A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level.

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

Differential Effect of Azetidine Substitution in Firefly Luciferin Analogues.

Replacing an N,N-dimethylamino group in a classical fluorophore with a four membered azetidine ring provides an improved luminescence quantum yield. Herein, we extended this strategy to bioluminescent firefly luciferin analogues and evaluated its general validity. For this purpose, four types of luciferin cores were employed, and a total of eight analogues were evaluated. Among these analogues, unexpectedly, only the benzothiazole core analogue benefited from an azetidine substitution and showed enhanced bioluminescence. In addition, fluorescence measurements revealed that an azetidine substitution improved the fluorescence quantum yield by 2.3-times compared to a N,N-dimethylamino group. These findings clarify the differential effects of azetidine substituents in luciferins and present one possible strategy for enhancing photon output in benzothiazole type luciferins through a synthetic approach.

Long-term single cell bioluminescence imaging with C-3 position protected coelenterazine analogues.

Bioluminescence is a powerful imaging modality for monitoring biological phenomena both in vitro and in vivo. Bioluminescence imagin (BLI) is becoming a seamless imaging technology covering the range from cells to organs of small animals. Long-term imaging at the single cell level would lead to a true understanding of the dynamics of life phenomena. This work presents a long-term single cell bioluminescence imaging technology accomplished with C-3 position protected furimazines (FMZs), a CTZ analogues, which generate intense blue emission when paired with a highly stable engineered luciferase, Nanoluc. Four types of FMZs protected at the C-3 position have been synthesized. The type and steric bulkiness of the protection group strongly contributed to storage stability and the kinetics of the bioluminescence reactions of the analogues in human living cells. In particular, two developed FMZ analogues resulted in significantly longer bioluminescence emission with higher S/N ratio than FMZ at single cell level. Long-term bioluminescence single cell imaging technology with the developed FMZ analogues will lead to seamless imaging in the range from cells to organs of small animals.

Ring-Fused Firefly Luciferins: Expanded Palette of Near-Infrared Emitting Bioluminescent Substrates.

Firefly bioluminescence is broadly applied as a noninvasive imaging modality in the biomedical research field. One limitation in firefly bioluminescence imaging is the limited variety of luciferins emitting in the near-infrared (NIR) region (650-900 nm), where tissue penetration is high. Herein, we describe a series of structure-inherent NIR emitting firefly luciferin analogues, NIRLucs, designed through a ring fusion strategy. This strategy resulted in pH-independent structure-inherent NIR emission with a native firefly luciferase, which was theoretically supported by quantum chemical calculations of the oxidized form of each luciferin. When applied to cells, NIRLucs displayed dose-independent improved NIR emission even at low concentrations where the native d-luciferin substrate does not emit. Additionally, excellent blood retention and brighter photon flux (7-fold overall, 16-fold in the NIR spectral range) than in the case of d-luciferin have been observed with one of the NIRLucs in mice bearing subcutaneous tumors. We believe that these synthetic luciferins provide a solution to the longstanding limitation in the variety of NIR emitting luciferins and pave the way to the further development of NIR bioluminescence imaging platforms.

Near-Infrared Fluorescent Probes for Imaging of Intracellular Mg2+ and Application to Multi-Color Imaging of Mg2+, ATP, and Mitochondrial Membrane Potential.

The magnesium ion (Mg2+) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg2+, there is a great need for the development of Mg2+-selective fluorescent probes. However, conventional Mg2+ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multi-color imaging, we designed and synthesized highly Mg2+-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged ß-diketone as a selective binding site for Mg2+ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg2+. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg2+. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg2+ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg2+ dynamics with NIR fluorescence. Moreover, it allows one to simultaneously visualize changes in Mg2+ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multi-color imaging of intracellular Mg2+.

Inkjet-printed pH-independent paper-based calcium sensor with fluorescence signal readout relying on a solvatochromic dye.

