Evidence for a liquid silicate layer atop the Martian core.

Seismic recordings made during the InSight mission1 suggested that Mars's liquid core would need to be approximately 27% lighter than pure liquid iron2,3, implying a considerable complement of light elements. Core compositions based on seismic and bulk geophysical constraints, however, require larger quantities of the volatile elements hydrogen, carbon and sulfur than those that were cosmochemically available in the likely building blocks of Mars4. Here we show that multiply diffracted P waves along a stratified core-mantle boundary region of Mars in combination with first-principles computations of the thermoelastic properties of liquid iron-rich alloys3 require the presence of a fully molten silicate layer overlying a smaller, denser liquid core. Inverting differential body wave travel time data with particular sensitivity to the core-mantle boundary region suggests a decreased core radius of 1,675 ± 30 km associated with an increased density of 6.65 ± 0.1 g cm-3, relative to previous models2,4-8, while the thickness and density of the molten silicate layer are 150 ± 15 km and 4.05 ± 0.05 g cm-3, respectively. The core properties inferred here reconcile bulk geophysical and cosmochemical requirements, consistent with a core containing 85-91 wt% iron-nickel and 9-15 wt% light elements, chiefly sulfur, carbon, oxygen and hydrogen. The chemical characteristics of a molten silicate layer above the core may be revealed by products of Martian magmatism.

Contribution of the human cytochrome P450 2C subfamily to the metabolism of and the interactions with endogenous compounds including steroid hormones.

The objectives of this study were as follows: 1) to compare the metabolic activities of endogenous compounds and their effects on dopamine formation and hydroxylation of steroid hormones, mediated by human cytochrome P450 (CYP), including CYP2C9.1 and CYP2C19, as well as the variants CYP2C9.2 (Arg144Cys) and CYP2C9.3 (Ile359Leu); and 2) to assess the effects of steroid hormones on the activities of CYP2C9.1, CYP2C9.2, and CYP2C19 to estimate the contribution of the CYP2C subfamily to metabolism and drug-drug interactions of endogenous compounds. Dopamine formation from p -tyramine and 6ß- and 21- (for progesterone) hydroxylation of testosterone, cortisol, and progesterone by CYP2C9 variants, CYP2C19, CYP2D6, and CYP3A4 were determined using HPLC. The effects of steroid hormones such as testosterone, cortisol, and progesterone on tolbutamide methyl hydroxylation mediated by CYP2C subfamily members were investigated. Only CYP2D6 catalyzed dopamine formation. The 6ß-hydroxylation activities of testosterone, cortisol, and progesterone catalyzed by CYP2C9 variants and CYP2D6 were less than 5% of those by CYP3A4. Although cortisol did not inhibit tolbutamide methyl hydroxylation catalyzed by CYP2C9.1, CYP2C9.2, or CYP2C19 and testosterone did not inhibit CYP2C19 activity, the reactions catalyzed by CY2C9.1 and CYP2C9.2 were inhibited by testosterone. The inhibition of progesterone by CYP2C19 was stronger than that by CYP2C9.1 and CYP2C9.2. CYP2C9.1 and CYP2C19 noncompetitively and competitively inhibited tolbutamide methyl hydroxylation with inhibition constants of 43.2 µM and 1.03 µM, respectively. Clinical interactions among endogenous compounds would vary within the CYP2C subfamily, although the contribution of the CYP2C subfamily may be of minor importance for dopamine formation and the detoxification (6ß-hydroxylation) of endogenous steroid hormones.

Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors.

