Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca2+. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca2+ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca2+, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks

Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance

SummaryAn increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA-dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.Video Abstrac

Deutschsprachige Literatur und Theater seit 1945 in den Metropolen Seoul, Tokio und Berlin Studien zur urbanen Kulturentwicklung unter komparatistischen und rezeptionsgeschichtlichen Perspektiven

Im Mittelpunkt der Studien stand die Frage nach der spezifischen Funktion der Literatur, insbesondere des Theaters, für die Entwicklung einer urbanen Kultur nach 1945. Die beteiligten Beiträge konzentrieren sich dabei auf die Aufnahme des dramatischen Werks deutschsprachiger Autoren (Bertolt Brecht, Heiner Müller, Peter Handke, Botho Strauß) in Berlin, Tokio und Seoul. Einen ergänzenden Untersuchungsaspekt bildet die Rezeption der japanischen Autorin Yoko Tawada und des koreanischen Dramaturgen und Regisseurs Kim Min-Gi in Deutschland. Die Konstellation der Metropolen Berlin-Tokio-Seoul unter kultur-komparatistischen und rezeptionsgeschichtlichen Gesichtspunkten bietet eine nicht nur reizvolle, sondern auch innovative Fragestellung, insbesondere im Hinblick auf Aspekte der urbanen Kulturentwicklung seit 1945. Das Ende des Zweiten Weltkriegs brachte für die Hauptstädte Deutschlands, Japans und Koreas gleichermaßen das Erfordernis einer radikalen Neuorientierung mit sich, die den gesamten kulturellen Bereich einbeziehen sollte. Dem Theater – und zwar der neu entstehenden Dramatik und ihren bedeutenden Autoren ebenso wie den bestehenden und den neu zu errichtenden Bühnen – kommt hierbei eine kulturpolitisch wie kulturgeschichtlich einzigartige Bedeutung zu

Simultaneous live cell imaging using dual FRET sensors with a single excitation light.

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+) and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility

The synthesis and structural characterization of boron-doped silicon-nanocrystals with enhanced electroconductivity

Boron (B)-doped silicon-nanocrystals (Si-NCs) with wavelength-tunable photoluminescence (PL) properties in the visible region are successfully prepared for the first time, leading to significant enhancement of electroconductivities. The B-doped Si-NCs are prepared on a p-type Si(100) substrate by co-deposition of p-type Si(100) chips/boron chips/silica disk targets. As the number of the B chips used as the target is increased, the amount of doped B content increases gradually. Here the amount of doped B content in the Si-NCs is controlled from 0 to 0.4, 0.7, 2.3 at.%. The B elemental states, compositional ratios, and surface condition of the obtained Si-NCs are fully characterized by high-resolution transmission electron microscopy (HRTEM) observations, micro-Raman scattering spectroscopic analysis, etc. Our B-doped Si-NCs possess both the continuous luminescence property in the visible region and enhanced electroconductivity. The red-shift of the PL peak is confirmed by the increase of the amount of doped B content. This paper should be very important from the viewpoint of application to optoelectronic devices and electroluminescent (EL) displays

A STUDY ON UNIFICATION OF WELDING CONSUMABLES IN CONSTRUCTION OF CHEMICAL CARGO TANKER MADE OF DUPLEX STAINLESS STEEL: EVALUATION OF STATIC STRENGTH OF WELDED JOINTT

Austenitic stainless steels such as SUS 316 LN and austenitic stainless clad steel are used in cargo holds of chemical tankers owing to their corrosion resistance. Recently, the use of duplex stainless steels has been increasing, owing to their better strength and corrosion resistance and lower content of expensive Ni, compared with those features of austenitic stainless steels. However, few duplex stainless clad steels have been approved by classification bodies. Furthermore, the application of duplex stainless steel is not yet mainstream as hull structural materials because a stable supply market has yet to be established. Therefore, when applying cladding steel to construction of chemical tankers, austenitic stainless clad steel is often used at present. The duplex stainless steel and the austenitic stainless clad steel are mixed at construction factories; hence, there is a risk of misuse of welding consumables. If misuse is suspected, it is not possible to judge the erroneous use from visual inspection after construction; therefore it is necessary to uniformly remove the weld and re-weld. However, if universal welding consumables were identified, this might avoid the problems of misuse and simplify the procurement of welding materials. / In this paper, we report on our studies to verify welding consumables for use in the hull structures, involving a mixture of duplex stainless steel and the austenitic stainless clad steel. The static strength of the welded joints is a particular focus of this study, from which we confirmed the validity and limitations of welding consumables.Paper No: OMAE2019-95818, V004T03A05

Nursing support for breathlessness in patients with cancer: a scoping review

Objective To identify nursing support provided for the relief of breathlessness in patients with cancer.Design A scoping review following a standard framework proposed by Arksey and O’Malley.Study selection Electronic databases (PubMed, CINAHL, CENTRAL and Ichushi-Web of the Japan Medical Abstract Society Databases) were searched from inception to 31 January 2022. Studies reporting on patients with cancer (aged ≥18 years), intervention for relief from breathlessness, nursing support and quantitatively assessed breathlessness using a scale were included.Results Overall, 2629 articles were screened, and 27 were finally included. Results of the qualitative thematic analysis were categorised into 12 nursing support components: fan therapy, nurse-led intervention, multidisciplinary intervention, psychoeducational programme, breathing technique, walking therapy, inspiratory muscle training, respiratory rehabilitation, yoga, acupuncture, guided imagery and abdominal massage.Conclusions We identified 12 components of nursing support for breathlessness in patients with cancer. The study results may be useful to understand the actual state of nursing support provided for breathlessness in patients with terminal cancer and to consider possible support that can be implemented

In vitro characterization of the human segmentation clock.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock1,2. Although this oscillator has been well-characterized in model organisms1,2, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans3. Here we show that human PSM cells derived in vitro-as well as those of the mouse4-recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling5. Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'clock and wavefront' models1. Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology