Ability of Nonswelling Polyethylene Glycol-Based Vitreous Hydrogel to Maintain Transparency in the Presence of Vitreous Hemorrhage.

Purpose: Postoperative vitreous hemorrhage is a vision-impacting complication of vitrectomy. This preclinical in vitro study assessed the potential ability of a nonswelling polyethylene glycol-based artificial vitreous hydrogel to maintain transparency in the vitreous cavity in the presence of vitreous hemorrhage. Methods: Samples (1 mL) of diluted blood at concentrations of 0.1%, 0.25%, 0.5%, and 1.25% were added to 1 mL samples of polymerized hydrogel in cuvettes (gel + blood group); 2 mL samples of diluted blood at the same concentrations were prepared as controls (blood only group). Spectral transmission curves for the hydrogel (gel + blood group) and diluted blood (blood only group) were obtained before and on days 1, 2, 5, 7, 14, and 28 of the experiment. Between-group comparisons were made using the Student's t-test. The percentage of transmittance in the visible light spectrum (400-700 nm) was measured at each time point. Results: Mean light transmittance was maintained at >90% until day 7 in the gel + blood group and was significantly greater in the gel + blood than in the blood only groups in samples containing blood diluted to 0.25%, 0.5%, and 1.25% during the 28-day study period (P < 0.05). Conclusions: A nonswelling polyethylene glycol-based artificial vitreous hydrogel maintained high optical transparency in the presence of blood through the study period. Injection of this hydrogel into the vitreous cavity at the end of surgery might help to prevent or mitigate vitreous hemorrhage-associated postoperative visual loss. Translational Relevance: The hydrogel may prevent visual loss due to postoperative vitreous hemorrhage.

In vivo imaging of intraperitoneally disseminated tumors in model mice by using activatable fluorescent small-molecular probes for activity of cathepsins.

It is difficult to completely remove carcinomas in unguided ablative surgery because they cannot be distinguished with the unaided human eye. Therefore, in order to precisely visualize tiny tumors and the borders between cancerous lesions and normal tissues, we have been developing fluorescence probes activatable only in cancer cells. We previously reported the hydroxymethylrhodamine green (HMRG)-based fluorescence probe gGlu-HMRG for γ-glutamyltransferase (GGT), which is overexpressed in a variety of cancer cells, and we showed that it enables in vivo rapid detection of human ovarian cancer SHIN-3 nodules with a high tumor-to-background (T/B) fluorescence ratio in model mice. However, cancer cell lines with low GGT expression could hardly be detected with gGlu-HMRG. Here we developed two new HMRG-based fluorescence probes for the cathepsin family of cysteine proteases, including cathepsin B (CatB) and cathepsin L (CatL), which show increased expression and/or activity, secretion, and altered localization in many kinds of cancer cells. The developed probes, Z-Phe-Arg-HMRG and Z-Arg-Arg-HMRG, are colorless and nonfluorescent at the physiological pH of 7.4, but are hydrolyzed to HMRG upon reaction with purified cathepsins, resulting in a more than 200-fold fluorescence increase. These probes could visualize human ovarian cancer cell lines SHIN-3, SK-OV-3, and OVCAR-3, of which the latter two were hardly detectable with gGlu-HMRG. Z-Phe-Arg-HMRG showed higher applicability than Z-Arg-Arg-HMRG for in vivo imaging, and we confirmed that 0.5-mm-sized SK-OV-3 tumor nodules disseminated on the mesentery in a mouse model could be rapidly visualized by Z-Phe-Arg-HMRG, with a T/B fluorescence ratio of 4.2. Further, intraperitoneally disseminated tumor could be visualized in real time in vivo by fluorescence endoscopy after spraying Z-Phe-Arg-HMRG, with a T/B ratio of 3. In conclusion, our HMRG-based activatable probes targeted to cathepsins have expanded the detectable range of cancers, and appear to be suitable for clinical application.

Design and synthesis of a FlAsH-type Mg2+ fluorescent probe for specific protein labeling.

