Predicting muscarinic receptor occupancy in human bladder mucosa from urinary concentrations of antimuscarinic agents for overactive bladder.

To assess the pharmacologically relevant and selective muscarinic receptor occupancy in the bladder mucosa, we considered not only plasma drug concentrations but also urinary drug concentrations. The purpose of this study was to predict muscarinic receptor occupancy in the human bladder mucosa based on urinary concentrations in response to clinical dosages of antimuscarinic agents used to treat overactive bladder. The calculated mean plasma or serum unbound steady state concentrations were 0.06-11 nM in clinical dosages of five antimuscarinic agents. Urinary concentrations calculated from the mean plasma or serum and renal clearance ranged between 19 nM and 2 µM, which were >10-fold higher than the Ki values for bladder muscarinic receptors excluding propiverine. Bladder mucosal muscarinic receptor occupancy estimated from the urinary concentrations and the Ki values was >90 % at a steady state in clinical dosages of five antimuscarinic agents. The bladder muscarinic receptor occupancy was higher than that in the parotid gland calculated based on the mean plasma or serum unbound concentrations and Ki values for muscarinic receptors in the parotid gland. These results suggest that sufficient and selective muscarinic receptor occupancy by antimuscarinic agents, to exert pharmacological effects, in the bladder mucosa can be predicted using urinary concentrations.

Phylogenetic analysis of the promoter element 2 of paramyxo- and filoviruses.

Paramyxo- and filovirus genomes are equipped with bipartite promoters at their 3' ends to initiate RNA synthesis. The two elements, the primary promoter element 1 (PE1) and the secondary promoter element 2 (PE2), are separated by a spacer region that must be precisely a multiple of 6 nucleotides (nts), indicating these viruses adhere to the "rule of six." However, our knowledge of PE2 has been limited to a narrow spectrum of virus species. In this study, a comparative analysis of 1,647 paramyxoviral genomes from a public database revealed that the paramyxovirus PE2 can be clearly categorized into two distinct subcategories: one marked by C repeats at every six bases (exclusive to the subfamily Orthoparamyxovirinae) and another characterized by CG repeats every 6 nts (observed in the subfamilies Avulavirinae and Rubulavirinae). This unique pattern collectively mirrors the evolutionary lineage of these subfamilies. Furthermore, we showed that PE2 of the Rubulavirinae, with the exception of mumps virus, serves as part of the gene-coding region. This may be due to the fact that the Rubulavirinae are the only paramyxoviruses that cannot propagate without RNA editing. Filoviruses have three to eight consecutive uracil repeats every six bases (UN5) in PE2, which is located in the 3' end region of the genome. We obtained PE2 sequences from 2,195 filoviruses in a public database and analyzed the sequence conservation among virus species. Our results indicate that the continuity of UN5 hexamers is consistently maintained with a high degree of conservation across virus species. IMPORTANCE: The genomic intricacies of paramyxo- and filoviruses are highlighted by the bipartite promoters-promoter element 1 (PE1) and promoter element 2 (PE2)-at their 3' termini. The spacer region between these elements follows the "rule of six," crucial for genome replication. By a comprehensive analysis of paramyxoviral genome sequences, we identified distinct subcategories of PE2 based on C and CG repeats that were specific to Orthoparamyxovirinae and Avulavirinae/Rubulavirinae, respectively, mirroring their evolutionary lineages. Notably, the PE2 of Rubulavirinae is integrated into the gene-coding region, a unique trait potentially linked to its strict dependence on RNA editing for virus growth. This study also focused on the PE2 sequences in filovirus genomes. The strict conservation of the continuity of UN5 among virus species emphasizes its crucial role in viral genome replication.

Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

Generation of a photocontrollable recombinant bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (BPIV3) is a promising vaccine vector against various respiratory virus infections, including the human PIV3, respiratory syncytial virus, and severe acute respiratory syndrome-coronavirus 2 infections. In this study, we combined the Magnet system and reverse genetic approach to generate photocontrollable BPIV3. An optically controllable Magnet gene was inserted into the H2 region of the BPIV3 large protein gene, which encodes an RNA-dependent RNA polymerase. The generated photocontrollable BPIV3 grew in specific regions of the cell sheet only when illuminated with blue light, suggesting that spatiotemporal control can aid in safe clinical applications of BPIV3.

