Growth prolongation of human induced pluripotent stem cell aggregate in three-dimensional suspension culture system by addition of botulinum hemagglutinin.

Human induced pluripotent stem cells (hiPSCs) can be used in regenerative therapy as an irresistible cell source, and so the development of scalable production of hiPSCs for three-dimensional (3D) suspension culture is required. In this study, we established a simple culture strategy for improving hiPSC aggregate growth using botulinum hemagglutinin (HA), which disrupts cell-cell adhesion mediated by E-cadherin. When HA was added to the suspension culture of hiPSC aggregates, E-cadherin-mediated cell-cell adhesion was temporarily disrupted within 24 h, but then recovered. Phosphorylated myosin light chain, a contractile force marker, was also recovered at the periphery of hiPSC aggregates. The cell aggregates were suppressed the formation of collagen type I shell-like structures at the periphery by HA and collagen type I was homogenously distributed within the cell aggregates. In addition, these cell aggregates retained the proliferation marker Ki-67 throughout the cell aggregates. The apparent specific growth rate with HA addition was maintained continuously throughout the culture, and the final cell density was 1.7-fold higher than that in the control culture. These cells retained high expression levels of pluripotency markers. These observations indicated that relaxation of cell-cell adhesions by HA addition induced rearrangement of the mechanical tensions generated by actomyosin in hiPSC aggregates and suppression of collagen type I shell-like structure formation. These results suggest that this simple and readily culture strategy is a potentially useful tool for improving the scalable production of hiPSCs for 3D suspension cultures.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer.

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

Tetrodotoxins in the flatworm Planocera multitentaculata.

The marine polyclad flatworm Planocera multitentaculata is known to possess high levels of tetrodotoxin (TTX), but the presence of TTX analogues in the species has been unexplored. In this study, TTX and several analogues such as 5,6,11-trideoxyTTX, monodeoxyTTXs, dideoxyTTXs, and 11-norTTX-6(S)-ol were identified in three adults and egg plates of P. multitentaculata using high resolution liquid chromatography-mass spectrometry (HR-LC/MS) for the first time.

Improvement in Cycle Life of Organic Lithium-Ion Batteries by In-Cell Polymerization of Tetrathiafulvalene-Based Electrode Materials.

Redox-active organic molecules are promising candidates for next-generation electrode materials. Nevertheless, finding low-molecular-weight organic materials with a long cycle life remains a crucial challenge. Herein, we demonstrate the application of tetrathiafulvalene and its vinyl analogue bearing triphenylamines as long-cycle-life electrodes for lithium-ion batteries (LIBs). These molecules were successfully synthesized using palladium-catalyzed C-H arylation. Electrochemical analysis revealed that a polymer formed on the electrode. LIBs comprising these molecules exhibited noteworthy charge-discharge properties with a long cycle life (the capacity after 100 cycles was greater than 90% of the discharge capacity in the third cycle) and a high utilization ratio (approximately 100%). "In-cell" polymerization during the first charge process is considered to contribute to the effect. This study indicates new avenues for the creation of organic materials for rechargeable batteries.

First Detection of Tetrodotoxins in the Cotylean Flatworm Prosthiostomum trilineatum.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.

Ligand-Dependent Site-Selective Suzuki Cross-Coupling of 4-Bromopyrazol-5-yl Triflates.

Ligand-dependent Suzuki cross-coupling of 4-bromopyrazol-5-yl triflates has been developed. This approach enabled selective introduction of an aryl substituent at the C4 or C5 position in pyrazoles. This protocol is the first example in which the cross-coupling proceeded predominantly at the C4 position in pyrazoles, which is generally recognized as the least reactive position. The selection of phosphine ligands switched the order of arylation. This method should be highly useful for preparing diverse poly-substituted pyrazole derivatives.

Discovery of Novel Pyrazole-Based Selective Aldosterone Synthase (CYP11B2) Inhibitors: A New Template to Coordinate the Heme-Iron Motif of CYP11B2.

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11ß-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Nav 1.1 to Nav 1.7).

BACKGROUND AND PURPOSE: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7. EXPERIMENTAL APPROACH: Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. KEY RESULTS: The inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. CONCLUSIONS AND IMPLICATIONS: The reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.

Total synthesis of chiriquitoxin, an analogue of tetrodotoxin isolated from the skin of a dart frog.

The first total synthesis of chiriquitoxin, the most structurally complex analogue of tetrodotoxin isolated from a Costa Rican dart frog, has been accomplished from a newly designed intermediate for a variety of tetrodotoxin derivatives. The synthesis includes the third total synthesis of tetrodotoxin in this laboratory, and its intermediate was transformed into chiriquitoxin by a stereocontrolled aldol reaction with a D-camphor-derived lactone for installation of the unique side chain, and a new deprotection of methylthiomethyl (MTM) ether by using a Pummerer rearrangement.