Extracellular loop C of NPC1L1 is important for binding to ezetimibe.

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [(3)H]AS. Chimeric and mutational studies indicate that high-affinity binding of [(3)H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [(3)H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [(3)H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

3D Printing Hydrogel-Based Soft and Biohybrid Actuators: A Mini-Review on Fabrication Techniques, Applications, and Challenges.

Stimuli-responsive hydrogels are candidate building blocks for soft robotic applications due to many of their unique properties, including tunable mechanical properties and biocompatibility. Over the past decade, there has been significant progress in developing soft and biohybrid actuators using naturally occurring and synthetic hydrogels to address the increasing demands for machines capable of interacting with fragile biological systems. Recent advancements in three-dimensional (3D) printing technology, either as a standalone manufacturing process or integrated with traditional fabrication techniques, have enabled the development of hydrogel-based actuators with on-demand geometry and actuation modalities. This mini-review surveys existing research efforts to inspire the development of novel fabrication techniques using hydrogel building blocks and identify potential future directions. In this article, existing 3D fabrication techniques for hydrogel actuators are first examined. Next, existing actuation mechanisms, including pneumatic, hydraulic, ionic, dehydration-rehydration, and cell-powered actuation, are reviewed with their benefits and limitations discussed. Subsequently, the applications of hydrogel-based actuators, including compliant handling of fragile items, micro-swimmers, wearable devices, and origami structures, are described. Finally, challenges in fabricating functional actuators using existing techniques are discussed.