Firefly luciferases usually emit green-yellow bioluminescence at physiological pH values. However, under acidic conditions, in the presence of heavy metals and, at high temperatures they emit red bioluminescence. To understand the structural origin of bioluminescence colors and pH-sensitivity, about 20 firefly luciferases have been cloned, sequenced and investigated. The proton and metal-binding site responsible for pH- and metal sensitivity in firefly luciferases was shown to involve the residues H310, E311 and E354 in firefly luciferases. However, it is still unclear how and why pH-sensitivity arose and evolved in firefly luciferases. Here, we cloned and characterized two novel luciferase cDNAs from the fat body and lanterns of the Brazilian firefly Aspisoma lineatum. The larval fat body isozyme (AL2) has 545 residues, and displays very slow luminescence kinetics and a pH-insensitive spectrum. The adult lantern isozyme (AL1) has 548 residues, displays flash-like kinetics and pH and metal sensitive bioluminescence spectra, and is at least 10 times catalytically more efficient than AL2. Thermostability and CD studies showed that AL2 is much more stable and rigid than the AL1 isozyme. Multialignment and modelling studies show that the E310Q substitution (E310 in AL2 and Q310 in AL1) may have been critical for the origin of pH-sensitivity in firefly luciferases. The results indicate that the lantern efficient flash-emitting pH-sensitive luciferases arose from less efficient glow-type pH-insensitive luciferases found in the fat body of ancestral larval fireflies by enzyme structure flexibilization and substitution at position 310.

Tissue-dependent DNA methylation of carp uncoupling protein 1 promoter.

DNA methylation of enhancers and promoters generally inhibits gene transcription. DNA methylation occurs predominantly at the dinucleotide CpG, a methyl group that is covalently bonded to cytosine. We have previously demonstrated tissue-restricted expression of the uncoupling protein 1 (Ucp1) in common carp. Here, we characterized the methylation status of the upstream region of the transcriptional initiation site of the carp Ucp1 gene in the liver, brain, kidney, skeletal muscle, and scales. In addition, we explored the direct role of methylation on Ucp1 transcription. Ucp1 expression was higher in the liver than that in other tissues including the kidney, skeletal muscle, and scales. The extent of methylation at nt -2178 and nt -2103 was lower in the liver and kidney than that in the brain, skeletal muscle, and scales. In addition, methylation at the upstream proximal-region of the Ucp1 gene was generally less frequent in the liver compared with that in the other organs. The transcriptional activation assay using the CpG-free luciferase-based reporter suggested that the methylation of the distal and proximal regions of the carp Ucp1 gene did not affect Ucp1 transcription. Unexpectedly, mutation of cytidylic acid to guanylic acid at nt -108 decreased Ucp1 promoter activity. The present results reveal that the status of DNA methylation of the upstream region of the carp Ucp1 gene is different among different tissues, but suggest that the DNA methylation do not directly repress the transcription of Ucp1.

Neural stimulations regulate the infiltration of immune cells into the CNS.

The systemic regulation of immune reactions by the nervous system is well studied and depends on the release of hormones. Some regional regulations of immune reactions, on the other hand, depend on specific neural pathways. Better understanding of these regulations will expand therapeutic applications for neuroimmune and organ-to-organ functional interactions. Here, we discuss one regional neuroimmune interaction, the gateway reflex, which converts specific neural inputs into local inflammatory outputs in the CNS. Neurotransmitters released by the inputs stimulate specific blood vessels to express chemokines, which serve as a gateway for immune cells to extravasate into the target organ such as the brain or spinal cord. Several types of gateway reflexes have been reported, and each controls distinct CNS blood vessels to form gateways that elicit local inflammation, particularly in the presence of autoreactive immune cells. For example, neural stimulation by gravity creates the initial entry point to the CNS by CNS-reactive pathogenic CD4+ T cells at the dorsal vessels of fifth lumbar spinal cord, while pain opens the gateway at the ventral side of blood vessels in the spinal cord. In addition, it was recently found that local inflammation by the gateway reflex in the brain triggers the activation of otherwise resting neural circuits to dysregulate organ functions in the periphery including the upper gastrointestinal tract and heart. Therefore, the gateway reflex represents a novel bidirectional neuroimmune interaction that regulates organ functions and could be a promising target for bioelectric medicine.

Analysis of the microstructure of bulk MgB2 using TEM, EBSD and t-EBSD.

