Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

Derivatives of a novel scaffold, C-phenyl 1-thio-D-glucitol, were prepared and evaluated for sodium-dependent glucose cotransporter (SGLT) 2 and SGLT1 inhibition activities. Optimization of substituents on the aromatic rings afforded five compounds with potent and selective SGLT2 inhibition activities. The compounds were evaluated for in vitro human metabolic stability, human serum protein binding (SPB), and Caco-2 permeability. Of them, (1S)-1,5-anhydro-1-[5-(4-ethoxybenzyl)-2-methoxy-4-methylphenyl]-1-thio-D-glucitol (3p) exhibited potent SGLT2 inhibition activity (IC(50) = 2.26 nM), with 1650-fold selectivity over SGLT1. Compound 3p showed good metabolic stability toward cryo-preserved human hepatic clearance, lower SPB, and moderate Caco-2 permeability. Since 3p should have acceptable human pharmacokinetics (PK) properties, it could be a clinical candidate for treating type 2 diabetes. We observed that compound 3p exhibits a blood glucose lowering effect, excellent urinary glucose excretion properties, and promising PK profiles in animals. Phase II clinical trials of 3p (TS-071) are currently ongoing.

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left-right asymmetric body plan in zebrafish, is generated from a cluster of ∼20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan.left-right patterning | ciliogenesis F ibroblast growth factor (FGF) signaling plays crucial roles in multiple morphogenetic processes of vertebrate development, including gastrulation movement, mesoderm formation, and leftright (LR) patterning (1-3). Because gain or loss of function of FGF signaling results in morphological changes in the embryo, some mechanism must ensure appropriate FGF signal levels in space and time for proper morphogenesis throughout development. FGF effectors acting as positive or negative regulators show a wide range of expression patterns and activities, contributing to the precise regulation of FGF signal activity (1, 4). Although most effectors identified to date act as negative regulators of FGF signaling, a few that positively regulate FGF activity have been reported (1, 4).We recently identified in zebrafish a positive regulator of FGF signaling named canopy1 (cnpy1), which is required for maintenance of the midbrain-hindbrain boundary (MHB) (5). Expression of cnpy1 was restricted to the MHB at late-somitogenesis stages, whereas cnpy1 was broadly distributed in earlier embryos (5) (SI Appendix, Fig. S1A), suggesting an additional role(s) for Cnpy1-mediated FGF signaling beyond the regulation of MHB formation. In this study, we characterize cnpy1 in detail during early zebrafish development and show that a Cnpy1-mediated positive feedback loop of FGF signaling promotes cell cluster formation between dorsal forerunner cells (DFCs) during gastrulation. We also demonstrate that the failure of DFCs to cluster when this FGF positive loop is disrupted eventually leads to Kupffer's vesicle (KV) malformation and randomization of LR asymmetric patterning. Results Positive Feedback Loop of FGF Signaling Mediated by Cnpy1 IsActivated Specifically in DFCs During Zebrafish Gastrulation. To reveal the role of Cnpy1-mediated FGF signaling in ...

The concept of drug-likeness, an important characteristic for any compound in a screening library, is nevertheless difficult to pin down. Based on our belief that this concept is implicit within the collective experience of working chemists, we devised a data set to capture an intuitive human understanding of both this characteristic and ease of synthesis, a second key characteristic. Five chemists assigned a pair of scores to each of 3980 diverse compounds, with the component scores of each pair corresponding to drug-likeness and ease of synthesis, respectively. Using this data set, we devised binary classifiers with an artificial neural network and a support vector machine. These models were found to efficiently eliminate compounds that are not drug-like and/or hard-to-synthesize derivatives, demonstrating the suitability of these models for use as compound acquisition filters.

In a previous paper, we reported the N-hydroxyformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor. Despite its attraction as a potential therapeutic agent for cerebral diseases, the preparation of an injectable formulation of HET0016 was limited by its poor solubility under neutral conditions and instability under acidic conditions. The instability of HET0016 in acidic conditions is due to the N-hydroxyformamidine moiety, which is considered to be essential for the potent and selective activity seen in our previous study. The activity was maintained when the N-hydroxyformamidine moiety was replaced by an imidazole ring (3a; IC(50) = 5.7 +/- 1.0 nM), but this was associated with a loss of selectivity for cytochrome p450s (CYPs). However, other azole derivatives such as isoxazole derivative 23 (IC(50) value 38 +/- 10 nM) and pyrazole derivative 24 (IC(50) value 23 +/- 12 nM) showed potent and selective activities with improved stability.

Intuition. -(TAKAOKA*, Y.; ENDO, Y.; YAMANOBE, S.; KAKINUMA, H.; OKUBO, T.; SHIMAZAKI, Y.; OTA, T.; SUMIYA, S.; YOSHIKAWA, K.; J. Chem.

Enzymes have evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. To evolve abzymes toward higher catalytic activity, we have reconstructed an enzyme-evolutionary process in vitro. Thus, a phage-displayed combinatorial library from a hydrolytic abzyme, 6D9, generated by the conventional in vivo method with immunization of the transition-state analog (TSA), was screened against a newly devised TSA to optimize the differential affinity for the transition state relative to the ground state. The library format successfully afforded evolved variants with 6- to 20-fold increases in activity (kcat) as compared with 6D9. Structural analysis revealed an advantage of the in vitro evolution over the in vivo evolution: an induced catalytic residue in the evolved abzyme arises from double mutations in one codon, which rarely occur in somatic hypermutation in the immune response.

Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.