Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV-1 Tat protein (Tat-PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat-PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C-terminally with the Tat-PTD. Compared with unmodified GALC, GALC bearing a Tat-PTD, a myc epitope and 6 consecutive His residues [GALC-TMH (Tat-PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC-TMH was found to be taken up by cells both via mannose-6-phosphate receptor (M6PR)-mediated endocytosis as well as by M6PR-independent mechanisms. GALC-TMH displayed increased M6PR-independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat-PTD. Immunocytochemical analyses revealed that Tat-modified GALC protein co-localized in part with the lysosome-associated membrane protein-1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC-TMH. Therefore, GALC-TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake. © 2007 The Authors

Dual-sized hollow particle incorporated fibroin thermal insulating coatings on catheter for cerebral therapeutic hypothermia

Selective endovascular hypothermia has been used to provide cooling-induced cerebral neuroprotection, but current catheters do not support thermally-insulated transfer of cold infusate, which results in an increased exit temperature, causes hemodilution, and limits its cooling efficiency. Herein, air-sprayed fibroin/silica-based coatings combined with chemical vapor deposited parylene-C capping film was prepared on catheter. This coating features in dual-sized-hollow-microparticle incorporated structures with low thermal conductivity. The infusate exit temperature is tunable by adjusting the coating thickness and infusion rate. No peeling or cracking was observed on the coatings under bending and rotational scenarios in the vascular models. Its efficiency was verified in a swine model, and the outlet temperature of coated catheter (75 μm thickness) was 1.8–2.0 °C lower than that of the uncoated one. This pioneering work on catheter thermal insulation coatings may facilitate the clinical translation of selective endovascular hypothermia for neuroprotection in patients with acute ischemic stroke

Broadband ground motions from 3D physics-based numerical simulations using artificial neural networks

In this article, a novel strategy to generate broadband earthquake ground motions from the results of 3D physics-based numerical simulations (PBSs) is presented. Physics-based simulated ground motions embody a rigorous seismic-wave propagation model (i.e., including source, path, and site effects), which is however reliable only in the long-period range (typically above 0.75–1 s), owing to the limitations posed both by computational constraints and by insufficient knowledge of the medium at short wavelengths. To cope with these limitations, the proposed approach makes use of Artificial Neural Networks (ANNs), trained on a set of strong-motion records, to predict the response spectral ordinates at short periods. The essence of the procedure is, first, to use the trained ANN to estimate the short-period response spectral ordinates using as input the long-period ones obtained by the PBS, and, then, to enrich the PBS time histories at short periods by scaling iteratively their Fourier spectrum, with no phase change, until their response spectrum matches the ANN target spectrum. After several validation checks of the accuracy of the ANN predictions, the case study of the 29 May 2012 Mw 6.0 Po Plain earthquake is illustrated as a comprehensive example of application of the proposed procedure. The capability of the proposed approach to reproduce in a realistic way the engineering features of earthquake ground motion, including the peak values and their spatial correlation structure, is successfully proven