Although the magnesium ion (Mg(2+)) is one of the most abundant divalent cations in cells and is known to play critical roles in many physiological processes, its mobilization and underlying mechanisms are still unknown. Here, we describe a novel fluorescent Mg(2+) probe, "KMG-104-AsH", composed of a highly selective fluorescent Mg(2+) probe, "KMG-104", and a fluorescence-recoverable probe, "FlAsH", bound specifically to a tetracysteine peptide tag (TCtag), which can be genetically incorporated into any protein. This probe was developed for molecular imaging of local changes in intracellular Mg(2+) concentration. KMG-104-AsH was synthesized, and its optical properties were investigated in solution. The fluorescence intensity of KMG-104-AsH (at λ(em/max) = 540 nm) increases by more than 10-fold by binding to both the TCtag peptide and Mg(2+), and the probe is highly selective for Mg(2+) (K(d/Mg) = 1.7 mM, K(d/Ca) ≫ 100 mM). Application of the probe for imaging of Mg(2+) in HeLa cells showed that this FlAsH-type Mg(2+) sensing probe is membrane-permeable and binds specifically to tagged proteins, such as TCtag-actin and mKeima-TCtag targeted to the cytoplasm and the mitochondrial intermembrane space. KMG-104-AsH bound to TCtag responded to an increase in intracellular Mg(2+) concentration caused by the release of Mg(2+) from mitochondria induced by FCCP, a protonophore that eliminates the inner membrane potential of mitochondria. This probe is expected to be a strong tool for elucidating the dynamics and mechanisms of intracellular localization of Mg(2+).

A near-infrared fluorescent calcium probe: a new tool for intracellular multicolour Ca2+ imaging.

We report a novel near-infrared fluorescent calcium probe (KFCA), which has good optical properties such as intense NIR fluorescence emission (670 nm, QY: 0.24), excellent ON/OFF ratio (120-fold), and good wavelength-compatibility with visible-light-emissive fluorophores (Fluo-4, DsRed2), and which is applicable for real-time dual-colour intracellular Ca(2+) imaging.

Newly developed Mg2+-selective fluorescent probe enables visualization of Mg2+ dynamics in mitochondria.

Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).

Disk hologram made from a computer-generated hologram.

We have been investigating disk holograms made from a computer-generated hologram (CGH). Since a general flat format hologram has a limited viewable area, we usually cannot see the other side of the reconstructed object. Therefore, we propose a computer-generated cylindrical hologram (CGCH) to obtain a hologram with a 360 deg viewable area. The CGCH has a special shape that is difficult to construct and calculation of such a hologram takes too much time. In contrast, a disk-type hologram is well known as a 360 deg viewable hologram. Since a regular disk hologram is a flat reflective type, the reconstruction setup is easy. However, there are just a few reports about creating a disk hologram by use of a CGH. Because the output device lacks spatial resolution, the hologram cannot provide a large diffraction angle. In addition, the viewing zone depends on the hologram size; the maximum size of the fringe pattern is decided on the basis of the special frequency of the output device. The calculation amount of the proposed hologram is approximately a quarter of that of a CGCH. In a previous study, a disk hologram made from a CGH was achieved. However, since the relation between the vertical viewing zone and reconstructed image size is a trade-off, the size of the reconstructed image and view zone is not enough for practical use. To improve both parameters, we modified a fringe printer to issue a high-resolution fringe pattern for a disk hologram. In addition, we propose a new calculation method for fast calculation.

Fast calculation method for computer-generated cylindrical holograms.

Since a general flat hologram has a limited viewable area, we usually cannot see the other side of a reconstructed object. There are some holograms that can solve this problem. A cylindrical hologram is well known to be viewable in 360 deg. Most cylindrical holograms are optical holograms, but there are few reports of computer-generated cylindrical holograms. The lack of computer-generated cylindrical holograms is because the spatial resolution of output devices is not great enough; therefore, we have to make a large hologram or use a small object to fulfill the sampling theorem. In addition, in calculating the large fringe, the calculation amount increases in proportion to the hologram size. Therefore, we propose what we believe to be a new calculation method for fast calculation. Then, we print these fringes with our prototype fringe printer. As a result, we obtain a good reconstructed image from a computer-generated cylindrical hologram.

Bioorganic studies on plant movement, from natural products to its receptor.

The chemical aspects of the circadian leaf movement known as "nyctinasty" are discussed in this paper. Each of the nyctinastic plants of five different genera so far examined contained a pair of factors, one of which induced leaf closure and another induced leaf opening. The relative contents of the closing and opening factors changed correlating with the nyctinastic leaf movement. The use of fluorescence-labeled and photoaffinity-labeled factors revealed that the factors bind to specific cells, the motor cells, present in the pulvini, and that the membrane fraction of the motor cells contained two proteins of 210 and 180 kDa, which can bind to the factors.