Fluorescent Molecular Logic Gates Driven by Temperature and by Protons in Solution and on Solid.

Temperature-driven fluorescent NOT logic is demonstrated by exploiting predissociation in a 1,3,5-trisubstituted Δ2 -pyrazoline on its own and when grafted onto silica microparticles. Related Δ2 -pyrazolines become proton-driven YES and NOT logic gates on the basis of fluorescent photoinduced electron transfer (PET) switches. Additional PASS 1 and YES+PASS 1 logic gates on silica are also demonstrated within the same family. Beside these small-molecule systems, a polymeric molecular thermometer based on a benzofurazan-derivatized N-isopropylacrylamide copolymer is attached to silica to produce temperature-driven fluorescent YES logic.

Cationic Fluorescent Nanogel Thermometers based on Thermoresponsive Poly(N-isopropylacrylamide) and Environment-Sensitive Benzofurazan.

Cationic nanogels of N-isopropylacrylamide (NIPAM), including NIPAM-based cationic fluorescent nanogel thermometers, were synthesized with a cationic radical initiator previously developed in our laboratory. These cationic nanogels were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and fluorescence spectroscopy, as summarized in the temperature-dependent fluorescence response based on the structural change in polyNIPAM units in aqueous solutions. Cellular experiments using HeLa (human epithelial carcinoma) cells demonstrated that NIPAM-based cationic fluorescent nanogel thermometers can spontaneously enter the cells under mild conditions (at 25 °C for 20 min) and can show significant fluorescence enhancement without cytotoxicity with increasing culture medium temperature. The combination of the ability to enter cells and non-cytotoxicity is the most important advantage of cationic fluorescent nanogel thermometers compared with other types of fluorescent polymeric thermometers, i.e., anionic nanogel thermometers and cationic/anionic linear polymeric thermometers.

Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter.

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c+ microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein α (SIRPα), a membrane protein, induced the emergence of CD11c+ microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRPα, and microglia-specific SIRPα-knockout mice exhibited the same phenotype, suggesting that an interaction between microglial SIRPα and CD47 on neighbouring cells suppressed the emergence of CD11c+ microglia. A lack of SIRPα did not cause detectable damage to the white matter, but resulted in the increased expression of genes whose expression is characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRPα. Thus, microglial SIRPα suppresses the induction of CD11c+ microglia that have the potential to accelerate the repair of damaged white matter.

A Cell-Targeted Non-Cytotoxic Fluorescent Nanogel Thermometer Created with an Imidazolium-Containing Cationic Radical Initiator.

A cationic fluorescent nanogel thermometer based on thermo-responsive N-isopropylacrylamide and environment-sensitive benzothiadiazole was developed with a new azo compound bearing imidazolium rings as the first cationic radical initiator. This cationic fluorescent nanogel thermometer showed an excellent ability to enter live mammalian cells in a short incubation period (10 min), a high sensitivity to temperature variations in live cells (temperature resolution of 0.02-0.84 °C in the range 20-40 °C), and remarkable non-cytotoxicity, which permitted ordinary cell proliferation and even differentiation of primary cultured cells.

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.

Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.

A fluorescent acrylamide-type monomer bearing an environment-sensitive methoxybenzocoumarin structure for the development of functional polymeric sensors.

A new fluorescent acrylamide-type monomer bearing a hydrogen bonding- and polarity-sensitive benzocoumarin fluorophore was synthesized. The absorption spectra, fluorescence spectra, and fluorescence lifetime of a model compound were measured in ten solvents with different hydrogen-bonding abilities and polarities to investigate the sensitivity of the fluorophore to the surrounding environment. These spectroscopic studies demonstrated that the fluorophore emits stronger fluorescence in more protic, polar environments. A fluorescent polymeric thermometer was prepared from N-isopropylacrylamide and the new fluorescent monomer, and it showed good functionality in aqueous solution (e.g., high sensitivity to temperature changes and high chemical stability), indicating the applicability of the herein developed fluorescent monomer for use in functional sensors.

A cationic fluorescent polymeric thermometer for the ratiometric sensing of intracellular temperature.

We developed new cationic fluorescent polymeric thermometers containing both benzothiadiazole and BODIPY units as an environment-sensitive fluorophore and as a reference fluorophore, respectively. The temperature-dependent fluorescence spectra of the thermometers enabled us to perform highly sensitive and practical ratiometric temperature sensing inside living mammalian cells. Intracellular temperatures of non-adherent MOLT-4 (human acute lymphoblastic leukaemia) and adherent HEK293T (human embryonic kidney) cells could be monitored with high temperature resolutions (0.01-1.0 °C) using the new cationic fluorescent polymeric thermometer.

Activation of electrophilicity of stable Y-delocalized carbamate cations in intramolecular aromatic substitution reaction: evidence for formation of diprotonated carbamates leading to generation of isocyanates.