A challenge for paper-based cation sensors relying on classical carrier-based ion-selective optodes (ISOs) is their pH-cross response caused by the use of H+-sensitive chromoionophores as optical signal transducers. This work demonstrates fully pH-independent fluorescence-based calcium detection with a paper-based plasticizer-free ISO. To achieve a pH-independent assay, a solvatochromic dye (SD) instead of a traditional H+-sensitive chromoionophore has been applied to the paper-based ISO by means of inkjet printing technology. The detection principle depends on an ionophore-driven phase-transfer ion-exchange reaction between target cations and the positively charged SD, which no longer involves H+ in the optical signal transduction process. The developed paper-based ISOs with the SD resulted in Ca2+ concentration-dependent response curves not affected by the sample pH (pH 6.0, 7.0, and 8.0). The dynamic range obtained for Ca2+ detection was from 10-5 to 1 mol L-1 with a detection limit of 19.3 µmol L-1. Additionally, excellent selectivity derived from the used ionophore has been confirmed. As a simple practical application, the determination of Ca2+ in mineral water has been achieved without the pH-buffering process required for conventional cation-exchange ISOs.

Near-Infrared Bioluminescence Imaging with a through-Bond Energy Transfer Cassette.

A coelenterazine (CTZ) analogue emitting near-infrared (NIR) bioluminescence was synthesized for through-bond energy transfer (TBET)-based imaging modalities. The analogue, named Cy5-CTZ, was prepared by conjugating cyanine-5 (Cy5) dye to CTZ through an acetylene linker. This novel derivative is intrinsically fluorescent and emits NIR-shifted luminescence upon reacting with an appropriate luciferase, the Renilla luciferase. This Cy5-CTZ substrate is optically stable in physiological samples and rapidly permeabilize through the plasma membrane into the cytosolic compartment of live cells.

Effect of hip angle on neuromuscular activation of the adductor longus and adductor magnus muscles during isometric hip flexion and extension.

PURPOSE: Neuromuscular activation of the adductor longus (AL) and adductor magnus (AM) muscles at different hip flexion angles during hip flexion and extension has not been clarified. This study aimed to compare the relationship between hip flexion angle and the electromyogram of the AL muscle with that of the AM muscle during isometric hip flexion and extension. METHODS: Fifteen healthy young men were included in this study. Participants performed maximal voluntary contractions during hip flexion and extension at six different hip flexion angles: - 20°, 0°, 20°, 40°, 60°, and 80°. The surface electromyograms of the AL and AM muscles were recorded. The root mean square (RMS) was calculated and normalized by the RMS during hip adduction for each individual muscle. RESULTS: The normalized RMS of the AL muscle was significantly higher than that of the AM muscle at a hip flexion angle of - 20° during hip flexion (P < 0.05). The mean normalized RMS of the AM muscle was significantly higher than that of the AL muscle during hip extension (P < 0.01). CONCLUSION: These results suggest that the AL muscle is recruited specifically at the hip-extended position during hip flexion, and that the AM muscle is recruited regardless of the hip position during hip extension. Thus, the AL and AM muscles may have different functional roles in different hip flexion angles.

Rational design of pH-responsive near-infrared spirocyclic cyanines: the effects of substituents and the external environment.

pH-responsive spirocyclic cyanine dyes were designed and synthesized. The equilibrium constant for cyclization (pKcycl) could be rationally controlled by changing the nucleophilic moiety and the side chains. Encapsulation in polymeric micelles inhibited the H-aggregation of the dye, and the pKcycl could be shifted according to the amphiphilic polymer employed.

The Short-term Outcomes of Physiotherapy for Patients with Acetabular Labral Tears: An Analysis according to Severity of Injury in Magnetic Resonance Imaging.

