Characterization, Preparation, and Promotion of Plant Growth of 1,3-Diphenylurea/ß-Cyclodextrin Derivatives Inclusion Complexes.

The study aimed to prepare inclusion complexes of 1,3-diphenylurea (DPU) with ß-cyclodextrin (ßCD) and 2-hydroxypropyl-ß-cyclodextrin (HP-ßCD) using a three-dimensional ground mixture (3DGM). Their physicochemical properties, intermolecular interactions, solubilities, and plant growth-promoting activities were investigated on broccoli sprouts. Phase-solubility diagrams indicated the stability constant (Ks) and complexation efficiency (CE) of ßCD/DPU were found to be K1/1 = 250 M-1, CE = 2.48× 10-3. The Ks and CEs of HP-ßCD/DPU were found to be K1/1 = 427 M-1, CE = 3.93 × 10-3 and K2/1 = 196 M-1, CE = 1.93 × 10-3 respectively. The powder X-ray diffraction results of 3DGM (ßCD/DPU = 2/1, HP-ßCD/DPU = 2/1) showed that the diffraction peaks originating from the DPU and ßCD disappeared, indicating a halo pattern. Differential scanning calorimetry results showed an endothermic peak at 244 °C derived from the melting point of DPU, but the endothermic peak disappeared in the 3DGM (ßCD/DPU = 2/1, HP-ßCD/DPU = 2/1). Near-infrared absorption spectra showed peak shifts in 3DGM (ßCD/DPU and HP-ßCD/DPU) at the -CH and -NH groups of DPU and the -OH groups of ßCDs and free water. In the dissolution test (after 5 min), the concentration of intact DPU was 0.083 µg/mL. However, the dissolution concentrations of DPU in the 3DGM (ßCD/DPU = 1/1), 3DGM (ßCD/DPU = 2/1), 3DGM (HP-ßCD/DPU = 1/1), and 3DGM (HP-ßCD/DPU = 2/1) were 3.27, 3.64, 5.70, and 7.03 µg/mL, respectively, indicating higher solubility than that of the intact DPU. Further, 1H-1H NOESY NMR spectroscopic measurements showed cross-peaks between H-A (7.32 ppm) and H-B (7.12 ppm) of DPU and H-6 (3.79 ppm) in the ßCD cavity of the 3DGM (ßCD/DPU = 2/1). A cross-peak was also observed among DPU H-A (7.32 ppm), H-B (7.11 ppm), and H-6 (3.78 ppm) in the ßCD cavity. The results of the broccoli sprout cultivation experiment showed that 3DGM (ßCD/DPU = 1/1), 3DGM (ßCD/DPU = 2/1), 3DGM (HP-ßCD/DPU = 1/1), and 3DGM (HP-ßCD/DPU = 2/1) increased the stem thickness compared with that of the control group (DPU). These results indicated that the ßCD/DPU and HP-ßCD/DPU inclusion complexes were formed by the three-dimensional mixing and milling method, which enhanced the solubility and plant growth-promoting effects.

Stability of enzyme immobilized on the nanofluidic channel surface.

The lifetime of an enzyme is critical to prevent system failure and optimize maintenance schedules in biological and analytical chemistry. The lifetime metrics of an enzyme can be evaluated from enzyme activity in terms of catalytic cycles per enzyme at various storage times. Trypsin, which is a gold-standard enzyme in proteomics, has been known to decrease activity due to self-digestion. To improve the activity of trypsin, enzyme reactors have developed by immobilizing in micro and nanospace. However, an evaluation method for the catalytic cycle has not been established due to major issues such as nonuniform space, unstable liquid transport, and self-digestion during immobilization in conventional work. To solve these issues, we have previously developed an ultra-fast enzyme reactor with a well-defined nanofabrication method, stable liquid transport, and partial enzyme modification. Here, we aimed to investigate catalytic cycles in a nanochannel. To extend enzyme lifetime efficiently, we have evaluated the optimal immobilization process and catalytic cycles of trypsin. As a result, immobilized enzyme densities by the trypsinogen immobilization process were increased at all concentrations compared to the trypsin immobilization process. To evaluate the lifetime of trypsin, the immobilized enzyme densities and activities were almost the same before and after 72 h of enzyme storage, and the calculated catalytic cycles were 1740. These results indicated that self-digestion of the immobilized enzyme was highly suppressed. Consequently, the reaction efficiency has been evaluated depending on the catalytic cycles from the substrate for the first time, while preventing self-digestion by trypsin.

