A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging.

Single-molecule localization microscopy is used to construct super-resolution images, but generally requires prior intense laser irradiation and in some cases additives, such as thiols, to induce on-off switching of fluorophores. These requirements limit the potential applications of this methodology. Here, we report a first-in-class spontaneously blinking fluorophore based on an intramolecular spirocyclization reaction.

An activatable, polarity dependent, dual-luminescent imaging agent with a long luminescence lifetime.

In this proof-of-concept study, a new activatable imaging agent based on two luminophores and two different quenching mechanisms is reported. Both partial and total activation of the luminescence signal can be achieved, either in solution or in vitro. Bond cleavage makes the compound suitable for luminescence lifetime imaging.

Torque generation mechanism of F1-ATPase upon NTP binding.

Molecular machines fueled by NTP play pivotal roles in a wide range of cellular activities. One common feature among NTP-driven molecular machines is that NTP binding is a major force-generating step among the elementary reaction steps comprising NTP hydrolysis. To understand the mechanism in detail,in this study, we conducted a single-molecule rotation assay of the ATP-driven rotary motor protein F1-ATPase using uridine triphosphate (UTP) and a base-free nucleotide (ribose triphosphate) to investigate the impact of a pyrimidine base or base depletion on kinetics and force generation.

Practical fluorescence detection of acrolein in human plasma via a two-step tethering approach.

Acrolein, a cytotoxic α,β-unsaturated aldehyde and disease biomarker, was determined in plasma by means of a novel tethering strategy using Michael addition of the compound to a fluorescent dye, followed by immobilization of the product on microbeads via the aldehyde moiety. Elevation of blood acrolein was detected in mice treated with an anticancer agent cyclophosphamide, which releases acrolein upon activation. This method should be suitable for high-throughput diagnostic and clinical application.

Visualization of phagosomal hydrogen peroxide production by a novel fluorescent probe that is localized via SNAP-tag labeling.

Hydrogen peroxide (H2O2), a member of reactive oxygen species (ROS), plays diverse physiological roles including host defense and cellular signal transduction. During ingestion of invading microorganisms, professional phagocytes such as macrophages release H2O2 specifically into the phagosome to direct toxic ROS toward engulfed microbes. Although H2O2 is considered to exert discrete effects in living systems depending on location of its production, accumulation, and consumption, there have been limitations of techniques for probing this oxygen metabolite with high molecular specificity at the subcellular resolution.

Selective ablation of β-galactosidase-expressing cells with a rationally designed activatable photosensitizer.

We have developed an activatable photosensitizer capable of specifically inducing the death of β-galactosidase-expressing cells in response to photoirradiation. By using a selenium-substituted rhodol scaffold bearing β-galactoside as a targeting substituent, we designed and synthesized HMDESeR-βGal, which has a non-phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β-galactosidase efficiently converted HMDESeR-βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form.

Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics.

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Diverse functions of 24(S)-hydroxycholesterol in the brain.

24(S)-hydroxycholesterol (24S-OHC) which is enzymatically produced in the brain plays important physiological roles in maintaining brain cholesterol homeostasis. We found that 24S-OHC at sub-lethal concentrations down-regulated amyloid precursor protein (APP) trafficking via enhancement of the complex formation of APP with up-regulated glucose-regulated protein 78, an endoplasmic reticulum chaperone. In accordance with this mechanism, 24S-OHC suppressed amyloid-β production in human neuroblastoma SH-SY5Y cells.

Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1.

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs.

Adaptive responses induced by 24S-hydroxycholesterol through liver X receptor pathway reduce 7-ketocholesterol-caused neuronal cell death.

Lipid peroxidation products have been known to induce cellular adaptive responses and enhance tolerance against subsequent oxidative stress through up-regulation of antioxidant compounds and enzymes. 24S-hydroxycholesterol (24SOHC) which is endogenously produced oxysterol in the brain plays an important role in maintaining brain cholesterol homeostasis. In this study, we evaluated adaptive responses induced by brain-specific oxysterol 24SOHC in human neuroblastoma SH-SY5Y cells.

Mechanistic background and clinical applications of indocyanine green fluorescence imaging of hepatocellular carcinoma.

Background: Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear.

Methods: In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.

Safety and efficacy of faldaprevir with pegylated interferon alfa-2a and ribavirin in Japanese patients with chronic genotype-1 hepatitis C infection.

Background & Aims: Faldaprevir (BI 201335) is a potent once-daily (QD) NS3/4A protease inhibitor for the treatment of patients with genotype-1 (GT-1) hepatitis C virus (HCV). The aim of this study was to evaluate the safety, pharmacokinetics and efficacy of faldaprevir plus pegylated interferon alfa-2a (PegIFN) and ribavirin (RBV) in Japanese patients infected with chronic GT-1 HCV.