Purpose: The aim of this study was to evaluate the short-term outcome of physiotherapy in patients with acetabular labral tears and to assess the effectiveness of physiotherapy according to the severity of the labral tear. Materials and Methods: Thirty-five patients who underwent physiotherapy for treatment of symptomatic acetabular labral tears were enrolled. We evaluated the severity of the acetabular labral tears, which were classified based on the Czerny classification system using 3-T MRI. Clinical findings of microinstability and extra-articular pathologies of the hip joint were also examined. The International Hip Outcome Tool 12 (iHOT12) was use for evaluation of outcome scores pre- and post-intervention. Results: The mean iHOT12 score showed significant improvement from 44.0 to 73.6 in 4.7 months. Compared with pre-intervention scores, significantly higher post-intervention iHOT12 scores were observed for Czerny stages I and II tears (all P<0.01). However, no significant difference was observed between pre-intervention and post-intervention iHOT12 scores for stage III tears (P=0.061). In addition, seven patients (20.0%) had positive microinstability findings and 22 patients (62.9%) had findings of extra-articular pathologies. Of the 35 patients, eight patients (22.9%) underwent surgical treatment after failure of conservative management; four of these patients had Czerny stage III tears. Conclusion: The iHOT12 score of patients with acetabular labral tears was significantly improved by physiotherapy in the short-term period. Improvement of the clinical score by physiotherapy may be poor in patients with severe acetabular labral tears. Determining the severity of acetabular labral tears can be useful in determining treatment strategies.

Treatment with hepatocyte transplantation in a novel mouse model of persistent liver failure.

Background and Aim: Cell-based transplantation therapy using hepatocytes, hepatic stem cells, hepatocyte-like cells induced from stem cells, etc. is thought to be an attractive alternative to liver transplantation, and have been studied to date. For its clinical application, however, it is extremely important to develop a model that reproduces the pathological conditions with indication for treatment and enables the study for the ideal treatment strategy. Methods: The transgenic mice which express the thymidine kinase (TK) gene of human herpes simplex virus (HSV) in their hepatocytes with normal immunity has been developed (designated as HSVtk). After ganciclovir (GCV) administration which injure TK-expressing hepatocytes, the primary hepatocytes (PHs) isolated from green fluorescent protein (GFP) transgenic mouse (GFP-tg) were transplanted to HSVtk intrasplenically, and replacement index (RI) with transplanted PHs in the liver, liver histology, and mRNA expressions in the liver were analyzed up to 8 weeks after transplantation. Results: HSVtk without PH transplantation after GCV administration developed persistent liver failure with degenerated hepatocytes, persistent elevation of ALT and hepatic p16 mRNA levels, suggesting the existence of cellular senescence in the base of the disease. When autologous GFP-PHs were transplanted to HSVtk, the transplanted cells were successfully engrafted in the liver. Eight weeks after transplantation, serum ALT levels and liver histology were almost normalized, while RIs varied from 19.8 to 73.8%. Since the hepatic p16 mRNA levels were decreased significantly in these mice, the senescence of hepatocytes associated with liver injury was thought to be resolved. On the other hand, allogenic GFP-PHs transplanted to HSVtk were eliminated as early as 1 week after transplantation. In these mice, hepatic p16 mRNA levels were significantly increased at 8 weeks after transplantation, suggesting the aggravation of hepatocyte senescence. FK506 administration to HSVtk protected the transplanted hepatocytes with allogenic background from rejection at 2 weeks after transplantation, but the condition of mice and the senescent status in the liver seemed worsened. Conclusions: The mouse model with HSVtk/GCV system was useful for studying the mechanism of liver regeneration and the immune rejection responses in the hepatocyte transplantation treatment. It may also be utilized to develop the effective remedies to avoid immune rejection.

Development of Near-Infrared Fluorescent Mg2+ Probe and Application to Multicolor Imaging of Intracellular Signals.

Recent extensive studies revealed that the intracellular concentration of magnesium ions (Mg2+) is one of the important factors to regulate cellular functions. To evaluate the impact of Mg2+ concentration changes on intracellular signals or events, simultaneous imaging of Mg2+ with those phenomena is a powerful technique. The present protocol describes the synthesis and evaluation of near-infrared (NIR) fluorescent Mg2+-selective probes, named KMG-500 series, and the application to simultaneous imaging of the corresponding intracellular signal transductions and molecular events. The present protocol for multicolor imaging using fluorescent probes in the NIR and visible ranges is highly useful to reveal how multiple molecular events are correlated each other in each single cell.

Quantitative Evaluation of Ankle Instability Using a Capacitance-Type Strain Sensor.

