Brain Structures in a Human Embryo Imaged with MR Microscopy.

PURPOSE: To delineate brain microstructures in human embryos during the formation of the various major primordia by MR microscopy, with different contrasts appropriate for each target. METHODS: We focused mainly on the internal structures in the cerebral cortex and the accessory nerves of the brain. To find appropriate sequence parameters, we measured nuclear magnetic resonance (NMR) parameters and created kernel density plots of T1 and T2 values. We performed T1-weighted gradient echo imaging with parameters similar to those used in the previous studies. We performed T2*-weighted gradient echo imaging to delineate the target structures with the appropriate sequence parameters according to the NMR parameter and flip angle measurements. We also performed high-resolution imaging with both T1- and T2*-weighted sequences. RESULTS: T1, T2, and T2* values of the target tissues were positively correlated and shorter than those of the surrounding tissues. In T1-weighted images with a voxel size of (30 µm)3 and (20 µm)3, various organs and tissues and the agarose gel were differentiated as in previous studies, and the structure of approximately 40 µm in size was depicted, but the detailed structures within the cerebral cortex and the accessory nerves were not delineated. In T2*-weighted images with a voxel size of (30 µm)3, the layered structure within the cerebral cortex and the accessory nerves were clearly visualized. Overall, T1-weighted images provided more information than T2*-weighted images, but important internal brain structures of interest were visible only in T2*-weighted images. Therefore, it is essential to perform MR microscopy with different contrasts. CONCLUSION: We have visualized brain structures in a human embryo that had not previously been delineated by MR microscopy. We discussed pulse sequences appropriate for the structures of interest. This methodology would provide a way to visualize crucial embryological information about the anatomical structure of human embryos.

High-resolution MRI for human embryos with isotropic 10 µm resolution at 9.4 T.

Magnetic resonance (MR) microscopy of human embryos has contributed significantly to the development of human embryology. Higher-resolution MR microscopy will have obvious benefits, for example, in visualizing small structures that are blurred or lost in lower-resolution images, providing detailed information on the development and growth of various organs, and improving the accuracy of MR volumetry. However, high-resolution MR microscopy has yet to be realized because of many technical challenges. In this study, therefore, we have performed high-resolution MR microscopy for human embryos with isotropic resolutions of (12 µm)3 at full sampling and (10 µm)3 at compressed sensing, which far exceeds the resolution of previous embryonic MR studies. The hardware and the pulse sequence were improved to achieve higher spatial resolution. Line profile, signal-to-noise ratio, and histogram analysis using phantom images were performed to verify that the resolution and the voxel size were identical. Comparison with optical microscopy images of embryo specimens at the same developmental stage was performed to confirm that the microstructures were well delineated. Our results show that imaging at this high resolution effectively depicts the microstructures of human embryos. This technology is the cornerstone for constructing an unprecedented high-quality atlas that will contribute to the development of human embryology.

FKBP51 and FKBP52 regulate androgen receptor dimerization and proliferation in prostate cancer cells.

The growth of prostate cancer is dependent on the androgen receptor (AR), which serves as a ligand-specific transcription factor. Although two immunophilins, FKBP51 and FKBP52, are known to regulate AR activity, the precise mechanism remains unclear. We found that depletion of either FKBP51 or FKBP52 reduced AR dimer formation, chromatin binding, and phosphorylation, suggesting defective AR signaling. Furthermore, the peptidyl-prolyl cis/trans isomerase activity of FKBP51 was found to be required for AR dimer formation and cancer cell growth. Treatment of prostate cancer cells with FK506, which binds to the FK1 domain of FKBPs, or with MJC13, an inhibitor of FKBP52-AR signaling, also inhibited AR dimer formation. Finally, elevated expression of FKBP52 was associated with a higher rate of prostate-specific antigen recurrence in patients with prostate cancer. Collectively, these results suggest that FKBP51 and FKBP52 might be promising targets for prostate cancer treatment through the inhibition of AR dimer formation.

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.

Development of a fluorescent probe for detection of citrulline based on photo-induced electron transfer.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.