Transcriptional heterogeneity of catabolic genes on the plasmid pCAR1 causes host-specific carbazole degradation.

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

Effect of dopamine receptor-related compounds on naïve common marmosets for auditory steady-state response.

Abnormalities of auditory steady-state responses (ASSRs) and the effects of antipsychotic drugs on ASSRs have been investigated in patients with schizophrenia. It is presumed that drugs do not directly affect ASSRs because its abnormalities are associated with schizophrenia. Therefore, to investigate the direct effect of drugs on ASSRs, we established an ASSR evaluation system for common marmosets in a naïve state. Dopamine D1 receptor stimulation (SKF-81297, 2 mg/kg ip) significantly increased evoked power (EP) at 40 Hz. The phase locking factor (PLF) was increased significantly at 20, 30, 40, and 80 Hz. However, administration of a dopamine D1 receptor antagonist (SCH-39166, 0.3 mg/kg ip) resulted in a significant decrease in EP and PLF at 30 Hz. Dopamine D2 receptor stimulation (quinpirole, 1 mg/kg im) tended to increase EP and induced power (IP) at all frequencies, and a significant difference was observed at 30 Hz IP. There was no change in PLF at all frequencies. In addition, dopamine D2 receptor blockade (raclopride, 3 mg/kg ip) reduced EP and PLF at 30 Hz. Subcutaneous administration of the serotonin dopamine antagonist, risperidone (0.3 mg/kg), tended to increase IP and decrease PLF, but not significantly. Taken together, it is possible to compare the differences in the mode of action of drugs on ASSRs using naïve nonhuman primates.NEW & NOTEWORTHY We measured the effects of dopamine receptor-related compounds on ASSR in marmosets. D1 receptor stimulation increased the phase locking factor (PLF) and evoked power (EP), and reduced the induced power (IP). D2 receptor stimulation increased the IP. D1 and D2 receptor blockers reduced the PLF and EP at 30 Hz. Different modes of action of various drugs related to psychiatric disorders were evaluated by administering antipsychotic drugs to naïve marmosets.

Risperidone on apomorphine-induced stereotyped behavior and auditory sensory gating in rhesus monkeys.

The ameliorating effect of risperidone on apomorphine-induced stereotyped behavior and inhibition of auditory sensory gating was investigated using rhesus monkeys. The total duration of the stereotyped behavior observed in the control group was 43.7 ± 23.0 s (n = 3) between 10 and 25 min after vehicle administration, whereas the duration in the apomorphine-treated (0.1 or 0.15 mg/kg i.m., n = 3) group was observed to be significantly prolonged to 413.0 ± 150.6 s. Administration of 0.01, 0.03, 0.1 mg/kg of risperidone 60 min before apomorphine, significantly reduced the duration of this apomorphine-induced stereotyped behavior to 327 ± 104.9 s (n = 3), 8.3 ± 4.2 s (n = 3), and 0.0 ± × 0.0 s (n = 3, t-test: p < 0.05), respectively. Next, the auditory sensory gating test/conditioning (T/C) ratio was used as a bio-marker. The T/C ratio was 0.598 ± 0.0802 in the vehicle-administered control group (n = 4) and was significantly increased to 2.098 ± 0.254 (n = 4) by apomorphine (0.15 mg/kg, i.m.). Administering of risperidone (0.1 mg/kg, s.c.) 30 min before apomorphine treatment significantly restricted the T/C ratio to 0.571 ± 0.0886 (n = 4), compared to the T/C ratio in the vehicle-administered control group. The above results demonstrate, not only behaviorally but also electrophysiologically, the ameliorating effect of risperidone on the induction of schizophrenia-like symptoms by apomorphine in non-human primates.

Towards HCP-Style macaque connectomes: 24-Channel 3T multi-array coil, MRI sequences and preprocessing.

Macaque monkeys are an important animal model where invasive investigations can lead to a better understanding of the cortical organization of primates including humans. However, the tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the smaller macaque brain, high spatial, temporal, and angular resolutions combined with high signal-to-noise ratio are required to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enables adaptation of the Human Connectome Project (HCP) image acquisition protocols to the in-vivo macaque brain. In addition, we adapted HCP preprocessing methods to the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provides the necessary high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold accelerations for dMRI and fMRI. Automated FreeSurfer segmentation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI all performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. Analyses of functional connectivity in fMRI revealed high sensitivity as compared with those from publicly shared datasets. Tractography-based connectivity estimates correlated with tracer connectivity similarly to that achieved using ex-vivo dMRI. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available in studies of the human brain.

Preventing Silica Scale Formation Using Hydroxide Ions Generated by Water Electrolysis.

The reaction of silica with various cations in a solution and with hydroxide ions generated by water electrolysis was investigated as a means of preventing the formation of silica scales in geothermal binary power generation. Through batch and continuous experiments, it was found that all silica in the cathode phase of a reaction device could be removed if the necessary amounts of magnesium and calcium were present. This occurs because a silica-magnesium-calcium compound is produced via a polymerization reaction with cations in a solution and with hydroxide ions generated by electrolysis. Analysis by inductively coupled plasma and energy dispersive X-ray spectroscopy shows that this material has the formula 2CaO-5MgO-8SiO2-H2O, and thus is likely generated by the reaction proposed by Sheikholeslami et al. (2019). Increasing the current sent through the reaction solution subsequently produces calcium carbonate. This technique for the separation of silica and calcium from aqueous solutions can be operated continuously without channel clogging, which indicates the possibility of practical applications. However, overly high currents promote the migration of protons from the anode to cathode phases, which inhibits the formation of precipitates due to a neutralization reaction. The proposed method is an effective approach for removing silica from a solution in geothermal binary power generation; although, a means of suppressing the effects of proton generation will be necessary if the process is also to be used to remove calcium ions.

