The role of osteocrin in memory formation during early learning, as revealed by visual imprinting in chicks.

Osteocrin (OSTN) is structurally associated with natriuretic peptides. Its expression in the brain, which has only been recognized in anthropoid primates, is induced by sensory stimuli and regulates the activity-dependent dendritic growth of neurons. However, details on the signaling mechanisms of OSTN and its function in plastic changes during learning and memory have yet to be elucidated.

BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing.

We developed a nanobody-based homogeneous bioluminescent immunosensor to achieve a one-pot detection for point-of-care testing (POCT). This immunosensor was named BRET nano Q-body as its emission color changes via bioluminescence resonance energy transfer (BRET) upon antigen addition. NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminus of the nanobody, which was labeled with a fluorescent dye via thiol-maleimide Michael addition.

Customizable OpenGUS immunoassay: A homogeneous detection system using β-glucuronidase switch and label-free antibody.

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a β-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system.

[Make the invisible visible-innovation in the single fluorescent protein-based indicators].

Biological phenomena are generated by the cooperative and hierarchical relationships between a variety of biomolecules, such as proteins, metabolites, signaling molecules, and ions. In many cases, however, these biomolecules do not have color, and it is difficult to observe them as they are. Therefore, it is necessary to "visualize" each molecule with color or fluorescence, and to analyze the functional relationships between them.

Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP.

In situ FRET measurement of cellular tension using conventional confocal laser microscopy in newly established reporter mice expressing actinin tension sensor.

FRET-based sensors are utilized for real-time measurements of cellular tension. However, transfection of the sensor gene shows low efficacy and is only effective for a short period. Reporter mice expressing such sensors have been developed, but sensor fluorescence has not been measured successfully using conventional confocal microscopy.

Mitochondrial ATP concentration decreases immediately after glucose administration to glucose-deprived hepatocytes.

Hepatocytes can switch their metabolic processes in response to nutrient availability. However, the dynamics of metabolites (such as lactate, pyruvate, and ATP) in hepatocytes during the metabolic switch remain unknown. In this study, we visualized metabolite dynamics in primary cultured hepatocytes during recovery from glucose-deprivation.

Efficient and rapid linker optimization with heterodimeric coiled coils improves the response of fluorescent biosensors comprising antibodies and protein M.

We developed a coiled Q-probe (CQ-probe), a fluorescent probe containing a coiled-coil peptide pair E4/K4, to convert antibodies into biosensors for homogeneous immunoassays. This probe consists of an antibody-binding protein, protein M (PM) with the E4 peptide and the K4 peptide with a fluorescent dye. Compared to PM Q-probes, which are generated by modifying the C-terminus of PM with a fluorescent dye, CQ-probe variants with various linkers are easy to prepare and therefore enable the establishment of biosensors with a significant fluorescence response by localizing the fluorescent dye at the optimal position for quenching and antigen-dependent release.

Development of green fluorescent protein-based cAMP indicators for covering a wide range of cAMP concentrations.

There is a wide range in the concentration of intracellular cyclic adenosine 3',5'-monophosphate (cAMP), which mediates specific effects as a second messenger in pathways affecting many physiological processes. Here, we developed green fluorescent cAMP indicators, named Green Falcan (Green fluorescent protein-based indicator visualizing cAMP dynamics) with various EC values (0.3, 1, 3, 10 μM) for covering the wide range of intracellular cAMP concentrations.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed.

Recent progress in homogeneous immunosensors based on fluorescence or bioluminescence using antibody engineering.

Homogeneous immunosensors integrate the advantages of both biosensors and immunoassays; they include speed, high sensitivity, and accuracy. They have been developed rapidly in the past few years and offer a cost-effective alternative technology with rapidity, sensitivity, and user-friendliness, which has been applied in a wide variety of applications. This review introduces the current directions of immunosensor development, focusing on fluorescent and bioluminescent immunosensors and highlighting the advantages, improvements, and key approaches to overcome the limitations of each.

Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.

Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells.

F-actin determines the time-dependent shift in docking dynamics of glucagon-like peptide-1 granules upon stimulation of secretion.

Although exocytosis can be categorized into several forms based on docking dynamics, temporal regulatory mechanisms of the exocytotic forms are unclear. We explored the dynamics of glucagon-like peptide-1 (GLP-1) exocytosis in murine GLUTag cells (GLP-1-secreting enteroendocrine L-cells) upon stimulation with deoxycholic acid (DCA) or high K to elucidate the mechanisms regulating the balance between the different types of exocytotic forms (pre-docked with the plasma membrane before stimulation; docked after stimulation and subsequently fused; or rapidly recruited and fused after stimulation, without stable docking). GLP-1 exocytosis showed a biphasic pattern, and we found that most exocytosis was from the pre-docked granules with the plasma membrane before stimulation, or granules rapidly fused to the plasma membrane without docking after stimulation.

The Patrol Yeast: A new biosensor armed with antibody-receptor chimera detecting a range of toxic substances associated with food poisoning.

Baker's yeast is an attractive host with established safety and stability characteristics. Many yeast-based biosensors have been developed, but transmembrane signal transduction has not been used to detect membrane-impermeable substances using antigen-antibody interactions. Therefore, we created Patrol Yeast, a novel yeast-based immunosensor of various targets, particularly toxic substances in food.

Intra Q-body: an antibody-based fluorogenic probe for intracellular proteins that allows live cell imaging and sorting.

Although intracellular biomarkers can be imaged with fluorescent dye(s)-labeled antibodies, the use of such probes for precise imaging of intracellular biomarkers in living cells remains challenging due to background noise from unbound probes. Herein, we describe the development of a conditionally active Fab-type Quenchbody (Q-body) probe derived from a monoclonal antibody (DO-1) with the ability to both target and spatiotemporally visualize intracellular p53 in living cells with low background signal. p53 is a key tumor suppressor and validated biomarker for cancer diagnostics and therapeutics.

Development of a red fluorescent protein-based cGMP indicator applicable for live-cell imaging.

Cyclic guanosine 3', 5'-monophosphate (cGMP) is a second messenger that regulates a variety of physiological processes. Here, we develop a red fluorescent protein-based cGMP indicator, "Red cGull". The fluorescence intensity of Red cGull increase more than sixfold in response to cGMP.

Creating a Thermostable β-Glucuronidase Switch for Homogeneous Immunoassay by Disruption of Conserved Salt Bridges at Diagonal Interfaces.

β-glucuronidase (GUS) has been used as a reporter enzyme in molecular biology and engineered as an enzyme switch for the development of homogeneous biosensors. In this study, we developed a thermostable GUS enzyme switch based on the thermostable GUS mutant TR3337 by disrupting a conserved salt bridge (H514-E523) between the diagonal subunits of its homotetramer. A combinatorial library (240 variants) was screened using a novel high-throughput strategy, which led to the identification of mutant DLW (H514D/M516L/Y517W) as a functional enzyme switch in a caffeine-recognizing immunosensor.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (). When is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell.

Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface.

Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required.

Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain.

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins. Mutations in the 14-3-3-binding motif in USP8 are related to Cushing's disease.

Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess.

With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O, etc.).