EBSD analysis can provide information about grain orientation, texture and grain boundary misorientation of bulk superconducting MgB2 samples intended for supermagnet applications. However, as the grain size of the MgB2 bulks is preferably in the 100-200 nm range, the common EBSD technique operating in reflection mode works only properly on highly dense samples. In order to achieve reasonably good Kikuchi pattern quality on all types of MgB2 samples, we apply here the newly developed transmission EBSD (t-EBSD) technique to spark-plasma sintered MgB2 samples. This method requires the preparation of TEM slices by means of focused ion-beam milling, which are then analysed within the SEM, operating with a custom-built sample holder. To obtain multiphase scans, we identified the Kikuchi pattern of the MgB4 phase which appears at higher reaction temperatures and may act as additional flux pinning sites. We present here for the first time EBSD mappings of multiple phases, which include MgB2 , MgB4 and MgO. LAY DESCRIPTION: The electron backscatter diffraction (EBSD) technique operating in the scanning electron microscope provides information on the crystallographic orientation the material by recording Kikuchi patterns. In polycrystalline samples, it becomes possible to analyse the orientations of the grains to each other. The metallic superconductor with the currently highest superconducting transition temperature, MgB2 with a Tc of 38.5 K, can be used in applications in polycrystalline form. One such application of interest are trapped field magnets or supermagnets, where the superconductor cooled in an applied magnetic field can trap the magnetic field as vortices at numerous flux pinning sites in the sample. When the external magnetic field is removed, the sample will stay magnetised as long as it is kept cool, and importantly, the trapped magnetic fields can be much higher as for any permanent magnet. However, the small size of the MgB2 grains in the 100-200 nanometre range requires a different approach when using the EBSD technique on such samples. The recently developed EBSD technique working in transmission mode (t-EBSD) helps considerably to image such materials. In this approach, a tiny TEM slice has to be milled out from the original sample by using focused ion beam milling. To understand the properties of the flux pinning in the spark-plasma sintered MgB2 sample, we had to identify the Kikuchi pattern of MgB4 , which is another, non-superconducting phase appearing at higher reaction temperatures required to compact the material. Using this information, we could perform EBSD scans using three different phases, MgB2 , MgB4 and MgO. The EBSD mappings enable to see where the secondary phase particles are located in the sample, and to judge if the particles could work as flux pinning sites.

Fluoride-fiber-based side-pump coupler for high-power fiber lasers at 2.8 µm.

A side-pump coupler made of fluoride fibers was fabricated and tested. The tested device had a coupling efficiency of 83% and was driven with an incident pump power of up to 83.5 W, demonstrating high-power operation. Stable laser output of 15 W at a wavelength of around 2.8 µm was achieved over 1 h when using an erbium-doped double-clad fiber as the active medium. To the best of our knowledge, this is the first time a fluoride-glass-fiber-based side-pump coupler has been developed. A test with two devices demonstrated further power scalability.

Factors related to dissociation between objective and subjective masticatory function in Japanese community-dwelling elderly adults.

Clinicians' evaluations of older adults sometimes reveal inconsistencies between objective and subjective dental status. This study investigated which factors contribute both to good objective masticatory function (OMF) and the poor subjective masticatory function (SMF) that often becomes a clinical issue. Study participants included 635 elderly community-dwelling Japanese adults who underwent a comprehensive geriatric health examination in 2012. SMF was assessed with a question from the Kihon Checklist on eating difficulties (poor or good). OMF was assessed by a colour-changing gum (poor or good). Also investigated were age, sex, depressive symptoms, instrumental activities of daily living (I-ADLs), number of people who joined the participant at dinner, grip strength, usual walking speed, number of remaining teeth, number of functional teeth and their occlusal force. The group with good OMF and good SMF, defined as group 1, and the group with good OMF but poor SMF, group 2, were compared. Logistic regression analyses confirmed that the number of remaining and functional teeth participants had was statistically unrelated to differences between OMF and SMF. Instead, differences were related to stronger depressive symptoms (OR = 1.67, CI = 1.14-2.44), less ability to conduct I-ADL activities (OR = 0.73, CI = 0.59-0.91), slower usual walking speeds (OR = 0.18, CI = 0.06-0.58) and less occlusal force (OR = 0.99, CI = 0.99-1.00). Depressive symptoms, I-ADLs, and physical function are shown to be significantly related to divergence between objective and subjective masticatory function in elderly Japanese. This suggests that dissociations between objective and subjective dental evaluations of elderly adults indicate need for assessment of their mental and physical function.

The comparative anatomy of the folds, fossae, and adhesions around the duodenojejunal flexure in mammals.

