We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.

1. Ca2+ liberation induced in Xenopus oocytes by a poorly metabolized derivative of inositol 1,4,5-trisphosphate (3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate; 3-F-InsP3) was visualized using a video-rate confocal microscope to image fluorescence signals reported by the indicator dye calcium green-1.2. Low (10-30 nM) intracellular concentrations of 3-F-InsP3 evoked Ca2+ release as localized transient 'puffs'. Progressively higher concentrations (30-60 nM) gave rise to abortive Ca2+ waves triggered by puffs, and then (> 60 nM) to a sustained elevation of Ca2+followed by the appearance of propagating Ca2+ waves. At concentrations up to that giving waves, the frequency of puffs increased as about the third power of [InsP3], whereas their amplitudes increased only slightly.3. The rise of cytosolic Ca2+ during a puff began abruptly, and peaked within about 50 ms. The peak free Ca2+ level was about 180 nm, and the total amount of Ca2+ liberated was several attomoles (10-18 mol), too much to be accounted for by opening of a single InsP3-gated channel. The subsequent decline of Ca2+ occurred over a few hundred milliseconds, determined largely by diffusion of Ca2+ away from the release site, rather than by resequestration.

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Three-dimensional (3D) printed models represent educational tools of high quality compared with traditional teaching aids. Colored skull models were produced by 3D printing technology. A randomized controlled trial (RCT) was conducted to compare the learning efficiency of 3D printed skulls with that of cadaveric skulls and atlas. Seventy-nine medical students, who never studied anatomy, were randomized into three groups by drawing lots, using 3D printed skulls, cadaveric skulls, and atlas, respectively, to study the anatomical structures in skull through an introductory lecture and small group discussions. All students completed identical tests, which composed of a theory test and a lab test, before and after a lecture. Pre-test scores showed no differences between the three groups. In post-test, the 3D group was better than the other two groups in total score (cadaver: 29.5 [IQR: 25–33], 3D: 31.5 [IQR: 29–36], atlas: 27.75 [IQR: 24.125–32]; p = 0.044) and scores of lab test (cadaver: 14 [IQR: 10.5–18], 3D: 16.5 [IQR: 14.375–21.625], atlas: 14.5 [IQR: 10–18.125]; p = 0.049). Scores involving theory test, however, showed no difference between the three groups. In this RCT, an inexpensive, precise and rapidly-produced skull model had advantages in assisting anatomy study, especially in structure recognition, compared with traditional education materials.