Although cations with three heteroatoms, such as monoprotonated guanidine and urea, are stabilized by Y-shaped conjugation and such Y-conjugated cations are sufficiently basic to be further protonated (or protosolvated) to dications in strongly acid media, only O-monoprotonated species have been detected in the case of carbamates even in magic acid. We found that the trifluoromethanesulfonic acid-catalyzed cyclization of arylethylcarbamates proceeds to afford dihydroisoquinolones in high yield. In strong acids, methyl carbamates are fully O-monoprotonated, and these monocations do not undergo cyclization even under heating. But, as the acidity of the reaction medium is further increased, the cyclization reaction of methyl phenethylcarbamates starts to proceed as a first-order reaction, with a linear relationship between rate and acidity. The sign and magnitude of the entropy of activation ΔS(‡) were found to be similar to those of other A(Ac)1 reactions. These results strongly support the idea that further protonation of the O-protonated carbamates is involved in the cyclization, but the concentration of the dications is very low and suggests that the rate-determining step is dissociation of methanol from the diprotonated carbamate to generate protonated isocyanate, which reacts with the aromatic ring. Therefore, O-protonated carbamates are weak bases in sharp contrast to other Y-shaped monocations.

Environment-sensitive fluorophores with benzothiadiazole and benzoselenadiazole structures as candidate components of a fluorescent polymeric thermometer.

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.

A cross-sectional analysis of quality of life in Japanese acne patients using the Japanese version of Skindex-16.

We measured the quality of life of Japanese patients with acne using the Japanese version of Skindex-16, a semantically equivalent and validated translation of the original version produced by Chren et al. A total of 210 acne patients were enrolled in this study. The results showed that patients with acne experienced more severe emotional effects from their skin disease than functional or symptomatic effects. The scores for emotional aspects were related to the severity of the patients' acne following a prescribed scale, but the scores were high even when the patients only had a few comedones. Akaike's information criterion showed that the scores for symptoms were related to the severity of the patients' acne, and the patients' emotions were related to the interval between clinic visits. To improve patients' quality of life, treatment of comedones should be fully respected. In addition, dermatologists should encourage patients to visit clinics regularly to help them improve the emotional aspects of their quality of life.

Measurement of the impact of atopic dermatitis on patients' quality of life: a cross-sectional and longitudinal questionnaire study using the Japanese version of Skindex-16.

The impact of atopic dermatitis on patients' quality of life was measured using the Japanese version of Skindex-16 in a cross-sectional and longitudinal questionnaire study. One hundred sixty-two adult patients completed Skindex-16 and were followed-up with a standard medical therapy. Three to six months after the initial testing, 135 (83.3%) of the patients again completed Skindex-16 and also answered a general question about whether their skin condition had improved, remained the same, or become worse. The scores of Skindex-16 of 162 patients with atopic dermatitis were significantly higher than those of patients with isolated lesions, particularly in the Symptoms and Emotions scales. Patients with severe atopic dermatitis showed significantly higher scores in the three scales (Symptoms, Emotions, and Functioning), and there was a significant positive correlation between the severity and the 3-scale scores. After the follow-up period, 78 of 135 patients (57.8%) reported that their skin condition had improved. Forty-six patients (34.1%) reported that their skin condition had remained the same, and 11 (8.1%) became worse. Among the patients who said their dermatitis had improved, the scores of Skindex-16 significantly decreased. On the other hand, patients who reported their dermatitis worse showed an increase in the scores. These findings suggest that Skindex-16 responsively measures the disease severity and clinical change in the estimation of the effects of atopic dermatitis on patients' quality of life. This practical and sensitive, skin-disease specific, quality-of-life instrument is valuable for assessing patients' outcomes, especially their response to therapy, and is useful to understanding and improving the quality of life of patients suffering with atopic dermatitis.

The Japanese version of Skindex-16: a brief quality-of-life measure for patients with skin diseases.

A practical quality-of-life measure applicable to patients with skin diseases is necessary. Recently developed dermatological quality-of-life measures must be translated and adapted for use in cultures other than the ones in which they were created. In this study, we translated and adapted culturally into Japanese a skin-disease-specific, brief quality-of-life measure, Skindex-16, and studied its reliability and validity. Forward-and back-translations of Skindex-16 were carried out. Six doubtful items as well as the term "skin condition" required a second forward- and back-translation to reach satisfactory agreement with the original instrument. Cross-cultural adaptation and cross-sectional questionnaire studies were then performed to evaluate the reliability and validity of the instrument. One hundred patients and 30 healthy adults responded to the Japanese version. The internal-consistency reliability of the final Japanese version of Skindex-16 was high (range of Cronbach's alpha for each scale, symptoms, emotions, and functioning, was 0.83-0.92). The Japanese version showed construct and content validity. As hypothesized, scores for dermatological patients were higher than those for healthy persons (mean global scores 36 +/- 23 vs 1 +/- 2, p < 0.001) and scores for patients with inflammatory diseases were higher than those for patients with isolated skin lesions (mean global scores 48 +/- 21 vs 22 +/- 17, p < 0.001), indicating a poorer quality of life. Most patients' responses to an open-ended question about their skin disease were similar to those of the American responders and were addressed according to the items. In conclusion, we have developed a semantically equivalent translation of Skindex-16 into Japanese. It is a reliable and valid measure of the effects of skin disease on the quality of life in Japanese patients.