BACKGROUND: Anatomical knowledge of the duodenojejunal flexure is necessary for abdominal surgeries, and also important for physiologic studies about the duodenum. But little is known about the anatomy of this region in mammals. Here, we examined comparative anatomy to understand the anatomical formation of the duodenojejunal flexure in mammals. MATERIALS AND METHODS: The areas around the duonenojejunal flexure were ob-served in mouse, rat, dog, pig, and human, and the anatomical structures around the duodenojejunal junction in the animals were compared with those in human. RESULTS: The superior and inferior duodenal folds, and the superior and inferior duodenal fossae were identified in all examined humans. In pig, the structures were not clearly identified because the duodenum strongly adhered to the retroperitoneum and to the mesocolon. In mouse, rat, and dog, only the plica duodenocolica, which is regarded as the animal counterpart of the superior duo-denal fold in human, was identified, and other folds or fossae were not observed, probably because the duodenum was not fixed to the parietal peritoneum in those animals. Transection of the plica duodenocolica could return the normally rotated intestine back to the state of non-rotation in rat. CONCLUSIONS: This study showed the anatomical similarities and dissimilarities of the duodenojejunal flexure among the mammals. Anatomical knowledge of the area is useful for duodenal and pancreatic surgeries, and for animal studies about the duodenum. (Folia Morphol 2018; 77, 2: 286-292).

Active site mapping of Loxosceles phospholipases D: Biochemical and biological features.

Brown spider phospholipases D from Loxosceles venoms are among the most widely studied toxins since they induce dermonecrosis, triggering inflammatory responses, increase vascular permeability, cause hemolysis, and renal failure. The catalytic (H12 and H47) and metal-ion binding (E32 and D34) residues in Loxosceles intermedia phospholipase D (LiRecDT1) were mutated to understand their roles in the observed activities. All mutants were identified using whole venom serum antibodies and a specific antibody to wild-type LiRecDT1, they were also analyzed by circular dichroism (CD) and differential scanning calorimetry (DSC). The phospholipase D activities of H12A, H47A, H12A-H47A, E32, D34 and E32A-D34A, such as vascular permeability, dermonecrosis, and hemolytic effects were inhibited. The mutant Y228A was equally detrimental to biochemical and biological effects of phospholipase D, suggesting an essential role of this residue in substrate recognition and binding. On the other hand, the mutant C53A-C201A reduced the enzyme's ability to hydrolyze phospholipids and promote dermonecrosis, hemolytic, and vascular effects. These results provide the basis understanding the importance of specific residues in the observed activities and contribute to the design of synthetic and specific inhibitors for Brown spider venom phospholipases D.

Ear Acupuncture for Immediate Pain Relief-A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

Objective: To systematically review the literature on the effectiveness of ear acupuncture (EA) for immediate pain relief. Data sources: AMED, CINAHL, Cochrane Reviews, Embase, PsycINFO, PubMed, Scopus Web of Science, from inception through March 2015. Study selection: English publications, randomized controlled trials on human subjects involving EA as a treatment for pain, with outcomes recorded within 48 hours. Data extraction and design: Two authors independently assessed trial eligibility, quality, results, and side effects, and extracted data; a third author checked final data. Effect size (d), mean difference (MD), and 95% confidence interval (CI) were calculated. The Physiotherapy Evidence Database (PEDro) scoring system was used to assess study quality. Meta-analysis was performed for two primary outcomes measures-pain intensity score and analgesic requirements. Results: Ten studies met inclusion criteria. Quality per PEDro scores: four excellent, four good, two fair. Based on their primary outcome measures, six studies showed EA being superior to its comparator, three showed no difference to comparators (which in all cases were analgesics), and one study showed significant pain decrease at the first time point and no significant decrease at the second. Meta-analysis was completed for the three studies that evaluated pain intensity as a primary outcome measure, and EA was superior to comparator (MD = -0.96, 95% CI = -1.82- -0.11), but the MD was small. Meta-analysis was completed for the six studies that evaluated analgesic requirements, and EA was superior (MD = -1.08, 95% CI = -1.78- -0.38]), again with a small MD. Six studies reported side effects; all were minor and transient. Conclusions: Ear acupuncture may be a promising modality to be used for pain reduction within 48 hours, with a low side effect profile. Rigorous research is needed to establish definitive evidence of a clinically significant difference from controls or from other pain treatments.

