Store operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-aminoethoxydiphenyl borate (2-APB) is a well described SOCE inhibitor but its action remains largely elusive. Here we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai Activating Region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound with STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We find that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB’s action on STIM1, and it is most likely mediated by gating residues in Orai1 N terminus. Overall, our finding of this two-direct-site inhibition may help better understanding of Orai1-activation by STIM1 and future drug design.

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable...

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase-Rabs, and clathrin, are identified by proteomics. A goethite-binding experiment coupled with proteomics and RNA-seq highlights six chitin-binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.

Human mesenchymal stem cells (hMSCs), which have the ability to differentiate into osteoblasts, show promise for bone tissue engineering and bone defect treatment. While there are a number of approaches currently available to accomplish this, e.g., utilizing biodegradable materials loaded with the synthetic glucocorticoid osteogenic inducer dexamethasone (DEX), there are still many disadvantages with the current technologies. Here, we generated light-responsive microgels that we showed are capable of loading and releasing DEX in a light-triggered fashion, with the released DEX being able to induce hMSC differentiation into osteoblasts. Specifically, light-responsive poly­(N-isopropylacrylamide-co-nitrobenzyl methacrylate) (pNIPAm-co-NBMA) microgels were synthesized via free radical precipitation polymerization and their size, morphology, and chemical composition were characterized. We then went on to show that the microgels could be loaded with DEX (via what we think are hydrophobic interactions) and released upon exposure to UV light. We went on to show that the DEX released from the microgels was still capable of inducing osteogenic differentiation of hMSCs using an alamarBlue assay and normalized alkaline phosphatase (ALP) activity assay. We also investigated how hMSC differentiation was impacted by intermittent DEX released from UV-exposed microgels. Finally, we confirmed that the microgels themselves were not cytotoxic to hMSCs. Taken together, the DEX-loaded light-responsive microgels reported here may find a use for niche clinical applications, e.g., bone tissue repair.

Three kinds of nozzles normally used in industrial production are numerically simulated, and the structure of nozzle with the best jetting performance out of the three nozzles is optimized. TheR90 nozzle displays the most optimal jetting properties, including the smooth transition of the nozzle’s inner surface. Simulation results of all sample nozzles in this study show that the helix nozzle ultimately displays the best jetting performance. Jetting velocity magnitude alongYandZcoordinates is not symmetrical for the helix nozzle. Compared to simply changing the jetting angle, revolving the jet issued from the helix nozzle creates a grinding wheel on the cleaning surface, which makes not only an impact effect but also a shearing action on the cleaning object. This particular shearing action improves the cleaning process overall and forms a wider, effective cleaning range, thus obtaining a broader jet width.