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Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.

Genotyping technologies for single nucleotide polymorphisms (SNPs) and other mutation types have evolved to become essential tools in various fields. Although high-throughput genotyping technologies occupy a key position in handling large amounts of SNP data, simple, low-cost, and conventional genotyping technologies remain in demand. Allele-specific (AS) polymerase chain reaction (PCR) and its related improved methods can effectively identify target SNPs and allele types using AS primers that introduce instability through mismatched bases at and around the SNP site. In this chapter, we present what is known from the literature on primer design with mismatches for AS-PCR and describe three cases of mutation detection (SNPs and insertions/deletions) associated with functional genes of crop species, which could be useful to guide future AS-PCR experiments., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Purpose: To evaluate the accuracy of a bone coordinate system constructed using MR image composing., Method: A femoral coordinate system constructed using image composing of MR images of a whole bovine femur was evaluated using CT images. The MR images were acquired by moving the table and were processed with 3D distortion correction and composing. To evaluate the reproducibility of the measurements, the same operator repeated the construction of the femoral coordinate system. In addition, distortions in the MR images were evaluated in comparison with those in the CT images., Result: The center position of the femoral coordinate system constructed using the MR image composing was 1.6±0.9 mm on the X-axis, 1.5±0.8 mm on the Y-axis, and 0.2±0.3 mm on the Z-axis, and the rotation of each axis was 1° or less. The distortion of the composed MR image was about 0.3%., Conclusion: The femoral coordinate system constructed using MR image composing had the same accuracy as a system constructed with CT images. The effect of MR image composing on the construction of the femoral coordinate system was small.

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo . We recently developed γ-glutamyl-[1- 13 C]glycine (γ-Glu-[1- 13 C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo . However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu-Gly scaffold and develop a deuterated probe, γ-Glu-[1- 13 C]Gly- d 2 , that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu-Gly and the magnetic properties of 13 C-enriched γ-Glu-Gly (γ-Glu-[1- 13 C]Gly and γ-[5- 13 C]Glu-Gly) to support the validity of γ-Glu-[1- 13 C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time ( T 1 ) of γ-Glu-[1- 13 C]Gly and γ-Glu-[1- 13 C]Gly- d 2 under various conditions (D 2 O, PBS, and serum) and confirmed that the T 1 of γ-Glu-[1- 13 C]Gly and γ-Glu-[1- 13 C]Gly- d 2 was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1- 13 C]Gly revealed a significant contribution of the dipole-dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T 1 . In fact, by using γ-Glu-[1- 13 C]Gly- d 2 as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Polycyclic aromatic hydrocarbons (PAHs) represent an emerging class of π-conjugated molecules in the area of optoelectronic devices and materials. Unprecedented synthetic routes to various PAHs from simple phenol derivatives by a palladium-catalyzed annulative dimerization of phenylene triflate through twofold inter- and intramolecular C-H activation have been established. The initially formed partially fused PAHs can be smoothly transformed into a variety of fully fused PAHs by the Scholl reaction. Furthermore, the reactions of phenanthrene-substituted aryl triflates proceeded regioselectively. The findings inspired the development of a rapid and efficient synthesis of polybenzoacene derivatives. This study not only allows transformation of phenyl triflates, but also discloses a new retrosynthetic strategy towards PAHs, especially polybenzoacenes., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)