The relationship between lip-closing force and dental arch morphology in patient with Angle Class I malocclusion.

Dental arch morphology and tooth position are affected by lip-closing force (LCF). This study aimed to quantitatively evaluate the relationships between the horizontal or vertical balance of the LCF generated during maximum voluntary pursing-like movements and dental arch length (DAL) or width (DAW) or the lingual inclination of the upper or lower 1st molars (LIUM, LILM) in patients with Angle Class I malocclusion. Sixteen subjects with Angle Class I malocclusion (median age: 23·4 ± 5·9 years) who had never undergone orthodontic treatment were randomly selected. LCF was measured in eight directions during maximum voluntary pursing-like lip-closing movements. Dental arch models were scanned and analysed to obtain DAW, DAL, LIUM and LILM measurements. Mandibular deviation was measured on posteroanterior cephalograms. A significant negative correlation was detected between maxillary DAL and upper LCF. Maxillary DAL, DAW and the DAL/DAW ratio displayed significant negative correlations with total LCF and upper LCF. However, no significant correlations were detected between any mandibular dental arch morphological parameter and LCF. The difference in the LIUM between the deviation and non-deviation sides exhibited a significant positive correlation with the difference in upper LCF between the deviation and non-deviation sides and was significantly negatively correlated with the difference in lower LCF between the deviation and non-deviation sides. These results suggest that upper LCF is related to maxillary DAL, and the horizontal balance of the LCF of the upper and lower lips is related to the LIUM during pursing-like lip-closing movements in patients with Angle Class I malocclusion.

Pregnancies with factor V deficiency: a case report and review of the literature.

CASE REPORT: A 30-year-old Japanese nulliparous woman visited for pregnancy at 33 weeks with a massive ovarian tumor located in the pouch of Douglas. By preoperative screening, her prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, and her FV activity was significantly decreased to 4.8%. After prophylactic FFP 20 ml/kg was administered and her FV factor was 19.3%, cesarean delivery was performed, and her perioperative course was uneventful. One year later, she underwent a dilatation and evacuation because of a missed abortion, although prophylactic FFP was not administered. During a third pregnancy, after prophylactic FFP 20 ml/kg was administered and FV activity increased to 21.1%, elective cesarean delivery was performed, and her postoperative course was uneventful. CONCLUSION: For surgical therapy or delivery, the goal of therapy is to maintain FV activity above 20%. It is particularly useful to administer prophylactic FFP.

NecroX-5 suppresses IgE/Ag-stimulated anaphylaxis and mast cell activation by regulating the SHP-1-Syk signaling module.

BACKGROUND: IgE/Ag-stimulated mast cells release various pro-allergic inflammatory mediators, including histamine, eicosanoids, and pro-inflammatory cytokines. NecroX-5, a cell permeable necrosis inhibitor, showed cytoprotective effects in both in vitro and in vivo models. However, the anti-allergic effect of NecroX-5 has not yet been investigated. The aims of this study were to evaluate the anti-allergic activity of NecroX-5 in vivo and to investigate the underlying mechanism in vitro. METHODS: The anti-allergic activity of NecroX-5 was evaluated in vitro using bone marrow-derived mast cells (BMMCs) and IgE receptor-bearing RBL-2H3 or KU812 cells and in vivo using a mouse model of passive anaphylaxis. The levels of histamine, eicosanoids (PGD2 and LTC4 ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured using enzyme immunoassay kits. The mechanism underlying the action of NecroX-5 was investigated using immunoblotting, immunoprecipitation, and gene knockdown techniques. RESULTS: NecroX-5 markedly inhibited mast cell degranulation and the synthesis of eicosanoids, TNF-α, and IL-6 by suppressing the activation of Syk, LAT, phospholipase Cγ1, MAP kinases, the Akt/NF-κB pathway, and intracellular Ca(2+) mobilization via the activation of phosphatase SHP-1. Oral administration of NecroX-5 effectively suppressed mast cell-dependent passive cutaneous and systemic anaphylactic reactions in a dose-dependent manner. CONCLUSIONS: NecroX-5 might be a potential candidate for the development of a novel anti-allergic agent that suppresses IgE-dependent mast cells signaling.

The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343).