BACKGROUND: Manual evaluation is an important method for assessing ankle instability, but it is not quantitative. Capacitance-type sensors can be used to measure the distance on the basis of the capacitance value. We applied the sensor to the noninvasive device for measuring ankle instability and showed its utility. METHODS: First, 5 ankles embalmed by Thiel's method were used in an experiment using a cadaver. The capacitance-type sensor was fixed alongside the anterior talofibular ligament (ATFL) of a specially made brace, and the anterior drawer test was performed. The test had been performed for the intact ankle, with the ATFL transected and with both the ATFL and calcaneofibular ligament (CFL) transected. The anterior drawer distance was calculated by the sensor. Intra- and interinvestigator reliability were also analyzed.Next, as a clinical study, a brace with a sensor was fitted to 22 ankles of 20 patients with a history of ankle sprain. An anterior drawer test at a load of 150 N was conducted using a Telos stress device. The anterior drawer distances measured by the sensor and based on radiographic images were then compared. RESULTS: The mean anterior drawer distances were 3.7 ± 1.0 mm for the intact cadavers, 6.1 ± 1.6 mm with the ATFL transected (P < .001), and 7.9 ± 1.8 mm with the ATFL and CFL transected (P < .001). The intrainvestigator intraclass correlation coefficients (ICCs) were 0.862 to 0.939, and the interinvestigator ICC was 0.815. In the experiments on patients, the mean anterior drawer distance measured by the sensor was 2.9 ± 0.9 mm, and it was 2.7 ± 0.9 mm for the radiographic images. The correlation coefficient between the sensor and the radiographic images was 0.843. CONCLUSION: We quantitatively evaluated anterior drawer laxity using a capacitance-type sensor and found it had high reproducibility and strongly correlated with stress radiography measurements in patients with ankle instability. Capacitance-type sensors can be used for the safe, simple, and accurate evaluation of ankle instability.

Inkjet-Printed Colorimetric Paper-Based Gas Sensor Arrays for the Discrimination of Volatile Primary Amines with Amine-Responsive Dye-Encapsulating Polymer Nanoparticles.

Arrays of gas sensors are of high interest for "electronic nose" applications. Here, we describe the fabrication of a colorimetric single-use gas sensor array made of paper allowing the discrimination of volatile primary amines based on their polarity. For this purpose, polymeric nanoparticles with different polarities containing an amine-sensitive chromogenic dye are deposited onto paper substrates by means of inkjet printing. Data processing is conducted by red-green-blue (RGB) color extraction, followed by principal component analysis (PCA) or agglomerative hierarchical clustering (AHC) analysis. The application to the discrimination of two cheese samples is demonstrated.

Highly Sensitive Bioluminescent Probe for Thiol Detection in Living Cells.

The sensitive detection of thiols including glutathione and cysteine is desirable owing to their roles as indispensable biomolecules in maintaining intracellular biological redox homeostasis. Herein, we report the design and synthesis of SEluc-1 (sulfinate ester luciferin), a chemoselective probe exhibiting a ratiometric and turn-on response towards thiols selectively in fluorescence and bioluminescence, respectively. The probe, which was designed based on the "caged" luciferin strategy, displays excellent selectivity, high signal/noise ratio (>240 in the case of bioluminescence), and a biologically relevant limit of detection (LOD, 80 nm for cysteine), which are all desirable traits for a sensitive bioluminescent sensor. SEluc-1 was further applied to fluorescence imaging of thiol activity in living human cervical cancer HeLa cell cultures, and was successfully able to detect fluctuations in thiol concentrations induced by oxidative stress in a bioluminescent assay utilizing African green monkey fibroblast COS-7 cells and human breast adenocarcinoma MCF-7 cells.

An allylated firefly luciferin analogue with luciferase specific response in living cells.

An allylated firefly luciferin was successfully synthesized and its bioluminescence properties were evaluated. When applied to cellular imaging in combination with Eluc, which is one of the commercially available luciferases, this analogue displayed a luciferase-specific bioluminescence signal with prolonged emission (>100 min).