Evaluation of Clinical Practice Guidelines for Rare Diseases in Japan.

Introduction: The insufficient quantity and quality of clinical epidemiological evidence in the field of rare diseases have posed methodological challenges to develop clinical practice guidelines (CPGs). Guideline development groups struggle to provide patients and their families with beneficial guidance, such as that for medical care and in complex circumstances. Motivated by the challenges, we focused on information on resources for supporting the daily and social life to improve the CPGs for users. We aimed to assess the methodological quality of CPGs for rare diseases in Japan and to evaluate information on resources to support the daily and social life in the CPGs. Methods: We conducted a systematic search using PubMed, three electronic Japanese databases, and two hand-searched sources in Japan. The Appraisal of Guidelines for Research and Evaluation (AGREE) II instrument with six domains was used to assess the methodological quality of the CPGs. A content analysis of the CPG text was conducted using five keywords as information on non-medical resources, e.g., "Intractable Disease Consultation Support Center," "Japan Intractable Disease Information Center," and "Patient Association." Results: A total of 55 CPGs met the inclusion criteria. Among four domains of AGREE II with low scores (Stakeholder Involvement, Rigor of Development, Applicability, and Editorial Independence), Rigor of Development had the lowest median score. As for information on non-medical resources, 41 CPGs included at least 1 of the 5 keywords, while 14 CPGs included none. Conclusions: At the Rigor of Development domain, methodological challenges may have resulted in an insufficient description of items regarding the translation evidence to recommendations. As the sufficiency of five keywords as information on non-medical resources could be improved, the information will be advocative as clues to provide pragmatic guidance, particularly for rare diseases with limited medical evidence.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Essential role of TMPRSS2 in SARS-CoV-2 infection in murine airways.

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

Efficacy of a novel in ovo-attenuated live vaccine and recombinant vaccine against a very virulent infectious bursal disease virus in chickens.

Infectious bursal disease (IBD) causes severe economic damage to the poultry industry worldwide. To prevent IBD virus (IBDV) infection, live virus vaccines have been widely used in chickens having wide-ranging levels of maternally derived antibodies. But, the risks of infection with other pathogens because of lesions related to atrophy of the bursa of Fabricius in vaccinated chickens are a concern. To resolve the problems, a recombinant turkey herpesvirus (HVT) vaccine expressing IBDV-VP2 protein (rHVT-IBD) has been developed. However, the induction of neutralizing antibodies by rHVT-IBD against a virulent IBDV might be delayed compared with that by the live IBD vaccine, leading to the high risks of IBDV infection for young chickens. To find the best selection of IBDV vaccine for the onset of immunity, we examine the protective efficacy of a novel in ovo-attenuated live IBDV (IBD-CA) vaccine and the rHVT-IBD vaccine in young chickens challenged with a very virulent IBDV (vvIBDV) strain. We show that the protective efficacy of IBD-CA vaccine was higher than that of the rHVT-IBD vaccine in 14-day-old chickens challenged with the vvIBDV strain, leading to the risk of IBDV infection for young chickens when vaccinated with rHVT-IBD. Our results suggest that farmers should select the best vaccines to maximize vaccine efficacy in consideration of the vaccine characteristics, prevalence levels of IBDV in the areas, and initial MDA levels of the chickens since the attenuated live and recombinant vaccines play a role in the different vaccine efficacies.

Replication kinetics of turkey herpesvirus in lymphoid organs and feather follicle epithelium in chickens.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes immunosuppression, T-cell lymphomas, and neuropathic disease in infected chickens. To protect chickens from MDV infection, an avirulent live vaccine of turkey herpesvirus (HVT) has been successfully used for chickens worldwide. Similar to MDV for natural infection in both chickens and turkeys, HVT also infects lung in the early stage of infection and then lymphocytes from lymphoid organs. Virus replication requires cell-to-cell contact for spreading and semi-productive lytic replication in T and B cells. Then, cell-free infectious virions matured in the feather follicle epithelium (FFE) are released and spread through the feather from infected turkeys or chickens. To understand the lifecycle of HVT in inoculated chickens via the subcutaneous route, we investigate the replication kinetics and tissue organ tropism of HVT in chickens by a subcutaneous inoculation which is a major route of MDV vaccination. We show that the progeny virus matured in lymphocytes from the thymus, spleen, and lung as early as 2 days post-infection (dpi) and bursa of Fabricius at 4 dpi, whereas viral maturation in the FFE was observed at 6 dpi. Furthermore, semi-quantitative reverse transcription-PCR experiments to measure viral mRNA expression levels revealed that the higher expression levels of the late genes were associated with viral maturation in the FFE. These data that tropism and replication kinetics of HVT could be similar to those of MDV through the intake pathway of natural infection from respiratory tracts.