Methods: Part 1 of this phase II study was a randomized, double-blind, placebo-controlled, dose-ascending study.

Suppression of amyloid-β production by 24S-hydroxycholesterol via inhibition of intracellular amyloid precursor protein trafficking.

Cholesterol can be converted to 24S-hydroxycholesterol (24SOHC) by neuronal cholesterol 24-hydroxylase. In mouse models of Alzheimer's disease (AD), increasing 24SOHC levels reduced AD pathology. However, mechanisms underlying the effects of 24SOHC on amyloid-β (Aβ) production have remained unclear.

Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe.

Background: Pancreatic fistula (PF) remains the most serious complication after digestive surgery. It is difficult to prevent because of the inability to visualize the leakage of pancreatic juice during surgery or to evaluate the protease activity of leaked fluid, which is responsible for PF formation.

Methods: The fluorescence intensities of a chymotrypsin probe (glutaryl-phenylalanine [corrected] hydroxymethyl rhodamine green with added trypsin) in pancreatic juice and in intestinal or abdominal fluids drained after pancreatic resection were evaluated.

[In vivo cancer detection with a newly designed fluorescent probe].

Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, and we have succeeded to construct several versatile rational design strategies for novel fluorescence probes based on the concept of photoinduced electron transfer and intramolecular spirocyclization. Very recently, we have succeeded to develop various novel protease probes which were applicable for living cell system.

TokyoGreen derivatives as specific and practical fluorescent probes for UDP-glucuronosyltransferase (UGT) 1A1.

TokyoGreen (TG) derivatives were found to be efficient and specific substrates of an important drug-metabolizing enzyme, UDP-glucuronosyltransferase (UGT) 1A1. A rapid, specific, and sensitive assay of the enzyme was achieved simply by monitoring the change in fluorescence intensity. We also designed and developed the first "turn-on" fluorescent probes for UGTs.

Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization.

We have synthesized and evaluated a series of hydroxymethyl rhodamine derivatives and found an intriguing difference of intramolecular spirocyclization behavior: the acetylated derivative of hydroxymethyl rhodamine green (Ac-HMRG) exists as a closed spirocyclic structure in aqueous solution at physiological pH, whereas HMRG itself takes an open nonspirocyclic structure. Ac-HMRG is colorless and nonfluorescent, whereas HMRG is strongly fluorescent. On the basis of these findings, we have developed a general design strategy to obtain highly sensitive fluorescence probes for proteases and glycosidases, by replacing the acetyl group of Ac-HMRG with a substrate moiety of the target enzyme.

Motion-parallax smoothness of short-, medium-, and long-distance 3D image presentation using multi-view displays.

The discontinuity of motion parallax offered by multi-view displays was assessed by subjective evaluation. A super multi-view head-up display, which provides dense viewing points and has short-, medium-, and long-distance display ranges, was used. The results showed that discontinuity perception depended on the ratio of an image shift between adjacent parallax images to a pixel pitch of three-dimensional (3D) images and the crosstalk between viewing points.

Novel live imaging techniques of cellular functions and in vivo tumors based on precise design of small molecule-based 'activatable' fluorescence probes.

Recently established rational design strategies for novel fluorescence probes, especially those based on photoinduced electron transfer and spirocyclization were reviewed. Based on these design strategies, various novel fluorescence probes were successfully developed including those for reactive oxygen species, reporter enzymes. Furthermore, in vivo cancer imaging techniques based on rationally designed activatable probes such as cancer-specific antibodies tagged with acidic-pH activatable fluorescence probes and peptidase activatable fluorescence probes were also discussed.

Structural determination of glucosylceramides in the distillation remnants of shochu, the Japanese traditional liquor, and its production by Aspergillus kawachii.

Shochu is traditional Japanese liquor produced from various crops and fungi Aspergillus kawachi or A. awamorii . The amount of unutilized shochu distillation remnants is increasing because of the recent prohibition of ocean dumping of these remnants.

Fluorescence endoscopic detection of murine colitis-associated colon cancer by topically applied enzymatically rapid-activatable probe.

Objectives: Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer.

Methods: Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry.

Highly activatable and environment-insensitive optical highlighters for selective spatiotemporal imaging of target proteins.

Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions.

[Highly sensitive intraoperative detection of tiny tumors with novel "activatable" fluorescence probes].

Several versatile and rational design strategies for novel fluorescence probes including those based on photoinduced electron transfer have been successfully established. Indeed, based on these design strategies, various novel fluorescence probes were successfully developed including those for reactive oxygen species and reporter enzymes. Furthermore, we have succeeded to visualize tiny tumors in living mice by using cancer-specific antibodies tagged with acidic-pH activatable fluorescence probes.