Neuronal circuits, which are essential for brain functions, are built up by neurons as a three-dimensional network, so tracing the three-dimensional neuronal network of human cerebral cortex is the first step to understanding the mechanism of human brain functions. The cortical microstructures were visualized by x-ray microtomographic (micro-CT) imaging of adult frontal-cortex tissue stained with metal impregnation. Skeletonized wire models were built by tracing the three-dimensional distribution of x-ray absorption coefficients. The operating mechanism of the resolved circuits is discussed on the basis of neurotransmission in the circuits.
The transmissive and less refractile nature of x-rays with respect to biological tissue enables three-dimensional radiographic analysis without any clearing procedure such as those required for light microscopy. Therefore, x-ray microtomography is a potential method of visualizing the neuronal circuits of brain, like x-ray crystallography in molecular biology. DOI PubMed Pdf Movies

To estimate the spatial resolution of microtomographs, a test object on the submicrometer scale was prepared by focused ion beam milling and subjected to microtomographic analysis. Since biological tissues are composed of cells and extracellular matrices with micrometer and submicrometer structures, it is important to investigate the three-dimensional spatial resolution of microtomographs used to visualize microstructures of biological tissues. The resolutions along the direction within the tomographic slice plane (in-plane resolution) and perpendicular to it (through-plane resolution) were determined from the modulation transfer function of square-wave patterns. The in-plane resolution of the projection microtomograph at the SPring-8 BL20XU beamline was estimated to be 1.2 um from the modulation transfer function of the non-zoomed image. In contrast, the zoomed image gave an improved in-plane resolution of 0.8 um. Although the two-dimensional radiographs were taken with the pixel width of half the x-ray optics resolution, these three-dimensional resolution analyses indicated that zoom reconstruction should be performed to achieve in-plane resolution comparable to the x-ray optics resolution. DOI PubMed preprint

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. A series of precursor recombinants bearing N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site and that each of their peptide bonds is in the trans conformation. A nucleophilic attack of the C284 SG atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is thought to be resolved into a thioester acyl group upon cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of N->S acyl shift at the junction between the N-extein and intein. PubMed PDB1JVA PDB1UM2