Positive memory increases cataplexy-like behaviors in narcolepsy mice as revealed using conditioned place preference test.

BACKGROUND: Cataplexy is a loss of muscle tone that can lead to postural collapse, disturbing the daily life of narcolepsy patients; it is often triggered by positive emotions such as laughter in human patients. Narcolepsy model mice also show cataplexy, and its incidence increases in response to positive emotion-inducing stimuli such as chocolate and female courtship. Although such observation indicates a positive emotion-related nature of cataplexy in narcolepsy mice, they also show cataplexy without any apparent triggering stimulus ~ (spontaneous cataplexy). Therefore, we hypothesized that some spontaneous cataplexy in narcoleptic mice might indicate the remembering of happy moments. RESULTS: To test our hypothesis, we did a conditioned place preference test on orexin/hypocretin neuron-ablated (ORX-AB) mice, one of the animal models of human narcolepsy, and counted the number of cataplexy-like behaviors. ORX-AB mice successfully remembered the chocolate-associated chamber, and the number of cataplexy-like behaviors significantly increased in the chocolate-associated chamber but not in the control chamber. In addition, ORX-AB mice remembered the aversive odor-associated chamber and avoided entering without affecting the number of cataplexy-like behaviors. Finally, similar activation of the nucleus accumbens, a positive emotion-related nucleus, was observed during both spontaneous and chocolate-induced cataplexy behaviors. CONCLUSIONS: These results support our hypothesis and will promote the usefulness of a narcolepsy mice model in emotion research and serve as a basis for a better understanding of cataplexy in narcolepsy patients.

Kinetics of Enzymatic Reactions at the Solid/Liquid Interface in Nanofluidic Channels.

Nanostructures can realize highly efficient reactions due to their structural advantages. However, the mechanism of accelerating enzyme reactions in a nanospace is still unknown from a kinetic perspective because it is difficult to control a well-defined nanospace, enzyme density, and reaction time. Here, we investigated kinetic parameters of an immobilized enzyme in micro- and nanochannels using nanofabrication, partial enzyme patterning, fluidic control, and a high sensitivity detection system. Devices with channel depths of 300 nm, 4.4 µm, and 13.6 µm were fabricated. Kinetic parameters were determined by the Michaelis-Menten model. Compared to the bulk reaction, all kcats for immobilized enzyme reactors were decreased, although the kcats were approximately the same for the immobilized enzyme reactors of different depths. An ultrafast enzyme reaction could overcome the drawback due to immobilization by an increase of the apparent [E]0 due to the decreased channel depth.

Analysis of Environmental and Pathogenic Bacteria Attached to Aerosol Particles Size-Separated with a Metal Mesh Device.

Metal mesh devices (MMDs) are novel materials that enable the precise separation of particles by size. Structurally, MMDs consist of a periodic arrangement of square apertures of characteristic shapes and sizes on a thin nickel membrane. The present study describes the separation of aerosol particles using palm-top-size collection devices equipped with three types of MMDs differing in pore size. Aerosols were collected at a farm located in the suburbs of Nairobi, Kenya; aerosol particles were isolated, and pathogenic bacteria were identified in this microflora by next-generation sequencing analysis. The composition of the microflora in aerosol particles was found to depend on particle size. Gene fragments were obtained from the collected aerosols by PCR using primers specific for the genus Mycobacterium. This analysis showed that Mycobacterium obuense, a non-tuberculous species of mycobacteria that causes lung diseases, was present in these aerosols. These findings showed that application of this MMD analytical protocol to aerosol particles can facilitate the investigation of airborne pathogenic bacteria.

Accelerated protein digestion and separation with picoliter volume utilizing nanofluidics.