Muscarinic receptor binding activity in rat tissues by vibegron and prediction of its receptor occupancy levels in the human bladder.

OBJECTIVES: To examine the effects of vibegron, a selective ß3 -adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. METHODS: Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N-methyl-3 H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. RESULTS: Vibegron (0.1-100 µmol/L) inhibited specific [N-methyl-3 H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315- and 3.5-fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51-91% until 24 h after its oral administration at 50 mg of vibegron. CONCLUSIONS: This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder.

Cell-to-Cell Transmission of Turkey Herpesvirus in Chicken Embryo Cells via Tunneling Nanotubes.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that causes immunosuppression, T cell lymphomas, and neuropathic disease in infected chickens. To protect chickens from MDV infection, an avirulent live vaccine of turkey herpesvirus (HVT) has been successfully used in chickens worldwide. Many vaccine manufacturers have used chicken embryo fibroblast (CEF) cells to produce the HVT vaccine. Generally, it has been suggested that HVT is a highly cell-associated herpesvirus that spread via cell-to-cell contact, but it is unclear how HVT is transmitted from infected cells to uninfected target cells. Here, we show via immunofluorescence analysis that nanotubes containing the actin cytoskeleton and HVT antigens from infected CEF cells were observed to contact neighboring cells. When the infected cells were treated with inhibitors for actin polymerization or depolymerization, the formation and extension of the nanotubes from infected cells were greatly inhibited and the intercellular contact was abolished, leading to a drastic reduction in plaque formation and viral titers of the cell-associated virus. Our data indicate that cell-to-cell contacts via nanotubes composed of actin filaments are essential for efficient viral spreading and replication. This finding might contribute to the further improvement of efficient HVT vaccine production.

Nota de investigación­Transmisión de célula a célula del virus herpes del pavo en células embrionarias de pollo a través de tunelización por nanotubos. El virus de la enfermedad de Marek (MDV) es un alfaherpesvirus oncogénico que causa inmunosupresión, linfomas de células T y enfermedad neuropática en pollos infectados. Para proteger a los pollos de la infección por MDV, se ha utilizado con éxito una vacuna viva avirulenta del virus herpes del pavo (HVT) en pollos de todo el mundo. Muchos fabricantes de vacunas han utilizado células de fibroblasto de embrión de pollo (CEF) para producir la vacuna HVT. En general, se ha sugerido que el HVT es un virus herpes muy asociado a células que se propaga mediante el contacto entre células, pero no está claro cómo se transmite el virus HVT a partir de las células infectadas a las células blanco no infectadas. Aquí, se demuestra mediante análisis de inmunofluorescencia que nanotubos que contienen el citoesqueleto de actina y los antígenos del HVT dentro las células de fibroblasto de embrión de pollo infectadas son lo que contactan con las células vecinas. Cuando las células infectadas se trataron con inhibidores para la polimerización o despolimerización de actina, la formación y extensión de los nanotubos de las células infectadas se inhibió en gran medida y se abolió el contacto intercelular, lo que llevó a una reducción drástica en la formación de placa y de los títulos virales de virus asociados a células. Estos datos indican que los contactos entre células a través de nanotubos compuestos de filamentos de actina son esenciales para la propagación y replicación viral eficiente. Este hallazgo podría contribuir a la mejora adicional de la producción eficiente de vacunas HVT.

Possible Involvement of Muscarinic Receptor Blockade in Mirabegron Therapy for Patients with Overactive Bladder.