Similar in vitro effects and pulp regeneration in ectopic tooth transplantation by basic fibroblast growth factor and granulocyte-colony stimulating factor.

OBJECTIVES: Granulocyte-colony stimulating factor (G-CSF) has been shown to have combinatorial trophic effects with dental pulp stem cells for pulp regeneration. The aim of this investigation is to examine the effects of basic fibroblast growth factor (bFGF) in vitro and in vivo compared with those of G-CSF and to assess the potential utility of bFGF as an alternative to G-CSF for pulp regeneration. MATERIALS AND METHODS: Five different types of cells were examined in the in vitro effects of bFGF on cell migration, proliferation, anti-apoptosis, neurite outgrowth, angiogenesis, and odontogenesis compared with those of G-CSF. The in vivo regenerative potential of pulp tissue including vasculogenesis and odontoblastic differentiation was also compared using an ectopic tooth transplantation model. RESULTS: Basic fibroblast growth factor was similar to G-CSF in high migration, proliferation and anti-apoptotic effects and angiogenic and neurite outgrowth stimulatory activities in vitro. There was no significant difference between bFGF and G-CSF in the regenerative potential in vivo. CONCLUSIONS: The potential utility of bFGF for pulp regeneration is demonstrated as a homing/migration factor similar to the influence of G-CSF.

Employment status among non-retired cancer survivors in Japan.

Employed cancer patients confront some challenges as they attempt to return to work after treatment. We aimed to identify correlates of return to work for cancer survivors in Japan, with an emphasis on employment status. Participants were 260 patients (aged <65 years) who had received a cancer diagnosis ≥ 1 year previously and who were employed at the time of diagnosis. Participants completed questionnaires at consultations at any Regional Cancer Center Hospitals in Yamagata, Japan between 28 November 2011 and 9 December 2011. Logistic regression analysis was used to identify correlates of return to work. Data cross-tabulation was used to evaluate relationships to workplace and income-changes by employment status. A high proportion of patients (75.8%) had returned to work. Non-regularly employed survivors were less likely to return to work (odds ratio = 5.03; 95% confidence interval, 1.18-21.35). Individuals with poor health, advanced-stage tumours, of advanced age and women were significantly less likely to return to work. Only 52.8% of non-regular employees continued to be employed, and their income decreased by as much as 61.1%. Social and financial support policies should be organised based on more intensive study of employment circumstances.

Species differences in the metabolism of ritobegron in vitro and assessment of potential interactions with transporters and cytochrome P450 enzymes.

Ritobegron, a selective ß3-adrenoceptor agonist, is the prodrug of the active compound, KUC-7322. We investigated species differences in its metabolism in vitro and the potential for drug-drug interactions with ritobegron. In rat, dog, monkey, and human liver microsomes, ritobegron was not metabolized by cytochrome P450 enzymes (CYPs). KUC-7322 was the only metabolite observed. Hydrolysis of ritobegron to KUC-7322 was likely catalyzed by carboxylesterases in human liver microsomes. The maximum velocity of the reaction (V(max))/Michaelis-Menten constant (K(m)) for hydrolysis of ritobegron to KUC-7322 was much higher in rat serum than those in other species. There were also species differences in the conjugation of KUC-7322. Sulfate conjugates of ritobegron were detected in all species, whereas glucuronide and glutathione conjugates of KUC-7322 were only observed in rat liver subcellular fractions. Ritobegron and KUC-7322 did not affect the CYP-mediated metabolism of probe substrates in human liver microsomes and organic anion transporter 1 (OAT1)-, OAT2-, OAT3-, organic cation transporter 2 (OCT-2)-, OCT3-, or organic cation/carnitine transporter 1 (OCTN1)-mediated uptake of probe substrates in S2 cells. Ritobegron, but not KUC-7322, inhibited P-glycoprotein-mediated digoxin transport in Caco-2 cells. Significant uptake of KUC-7322 was observed in OAT3-expressing S2 cells. Therefore, CYP-mediated drug-drug interactions are not likely when ritobegron is administered with CYP substrates or inhibitors. Ritobegron may increase the plasma concentrations of P-glycoprotein substrates, such as digoxin, and the plasma concentration of KUC-7322 may increase when it is administered in combination with OAT inhibitors such as probenecid.