persistently increased incomplete autophagy and phase-lucent vacuoles at the perinuclear region, which were identified as enlarged and alkalinized late-endosomes. Screening of a panel of lipid kinases revealed that MPT0L145 strongly inhibits PIK3C3 with a value of 0.53 nmol/L. Ectopic expression of PIK3C3 reversed MPT0L145-increased cell death and incomplete autophagy. Four residues (Y670, F684, I760, D761) at the ATP-binding site of PIK3C3 are important for the binding of MPT0L145. In addition, MPT0L145 promotes mitochondrial dysfunction, ROS production, and DNA damage, which may in part, contribute to cell death. ATG5-knockout rescued MPT0L145-induced cell death, suggesting simultaneous induction of autophagy is crucial to its anticancer activity. Finally, our data demonstrated that MPT0L145 is able to overcome cisplatin resistance in bladder cancer cells. MPT0L145 is a first-in-class PIK3C3/FGFR inhibitor, providing an innovative strategy to design new compounds that increase autophagy, but simultaneously perturb its process to promote bladder cancer cell death. .

The effect of cupric (Cu 2+ ) ion as an additive in the acidic electroless nickel plating (ENP) bath on the characteristics of the resulting nickel-phosphorus (Ni-P) alloys was investigated mainly with X-ray diffraction, scanning electron microscopy, and energy-dispersive X-ray (EDX) spectrometry. Cupric ions in the acidic ENP bath using hypophosphite anions as reductants have been reported ambiguously as stabilizers or codeposit constituents. In this work, the critical concentration of added copper salts, CuSO 4 ‚ 5H 2 O, that starts to inhibit the ENP process was determined to be ca. 536 mg/L. In general, the deposition rate, surface morphology, and pit formation on the surface of as-deposits are significantly improved with Cu 2+ addition at concentrations less than the critical value. The electroless nickel alloys were shown as a mixture of an amorphous deposit and a crystalline copper metal rather than as amorphous alloys alone. 1,2 During the initial stage of the electroless plating process, the copper contents in as-deposits were found to decrease rapidly with plating time from the EDX analyses. The X-ray photoelectron spectroscopy result also confirms that copper is the preferred deposited species during the initial stage of the ENP process. The theoretical model 3 is revised by taking into account the effect of adsorbed cupric ions on the shift in the depth of the net nuclear potential of the electroless nickel frontier, and successfully predicts the deposition rates. Moreover, as predicted by the revised model, the adsorbed cupric ions on the justdeposited Ni-Cu-P frontier could enhance the adsorption of hypophosphite anions and, accordingly, the deposition rates.