The selective ß 3-adrenoceptor agonist mirabegron, an established alternative to antimuscarinic therapy for patients with overactive bladder, induces additional effects against receptors, transporters, and hepatic enzymes. The present study aimed to elucidate the effects of mirabegron on muscarinic receptors in the rat bladder using radioligand binding and functional assays. Mirabegron (0.1-100 µM) inhibited specific [N-methyl-3H]scopolamine methyl chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. Binding affinity in the bladder was similar to that in the heart and significantly higher than those in the submaxillary gland and brain. Mirabegron induced the concentration-dependent relaxation of carbachol-induced contractions in the rat isolated bladder. Further analyses using a two-site model revealed that the relative quantities of high- and low-affinity components for mirabegron were 44.5% and 55.5%, respectively. Respective pEC50 values were 7.06 and 4.97. Based on the receptor binding affinity and pharmacokinetics of mirabegron, muscarinic receptor occupancy in the human bladder for 24 hours after the administration of a single oral dose of 50 mg mirabegron was 37%-76%. The present results demonstrate for the first time that mirabegron may relax the detrusor smooth muscle not only by ß 3-adrenoceptor activation but also muscarinic receptor blockade. SIGNIFICANCE STATEMENT: Mirabegron, the first selective ß 3-adrenoceptor agonist, represents an alternative to antimuscarinic agents for management of overactive bladder (OAB). The present study aimed to clarify whether mirabegron directly binds to muscarinic receptors and affects cholinergic agonist-induced contractions in rat urinary bladder and to predict muscarinic receptor occupancy in human bladder after oral administration of mirabegron. The results demonstrated that mirabegron therapy for patients with OAB may be due not only to ß 3-adrenoceptor activation but also muscarinic receptor blockade.

First episode of ventricular fibrillation in an 84-year-old man with long-QT type 2 syndrome: A case report.

Congenital long QT syndrome (LQTS) is associated with ventricular arrhythmia and an increased risk of sudden cardiac death in young people. However, it is extremely rare for an elderly man to experience ventricular fibrillation (VF) due to congenital LQTS as a first episode. We describe the case of an 84-year-old man who experienced syncope after urination. He had a medical history of hypertension and asthma, but no history of syncope. Electrocardiographic findings in 2017 showed QT prolongation (corrected QT = 505 ms). No medication that could induce QT prolongation was administered. Blood test results on admission showed no electrolyte abnormalities, and there were no abnormal findings on echocardiography. The second episode of loss of consciousness occurred during hospitalization, and electrocardiography revealed incessant torsade de pointes, caused by R-on-T with short-long-short (SLS) sequences due to bradyarrhythmia. Coronary angiography did not detect myocardial ischemia, and an implantable cardioverter-defibrillator was implanted for secondary prevention. Genetic testing revealed a mutation of the KCNH2 gene, indicating LQTS type 2. In summary, we report a rare case of prolonged QT interval with SLS sequences due to sick sinus syndrome triggering VF as the first attack in an elderly patient with LQTS type 2. .

An educational intervention improved knowledge of dietary supplements in college students.

BACKGROUND: We have previously reported on the prevalence of dietary supplements among college students; it was deduced that their intake of supplements increased according to their grade (i.e., 13.1% in the first grade to 20.5% in the sixth grade). We also reported that some students had experienced adverse events in Japan due to their intake of these supplements. However, awareness of dietary supplements among college students remains limited, even among pharmaceutical students. Being appropriately educated about them is important for pharmaceutical students, both for themselves as well as for their future careers as pharmacists. METHODS: We conducted a lecture-based educational intervention about dietary supplements on 328 college students in Japan-184 from pharmaceutical science and 144 from environmental science or food and life science disciplines. The purpose of this study was to evaluate the effects of an educational intervention on college students' understanding of dietary supplements. The intervention involved a lecture that covered the quality of dietary supplements, how they differed from drugs, and a summary of their adverse events. The lecture was evaluated using a 14-question questionnaire. We then compared the pre- and post-intervention responses to the same questionnaire using a Wilcoxon signed-rank test. The questions were assessed using a Likert scale that ranged from "strongly agree" to "strongly disagree"; the latter being the preferred answer. RESULTS: Before the intervention had taken place, the students' understanding of dietary supplements was shown to be deficient. Conversely, post-intervention, their knowledge levels had significantly improved, especially concerning agreement on whether "Dietary supplements are safe because they are just food items". Pre-intervention, 2.7% strongly agreed and 37.5% agreed; post-intervention, 1.2% strongly agreed and 15.6% agreed. On whether "Dietary supplements made from natural ingredients or herbs are safe", at the pre-intervention stage 2.8% strongly agreed and 44.0% agreed and post-intervention, 2.2% strongly agreed and 16.9% agreed. On whether "Dietary supplements made from food items are safe", 4.0% strongly agreed and 43.6% agreed pre-intervention and 0.9% strongly agreed and 16.6% agreed post-intervention. Despite there being a greater number of pharmaceutical students who had a correct understanding of dietary supplements before the intervention, these students still showed improvement after the lecture. CONCLUSION: An intervention in the form of a single educational lecture has the capacity to improve college students' understanding of dietary supplements. It is important for pharmacists to be appropriately educated about dietary supplements when they consult with patients. We will evaluate the long-term effects of the intervention on the alumni (pharmacists) in a subsequent study.

Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation.

Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L-P complex from parainfluenza virus 5 (PIV5) at 4.3-Šresolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Šresolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5' end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

Diagnostic Value of Atrial Fibrillation by Built-in Electrocardiogram Technology in a Blood Pressure Monitor.

Background: Hypertension in patients with atrial fibrillation (AF) is a known independent risk factor for stroke. The Complete blood pressure (BP) monitor (Omron Healthcare, Kyoto, Japan) was developed as the first BP monitor with electrocardiogram (ECG) capability in a single device to simultaneously monitor ECG and BP readings. This study investigated whether the Complete can accurately differentiate sinus rhythm (SR) from AF during BP measurement. Methods and Results: Fifty-six consecutive patients with persistent AF admitted for catheter ablation were enrolled in the study (mean age 65.8 years; 83.9% male). In all patients, 12-lead ECGs and simultaneous Complete recordings were acquired before and after ablation. The Complete interpretations were compared with physician-reviewed ECGs, whereas Complete recordings were reviewed by cardiologists in a blinded manner and compared with ECG interpretations. Sensitivity, specificity, and κ coefficient were also determined. In all, 164 Complete and ECG recordings were simultaneously acquired from the 56 patients. After excluding unclassified recordings, the Complete automated algorithm performed well, with 100% sensitivity, 86% specificity, and a κ coefficient of 0.87 compared with physician-interpreted ECGs. Physician-interpreted Complete recordings performed well, with 99% sensitivity, 85% specificity, and a κ coefficient of 0.85 compared with physician-interpreted ECGs. Conclusions: The Complete, which combines BP and ECG monitoring, can accurately differentiate SR from AF during BP measurement.

Effects of Anticholinergic Drugs Used for the Therapy of Overactive Bladder on P-Glycoprotein Activity.

We evaluated the effects of anticholinergic drugs principally used for the therapy of overactive bladder (OAB) on the activity of P-glycoprotein, an efflux transport protein, in Caco-2 cells. The time-dependent changes in the fluorescence of residual rhodamine 123, a P-glycoprotein activity marker, in the apical region of Caco-2 cells were measured in the presence of anticholinergic drugs using time-lapse confocal laser scanning microscopy. The effect of anticholinergic drugs on human P-glycoprotein ATPase activity was also measured. The fluorescence of residual rhodamine 123 in untreated Caco-2 cells decreased over time. The gradual decrease in the fluorescence was significantly inhibited by treatment with cyclosporine A, darifenacin, and trospium. In contrast, oxybutynin, N-desethyl-oxybutynin (DEOB), propiverine, and its active metabolites (M-1, M-2), imidafenacin, solifenacin, or tolterodine had little effect on the efflux of rhodamine 123. P-Glycoprotein ATPase activity was increased by darifenacin. Darifenacin and trospium reduced the rhodamine 123 transfer across the apical cell membrane. These data suggest that darifenacin and trospium interact with P-glycoprotein. Additionally, darifenacin influenced P-glycoprotein ATPase activity. These results suggest that darifenacin may be a substrate of P-glycoprotein. This study is the first paper to test simultaneously the effects of 10 anticholinergic drugs used currently for the therapy of OAB, on the P-glycoprotein.

Apical Trafficking Pathways of Influenza A Virus HA and NA via Rab17- and Rab23-Positive Compartments.