Single cell analyses can provide critical biological insight into cellular heterogeneity. In particular, the proteome, which governs cell functions, is much more difficult to analyze because it is principally impossible to amplify proteins compared to nucleic acids. The most promising approach to single cell proteomics is based on the liquid chromatography mass spectrometry (LC-MS) platform. However, pretreatments before MS detection have two critical issues for single cell analysis: analyte loss as a result of adsorption and artifacts due to the duration of analysis. This is a serious problem because single cells have a limited number of protein molecules and a small volume. To solve these issues, we developed an integrated nanofluidic device to manipulate samples on a femtoliter to picoliter (fL-pL) scale to achieve high-throughput analysis via suppressing analyte loss. This device can perform tryptic digestion, chromatographic separation, and non-labeled detection with high consistency. In addition, we introduced an open/close valve by physical deformation of glass on a nanometer scale to independently modify the nanochannel surfaces and control sample aliquots. The injection system equipped with this valve achieved an injection volume of 1.0 ± 0.1 pL. By using this integrated device, we found that the chromatogram of bulk-digestion for 12 hours resembled that of 15 min-digestion in the nanochannel, which indicated that these conditions reached a similar state of digestion. Therefore, an integrated device for ultra-fast protein analysis was developed on a 1 pL scale for the first time.

Picoliter enzyme reactor on a nanofluidic device exceeding the bulk reaction rate.

Single-cell analyses have recently become important to understand cell heterogeneity, the mechanism of cell function, and diseases. In contrast to single-cell analyses that target nucleic acids, single-cell protein analyses still pose challenges. We have proposed a general concept of integration and extended this concept to the 10-1000 nm scale with femtoliter-picoliter volumes which are smaller than the volume of a single cell exploring ultimate analytical performances (e.g. single-cell target proteomics). However, single-cell shotgun proteomics, which is used to analyze even unknown proteins, is still challenging because there is no digestion column with picoliter volume. The issues were long reaction time (overnight) and much larger reaction volume (microliter) in the conventional bulk method. In this study, an ultra-fast picoliter enzyme reactor using a nanochannel was developed. A device with a channel depth of 300 nm and a volume of 32.4 pL was fabricated. To prevent the self-digestion of trypsin (enzyme), the picoliter enzyme reactor was prepared by immobilizing trypsinogen which was activated to trypsin by enterokinase. The enzyme density obtained by the trypsinogen immobilization process was 2.5 times higher than that obtained by the conventional trypsin immobilization process. Furthermore, the apparent enzyme concentration was 36 times higher due to an extremely high surface-to-volume ratio of the nanochannel, compared to the limit concentration in the bulk. Finally, the enzyme reaction in the picoliter enzyme reactor was accelerated 25 times compared to that in the bulk. Using the picoliter enzyme reactor, protein solution with picoliter volume will be digested without self-digestion and artificial modification, which will greatly contribute to single-cell shotgun proteomics.

Peripheral administration of SB223412, a selective neurokinin-3 receptor antagonist, suppresses pulsatile luteinizing hormone secretion by acting on the gonadotropin-releasing hormone pulse generator in estrogen-treated ovariectomized female goats.

Accumulating evidence suggests that KNDy neurons located in the hypothalamic arcuate nucleus (ARC), which are reported to express kisspeptin, neurokinin B, and dynorphin A, are indispensable for the gonadotropin-releasing hormone (GnRH) pulse generation that results in rhythmic GnRH secretion. The aims of the present study were to investigate the effects of peripheral administration of the neurokinin 3 receptor (NK3R/TACR3, a receptor for neurokinin B) antagonist, SB223412, on GnRH pulse-generating activity and pulsatile luteinizing hormone (LH) secretion in ovariectomized Shiba goats treated with luteal phase levels of estrogen. The NK3R antagonist was infused intravenously for 4 h {0.16 or 1.6 mg/(kg body weight [BW]·4 h)} during which multiple unit activity (MUA) in the ARC was recorded, an electrophysiological technique commonly employed to monitor GnRH pulse generator activity. In a separate experiment, the NK3R antagonist (40 or 200 mg/[kg BW·day]) was administered orally for 7 days to determine whether the NK3R antagonist could modulate pulsatile LH secretion when administered via the oral route. Intravenous infusion of the NK3R antagonist significantly increased the interval of episodic bursts of MUA compared with that of the controls. Oral administration of the antagonist for 7 days also significantly prolonged the interpulse interval of LH pulses. The results of this study demonstrate that peripheral administration of an NK3R antagonist suppresses pulsatile LH secretion by acting on the GnRH pulse generator, suggesting that NK3R antagonist administration could be used to modulate reproductive functions in ruminants.

