Fuminori Sugihara

Real-Time Background-Free Selective Imaging of Fluorescent Nanodiamonds in Vivo

Recent developments of imaging techniques have enabled fluorescence microscopy to investigate the localization and dynamics of intracellular substances of interest even at the single-molecule level. However, such sensitive detection is often hampered by autofluorescence arising from endogenous molecules. Those unwanted signals are generally reduced by utilizing differences in either wavelength or fluorescence lifetime; nevertheless, extraction of the signal of interest is often insufficient, particularly for in vivo imaging. Here, we describe a potential method for the selective imaging of nitrogen-vacancy centers (NVCs) in nanodiamonds. This method is based on the property of NVCs that the fluorescence intensity sensitively depends on the ground state spin configuration which can be regulated by electron spin magnetic resonance. Because the NVC fluorescence exhibits neither photobleaching nor photoblinking, this protocol allowed us to conduct long-term tracking of a single nanodiamond in both Caenorhabditis elegans and mice, with excellent imaging contrast even in the presence of strong background autofluorescence.

Dual‐Function Probe to Detect Protease Activity for Fluorescence Measurement and 19F MRI

Dynamic duo: Magnetic resonance imaging (MRI) can visualize deep regions of living bodies, whereas fluorescence measurement offers excellent sensitivity. These methods thus offer signal enhancement potential for detecting enzyme activities. A dual-mode off/on probe to detect caspase-3 activity by fluorescence and (19)F MRI is presented.

Identification of an atypical monocyte and committed progenitor involved in fibrosis

Monocytes and macrophages comprise a variety of subsets with diverse functions. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6C−F4/80−Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb−/− haematopoietic cells. Adoptive transfer of SatM into Cebpb−/− mice resulted in fibrosis. Notably, SatM are derived from Ly6C−FcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6C−FcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.

Multifunctional Core-Shell Silica Nanoparticles for Highly Sensitive19F Magnetic Resonance Imaging

19F magnetic resonance imaging (19F MRI) is useful for monitoring particular signals from biological samples, cells, and target tissues, because background signals are missing in animal bodies. Therefore, highly sensitive 19F MRI contrast agents are in great demand for their practical applications. However, we have faced the following challenges: 1) increasing the number of fluorine atoms decreases the solubility of the molecular probes, and 2) the restriction of the molecular mobility attenuates the 19F MRI signals. Herein, we developed novel multifunctional core–shell nanoparticles to solve these issues. They are composed of a core micelle filled with liquid perfluorocarbon and a robust silica shell. These core–shell nanoparticles have superior properties such as high sensitivity, modifiability of the surface, biocompatibility, and sufficient in vivo stability. By the adequate surface modifications, gene expression in living cells and tumor tissue in living mice were successfully detected by 19F MRI.

Design of chemical shift-switching 19F magnetic resonance imaging probe for specific detection of human monoamine oxidase A.

Monoamine oxidase (MAO) A is a flavoenzyme that catalyzes the oxidation of biologically important monoamines and is thought to be associated with psychiatric disorders. Here, we report a strategy for rationally designing a (19)F magnetic resonance imaging probe for the specific detection of human MAO-A (hMAO-A) activity. Our designed (19)F probe was oxidized expeditiously by hMAO-A to produce 2-fluoro-4-nitrophenol via a spontaneous β-elimination mechanism. Concomitant with the structural change of the probe to the product, the (19)F chemical shift changed by 4.2 ppm, which was enough to visualize the probe and enzymatic product separately. Importantly, our probe achieved excellent discrimination of hMAO-A from its isoform hMAO-B.

19F MRI detection of β-galactosidase activity for imaging of gene expression

Imaging of gene expression by magnetic resonance imaging (MRI) yields direct information regarding living systems that cannot be obtained via other methods. In this study, we report the rational design and synthesis of a novel 19F MRI probe that detects β-galactosidase (β-gal) activity, enabling the imaging of gene expression in cells. The 19F MRI signal of the probe was quenched by the intramolecular paramagnetic resonance enhancement from a Gd3+ ion. A contrivance was made in the probe structure to recover the 19F MRI signal after hydrolysis by β-gal with a following self-immolative reaction. This 19F MRI signal change was observed in the physiological aqueous condition. The probe could also detect β-gal activity in fixed HEK293T cells. In conclusion, this new probe enables the 19F MRI detection of cellular gene expression. The probe design strategy is also expected to lead to the development of MRI probes for a wide variety of hydrolase activities.

Highly redundant function of multiple AT-rich sequences as core promoter elements in the TATA-less RPS5 promoter of Saccharomyces cerevisiae

In eukaryotes, protein-coding genes are transcribed by RNA polymerase II (pol II) together with general transcription factors (GTFs). TFIID, the largest GTF composed of TATA element-binding protein (TBP) and 14 TBP-associated factors (TAFs), plays a critical role in transcription from TATA-less promoters. In metazoans, several core promoter elements other than the TATA element are thought to be recognition sites for TFIID. However, it is unclear whether functionally homologous elements also exist in TATA-less promoters in Saccharomyces cerevisiae. Here, we identify the cis-elements required to support normal levels of transcription and accurate initiation from sites within the TATA-less and TFIID-dependent RPS5 core promoter. Systematic mutational analyses show that multiple AT-rich sequences are required for these activities and appear to function as recognition sites for TFIID. A single copy of these sequences can support accurate initiation from the endogenous promoter, indicating that they carry highly redundant functions. These results show a novel architecture of yeast TATA-less promoters and support a model in which pol II scans DNA downstream from a recruited site, while searching for appropriate initiation site(s).

Activatable 19F MRI Nanoparticle Probes for the Detection of Reducing Environments

(19)F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the (19)F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction-responsive PFC-encapsulated nanoparticle probe, FLAME-SS-Gd(3+) (FSG). This is the first example of an activatable PFC-encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd(3+) complexes per nanoparticle was more than approximately 5.0×10(2), resulting in the high signal augmentation.

A novel magnetic resonance-based method to measure gene expression in living cells

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well.

19F MRI Monitoring of Gene Expression in Living Cells through Cell-Surface β-Lactamase Activity

Magnetic resonance imaging provides important intravital information on deep tissues that cannot be visualized by other methods. Although we had previously developed an off/on switching 19F MRI probe to monitor reporter enzyme activity on the basis of the paramagnetic relaxation enhancement effect, it was difficult to monitor biological events in living cells because the 19F MRI probe did not permeate living cell membrane. In this study, we have developed a new 19F MRI system for monitoring gene expression in living cells by exploiting cell‐surface‐displayed β‐lactamase and the specifically designed 19F MRI probe. By using this system, cellular gene expression was successfully detected by 19F MRI without cell fixation. This imaging strategy shows promise for monitoring in vivo gene expression, and therefore it could lead to useful technologies for the diagnosis and therapy of various diseases.