The envelope proteins of influenza A virus, hemagglutinin (HA) and neuraminidase (NA), play critical roles in viral entry to host cells and release from the cells, respectively. After protein synthesis, they are transported from the trans-Golgi network (TGN) to the apical plasma membrane (PM) and assembled into virus particles. However, the post-TGN transport pathways of HA and NA have not been clarified. Temporal study by confocal microscopy revealed that HA and NA colocalized soon after their synthesis, and relocated together from the TGN to the upper side of the cell. Using the Rab family protein, we investigated the post-TGN transport pathways of HA and NA. HA partially colocalized with AcGFP-Rab15, Rab17, and Rab23, but rarely with AcGFP-Rab11. When analyzed in cells stably expressing AcGFP-Rab, HA/NA colocalized with Rab15 and Rab17, markers of apical sorting and recycling endosomes, and later colocalized with Rab23, which distributes to the apical PM and endocytic vesicles. Overexpression of the dominant-negative (DN) mutants of Rab15 and Rab17, but not Rab23, significantly delayed HA transport to the PM. However, Rab23DN impaired cell surface expression of HA. Live-cell imaging revealed that NA moved rapidly with Rab17 but not with Rab15. NA also moved with Rab23 in the cytoplasm, but this motion was confined at the upper side of the cell. A fraction of HA was localized to Rab17 and Rab23 double-positive vesicles in the cytoplasm. Coimmunoprecipitation indicated that HA was associated with Rab17 and Rab23 in lipid raft fractions. When cholesterol was depleted by methyl-ß-cyclodextrin treatment, the motion of NA and Rab17 signals ceased. These results suggest that HA and NA are incorporated into lipid raft microdomains and are cotransported to the PM by Rab17-positive and followed by Rab23-positive vesicles.

Prostaglandin Transporter OATP2A1/SLCO2A1 Is Essential for Body Temperature Regulation during Fever.

Prostaglandin E2 (PGE2) in the hypothalamus is a principal mediator of the febrile response. However, the role of organic anion transporting polypeptide 2A1 (OATP2A1/SLCO2A1), a prostaglandin transporter, in facilitating this response is unknown. Here, we investigated the effect of Slco2a1 deficiency on the body core temperature (Tc) and on the PGE2 concentration in hypothalamus interstitial fluid (Cisf) and CSF (Ccsf) of lipopolysaccharide (LPS; 100 µg/kg, i.p.)-treated mice of both sexes. Slco2a1-/- mice did not develop a febrile response. Ccsf was increased in Slco2a1+/+ and Slco2a1-/- mice, and Ccsf of Slco2a1-/- mice was well maintained at 5 h after LPS injection (1160 pg/ml) compared with Slco2a1+/+ mice (316 pg/ml). A microdialysis study revealed that Cisf peaked at 2 h after LPS injection in Slco2a1+/+ mice (841 pg/ml), whereas the increase in Cisf was negligible in Slco2a1-/- mice. The PGE2 plasma concentration in Slco2a1-/- mice (201 pg/ml) was significantly higher than that in Slco2a1+/+ mice (54 pg/ml) at 1 h after LPS injection, whereas the two groups showed similar PGE2 concentrations in the hypothalamus. Strong Oatp2a1 immunoreactivity was observed in F4/80-positive microglia and perivascular cells and in brain capillary endothelial cells. The changes in Tc and Cisf seen in LPS-injected Slco2a1+/+ mice were partially attenuated in monocyte-/macrophage-specific Slco2a1-/- (Slco2a1Fl/Fl/LysMCre/+) mice. Thus, OATP2A1 facilitates the LPS-induced febrile response by maintaining a high level of Cisf, possibly by regulating PGE2 secretion from F4/80-positive glial cells and/or facilitating PGE2 transport across the blood-brain barrier. These findings suggest that OATP2A1 is a useful therapeutic target for neuroinflammation.SIGNIFICANCE STATEMENT Fever is a physiological response caused by pyrogen-induced release of prostaglandin E2 (PGE2) in the hypothalamus, which plays a central role in regulating the set-point of body temperature. However, it is unclear whether the prostaglandin transporter OATP2A1/SLCO2A1 is involved in this response. We show here that LPS-induced fever is associated with increased PGE2 concentration in hypothalamus interstitial fluid (Cisf), but not in CSF (Ccsf), by means of a microdialysis study in global Slco2a1-knock-out mice and monocyte-/macrophage-specific Slco2a1-knock-out mice. The results suggest that OATP2A1 serves as a regulator of Cisf in F4/80-positive glial cells. OATP2A1 was detected immunohistochemically in brain capillary endothelial cells and, therefore, may also play a role in PGE2 transport across the blood-brain barrier.