Controlled Deposition Number of Organic Molecules Using Quartz Crystal Microbalance Evaluated by Scanning Tunneling Microscopy Single-Molecule-Counting.

Precise control of organic molecule deposition on a substrate is quite important for fabricating single-molecule-based devices. In this study, we demonstrate whether a quartz-crystal microbalance (QCM) widely used for a film growth calibration has the ability to precisely measure the number of organic molecules adsorbed on a substrate. The well-known Sauerbrey's equation is extended to formulate the relation between QCM resonant frequency shift and the number of adsorbed molecules onto the QCM surface. The formula is examined by QCM measurements of sublimation of π-conjugated organic molecules and direct counting of the deposited molecules one by one onto metal substrates, using ultrahigh vacuum low-temperature scanning tunneling microscopy (STM). It is revealed that the number of adsorbed molecules evaluated by QCM ( NQCM) show good agreement with those counted from the STM images ( NSTM) within the error of ±25%. The results ensure the QCM capability for controlling the deposition number of organic molecules with high accuracy, that is, if one needs to deposit 100 molecules on the substrate, QCM control promises deposition of 100 ± 25 molecules.

Quantification of receptor activation by oxytocin and vasopressin in endocytosis-coupled bioluminescence reduction assay using nanoKAZ.

Oxytocin (OXT) and arginine vasopressin (AVP) are structurally similar neuropeptide hormones that function as neurotransmitters in the brain, and have opposite key roles in social behaviors. These peptides bind to their G protein-coupled receptors (OXTR and AVPRs), inducing calcium ion-dependent signaling pathways and endocytosis of these receptors. Because selective agonists and antagonists for these receptors have been developed as therapeutic and diagnostic agents for diseases such as psychiatric disorders, facile methods are in demand for the evaluation of selectivity between these receptors. In this study, we developed a quantitative assay for OXT- and AVP-induced endocytosis of their receptors. The mutated Oplophorus luciferase, nanoKAZ, was fused to OXTR and AVPRs to enable rapid quantification of agonist-induced endocytosis by bioluminescence reduction. Agonist stimulation significantly decreases bioluminescence of nanoKAZ-fused receptors in living cells. Using this system, we evaluated clinically used OXTR antagonist atosiban and a reported pyrazinyltriazole derivative, hereby designated as PF13. Atosiban acted as an antagonist of AVPR1a, as well as an agonist for AVPR1b, whereas PF13 antagonized OXTR more selectively than atosiban, as reported previously. This paper shows a strategy for quantification of agonist-induced endocytosis of OXTR and AVPRs, and confirms its potent utility in the evaluation of agonists and antagonists.

Visualization of drug translocation in the nasal cavity and pharmacokinetic analysis on nasal drug absorption using positron emission tomography in the rat.

We performed positron emission tomography (PET) using 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) to evaluate the pharmacokinetics of nasal drug absorption in the rat. The dosing solution of [(18)F]FDG was varied in volume (ranging from 5 to 25 µl) and viscosity (using 0% to 3% concentrations of hydroxypropylcellulose). We modeled the pharmacokinetic parameters regarding the nasal cavity and pharynx using mass balance equations, and evaluated the values that were obtained by fitting concentration-time profiles using WinNonlin® software. The regional nasal permeability was also estimated using the active surface area derived from the PET images. The translocation of [(18)F]FDG from the nasal cavity was visualized using PET. Analysis of the PET imaging data revealed that the pharmacokinetic parameters were independent of the dosing solution volume; however, the viscosity increased the absorption rate constant and decreased the mucociliary clearance rate constant. Nasal permeability was initially higher but subsequently decreased until the end of the study, indicating regional differences in permeability in the nasal cavity. We concluded that the visualization of drug translocation in the nasal cavity in the rat using PET enables quantitative analysis of nasal drug absorption, thereby facilitating the development of nasal formulations for human use.

Effects of intracanal carbon dioxide laser irradiation on cultured human fibroblasts.

OBJECTIVE: The purpose of this study was to evaluate, under various conditions, the damage that might occur to viable cells after CO2 laser irradiation of the root canal. STUDY DESIGN: A laser tip was placed within the root canal of extracted human teeth that were positioned above cultured fibroblasts (NB cells), and then irradiation was applied. The irradiation modes used were either pulse (40 pps) or super-pulse (151 pps). The laser energy was set at 32 J in both modes, and the tip of the laser beam was positioned to a point 2 mm from the fibroblasts. RESULTS: When the pulse mode irradiation was applied, there was significant difference (P < .05) in the percentage of viable cells between teeth with closed apical foramen (1-mm wall between root canal and root apex) and the #15 group (No. 15 K-file used), #30 group (No. 30 K-file), or #50 group (No. 50 K-file). The difference between the #30 group and the #50 group (P < .05) was also significant. The larger the diameter of the apical foramen became, the lower was the percentage of viable cells. CONCLUSION: If a CO2 laser irradiates a root canal system enlarged to within 1 mm of the root canal length, little damage to the periapical tissues would be expected to occur.