Morphological basis of the lung adenocarcinoma subtypes.

Lung adenocarcinoma (LUAD), which accounts for a large proportion of lung cancers, is divided into five major subtypes based on histologic characteristics. The clinical characteristics, prognosis, and responses to treatments vary among subtypes. Here, we demonstrate that the variations of cell-cell contact energy result in the LUAD subtype-specific morphogenesis.

Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB).

An integrated microfluidic bubble pocket for long-term perfused three-dimensional intestine-on-a-chip model.

Perfused three-dimensional (3D) cultures enable long-term growth and monitoring of 3D organoids making them well-suited for investigating organoid development, growth, and function. One of the limitations of this long-term on-chip perfused 3D culture is unintended and disruptive air bubbles. To overcome this obstacle, we invented an imaging platform that integrates an innovative microfluidic bubble pocket for long-term perfused 3D culture of gastrointestinal (GI) organoids.

[Evaluation of Hygiene in Dry Powder Steroid Drug Inhalers Used by Elderly Asthma Patients].

Dry powder inhalers (DPI) are frequently used by asthmatic patients, and the usage rate increases every year. The pharmacists at our hospital provided initial inhalation instructions on how the inhaler must be used but did not elaborate on the cleaning of the device. Therefore, the cleaning status of the inhaler is unknown, and there is a possibility of bacterial growth.

Inositol 1,4,5-Trisphosphate Receptor Type 3 Regulates Neuronal Growth Cone Sensitivity to Guidance Signals.

During neurodevelopment, the growth cone deciphers directional information from extracellular guidance cues presented as shallow concentration gradients via signal amplification. However, it remains unclear how the growth cone controls this amplification process during its navigation through an environment in which basal cue concentrations vary widely. Here, we identified inositol 1,4,5-trisphosphate (IP) receptor type 3 as a regulator of axonal sensitivity to guidance cues in vitro and in vivo.

Dual-FRET imaging of IP and Ca revealed Ca-induced IP production maintains long lasting Ca oscillations in fertilized mouse eggs.

In most species, fertilization induces Ca transients in the egg. In mammals, the Ca rises are triggered by phospholipase Cζ (PLCζ) released from the sperm; IP generated by PLCζ induces Ca release from the intracellular Ca store through IP receptor, termed IP-induced Ca release. Here, we developed new fluorescent IP sensors (IRIS-2s) with the wider dynamic range and higher sensitivity (Kd = 0.

WNT Takes Two to Tango: Molecular Links between the Circadian Clock and the Cell Cycle in Adult Stem Cells.

Like two dancers, the circadian clock and cell cycle are biological oscillators engaged in bidirectional communication, resulting in circadian clock-gated cell division cycles in species ranging from cyanobacteria to mammals. The identified mechanisms for this phenomenon have expanded beyond intracellular molecular coupling components to include intercellular connections. However, detailed molecular mechanisms, dynamics, and physiological functions of the circadian clock and cell cycle as coupled oscillators remain largely unknown.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition.

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles.

Attenuation of teratoma formation by p27 overexpression in induced pluripotent stem cells.

Background: Pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells, have a great potential for regenerative medicine. Induced pluripotent stem cells, in particular, are suitable for replacement of tissue by autologous transplantation. However, tumorigenicity is a major risk in clinical application of both embryonic stem cells and induced pluripotent stem cells.

Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid.

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration.

Efficient gene editing in with CRISPR technology.

Background: Efficient gene editing is a critical tool for investigating molecular mechanisms of cellular processes and engineering organisms for numerous purposes ranging from biotechnology to medicine. Recently developed RNA-guided CRISPR/Cas9 technology has been used for efficient gene editing in various organisms, but has not been tested in a model filamentous fungus, .

Findings: In this report, we demonstrate efficient gene replacement in a model filamentous fungus, , with the CRISPR/Cas9 system.

Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine.

Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial 'miniguts'), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells.

Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+) signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+) oscillations in terms of the time constant of Ca(2+) spike amplitude decay and the Ca(2+) oscillation frequency.

Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation.

Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca²+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca²+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca²+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca²+ responses.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits.

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells.

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown.

Spontaneous network activity visualized by ultrasensitive Ca(2+) indicators, yellow Cameleon-Nano.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60.

Auto-luminescent genetically-encoded ratiometric indicator for real-time Ca2+ imaging at the single cell level.

Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination.

Methodology/principal Findings: We applied BRET to develop an autoluminescent Ca(2+) indicator, BRAC, which is composed of Ca(2+)-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far.

G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1,4,5-triphosphate receptor-mediated Ca2+ signal regulation.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated calcium (Ca(2+)) release channel and plays important roles in regulation of numerous Ca(2+)-dependent cellular responses. Many intracellular modulators and IP(3)R-binding proteins regulate the IP(3)R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP(3)R-binding proteins.

Control of neuronal growth cone navigation by asymmetric inositol 1,4,5-trisphosphate signals.

Inositol 1,4,5-trisphosphate (IP(3)) is generally viewed as a global messenger that increases cytosolic calcium ion (Ca(2+)) concentration. However, the spatiotemporal dynamics of IP(3) and the functional significance of localized IP(3) production in cell polarity remain largely unknown. Here, we demonstrate the critical role of spatially restricted IP(3) signals in axon guidance.

Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities.

We developed a simple algorithm, i-Score (inhibitory-Score), to predict active siRNAs by applying a linear regression model to 2431 siRNAs. Our algorithm is exclusively comprised of nucleotide (nt) preferences at each position, and no other parameters are taken into account. Using a validation dataset comprised of 419 siRNAs, we found that the prediction accuracy of i-Score is as good as those of s-Biopredsi, ThermoComposition21 and DSIR, which employ a neural network model or more parameters in a linear regression model.

Influence of visual information and test paradigm on clinical olfactory test results.

Objective: The goal of this study was to determine if visual information and test paradigms affect clinical olfactory test results.

Methods: Three hundred and ninety-seven Japanese patients with complaints of olfactory dysfunction were administered both a new clinical olfactory test, the Odor Stick Identification Test for Japanese (OSIT-J), and the Japanese benchmark olfactory test, T&T olfactometry. Four different methods were used to administer the OSIT-J combining paradigms using word or picture-word alternatives with the four-plus alternative method based on a top-down strategy or the two-step identification method based on a bottom-up strategy.