Vapor-phase Synthesis of Bimetallic Plasmonic Nanoparticles.

Regulating the nanostructure of composite nanoparticles is essential for making them suitable for various applications. In general, the chemical reduction method is employed for preparing metal nanoparticles in the liquid phase. However, complicated techniques are required to control the nanostructure during particle synthesis. The evaporation/condensation method is used for synthesizing nanoparticles in the vapor-phase. Although this method produces impurity-free particles without aggregation, very few studies have been carried out on the synthesis of composite particles in the vapor-phase. In this study, we synthesized composite nanoparticles in the vapor-phase by using the evaporation/condensation method. The results showed that bimetallic nanoparticles are produced by this method. Moreover, it was indicated that the nanostructure of the synthesized nanoparticles is influenced by the order of the electric furnace with different temperatures.

Scaffold hopping of fused piperidine-type NK3 receptor antagonists to reduce environmental impact.

Neurokinin-3 receptor (NK3R) plays a pivotal role in the release of gonadotropin-releasing hormone in the hypothalamus-pituitary-gonadal (HPG) axis. To develop novel NK3R antagonists with less environmental toxicity, a series of heterocyclic scaffolds for the triazolopiperazine substructure in an NK3R antagonist fezolinetant were designed and synthesized. An isoxazolo[3,4-c]piperidine derivative exhibited moderate NK3R antagonistic activity and favorable properties that were decomposable under environmental conditions.

Synthesis of Triazolo- and Oxadiazolopiperazines by Gold(I)-Catalyzed Domino Cyclization: Application to the Design of a Mitogen Activated Protein (MAP) Kinase Inhibitor.

An efficient method for the synthesis of [1,2,4]triazolo[4,3- a]piperazine derivatives was established based on a gold(I)-catalyzed domino cyclization of an amidrazone substrate with a terminal alkyne. The amidoxime congeners were converted into [1,2,4]oxadiazolo[4,5- a]piperazine derivatives in the presence of a gold catalyst. The oxadiazolopiperazine is a promising scaffold for the design of novel inhibitors against p38 mitogen activated protein kinase (MAP kinase).

Elimination of a signal sequence-uncleaved form of defective HLA protein through BAG6.

A portion of newly synthesized transmembrane domain proteins tend to fail to assemble correctly in the lumen of the endoplasmic reticulum, thus resulting in the production of a signal sequence-uncleaved form of the defective species. Although the efficient degradation of these mistargeted polypeptides is crucial, the molecular mechanism of their elimination pathway has not been adequately characterized. In this study, we focused on one such cryptic portion of a defective transmembrane domain protein, HLA-A, and show that a part of HLA-A is produced as a signal sequence-uncleaved labile species that is immediately targeted to the degradation pathway. We found that both BAG6 and proteasomes are indispensable for elimination of mislocalized HLA-A species. Furthermore, defective HLA-A is subjected to BAG6-dependent solubilization in the cytoplasm. These observations suggest that BAG6 acts as a critical factor for proteasome-mediated degradation of mislocalized HLA-A with a non-cleaved signal sequence at its N-terminus.

SB223412, a neurokinin-3 receptor-selective antagonist, suppresses testosterone secretion in male guinea pigs.

Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species.

Development of novel NK3 receptor antagonists with reduced environmental impact.

The neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling positively regulates the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective antagonists may suppress the reproductive functions of mammals. For development of novel NK3R antagonists with reduced environmental toxicity, a structure-activity relationship study of an NK3R antagonist, talnetant, was carried out. Among several talnetant derivatives with labile functional groups in the natural environment, 3-mercaptoquinoline 2f exhibited a comparable biological activity to that of the parent talnetant. Additionally, compound 2f was converted into the disulfide 3f or isothiazolone 8 by air-oxidation, both of which showed no binding affinity to NK3R.

Development of novel neurokinin 3 receptor (NK3R) selective agonists with resistance to proteolytic degradation.

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.