The apiosyltransferase celery UGT94AX1 catalyzes the biosynthesis of the flavone glycoside apiin.

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery (Apium graveolens) and parsley (Petroselinum crispum) in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified UGT94AX1 as an A. graveolens apiosyltransferase (AgApiT) responsible for catalyzing the last sugar modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. Identification of this plant apiosyltransferase gene will enhance our understanding of the physioecological functions of apiose and apiose-containing compounds.

Broken Screw Rotational Symmetry in the Near-Surface Electronic Structure of AB-Stacked Crystals.

We investigate the electronic structure of 2H-NbS_{2} and h-BN by angle-resolved photoemission spectroscopy (ARPES) and photoemission intensity calculations. Although in bulk form, these materials are expected to exhibit band degeneracy in the k_{z}=π/c plane due to screw rotation and time-reversal symmetries, we observe gapped band dispersion near the surface. We extract from first-principles calculations the near-surface electronic structure probed by ARPES and find that the calculated photoemission spectra from the near-surface region reproduce the gapped ARPES spectra. Our results show that the near-surface electronic structure can be qualitatively different from the bulk electronic structure due to partially broken nonsymmorphic symmetries.

Coronary anomalies associated with ventricular septal defect.

Coronary anomalies encompass different conditions in terms of anomalous origin, coursing, and branching, which are occasionally associated with congenital heart disease. This study aimed to explore coronary anomalies associated with ventricular septal defect that was a stereotypical congenital heart disease. We retrospectively reviewed angiographic findings in patients with ventricular septal defect who required corrective surgery, and identified coronary abnormalities, including anomalous origin, coursing, and branching of coronary arteries based on angiography. We studied the prevalence and types of coronary anomalies among them. A total of 998 patients with ventricular septal defect was studied. Age and weight were 2.0 (2.1-15.2) months and 5.7 (4.1-8.7) kg, respectively. There were 115 patients (12%) with syndrome or genetic disorder, and 34 patients (3%) with extracardiac major organ anomalies. The overall prevalence of coronary anomalies was 6.2%. All coronary anomalies were anomalous origin and coursing, among 3 patients accompanied anomalous intrinsic coronary anatomy. Originating within aortic root above the Valsalva sinus was the most common coronary anomaly. Coronary anomalies were independently correlated with bicuspid aortic valve (odds ratio [OR]: 8.02, 95% confidence interval [CI]: 2.34-23.4) and persistent left superior caval vein (OR: 5.02, 95% CI: 1.93-11.7). We showed the possibility that minor cardiac variants, such as bicuspid aortic valve and persistent left superior caval vein, contribute to higher prevalence of coronary anomalies in patients with ventricular septal defect.

Biochemical Characterization of Parsley Glycosyltransferases Involved in the Biosynthesis of a Flavonoid Glycoside, Apiin.

The flavonoid glycoside apiin (apigenin 7-O-[ß-D-apiosyl-(1→2)-ß-D-glucoside]) is abundant in apiaceous and asteraceous plants, including celery and parsley. Although several enzymes involved in apiin biosynthesis have been identified in celery, many of the enzymes in parsley (Petroselinum crispum) have not been identified. In this study, we identified parsley genes encoding the glucosyltransferase, PcGlcT, and the apiosyltransferase, PcApiT, that catalyze the glycosylation steps of apiin biosynthesis. Their substrate specificities showed that they were involved in the biosynthesis of some flavonoid 7-O-apiosylglucosides, including apiin. The expression profiles of PcGlcT and PcApiT were closely correlated with the accumulation of flavonoid 7-O-apiosylglucosides in parsley organs and developmental stages. These findings support the idea that PcGlcT and PcApiT are involved in the biosynthesis of flavonoid 7-O-apiosylglucosides in parsley. The identification of these genes will elucidate the physiological significance of apiin and the development of apiin production methods.

Evidence for a higher-order topological insulator in a three-dimensional material built from van der Waals stacking of bismuth-halide chains.

Low-dimensional van der Waals materials have been extensively studied as a platform with which to generate quantum effects. Advancing this research, topological quantum materials with van der Waals structures are currently receiving a great deal of attention. Here, we use the concept of designing topological materials by the van der Waals stacking of quantum spin Hall insulators. Most interestingly, we find that a slight shift of inversion centre in the unit cell caused by a modification of stacking induces a transition from a trivial insulator to a higher-order topological insulator. Based on this, we present angle-resolved photoemission spectroscopy results showing that the real three-dimensional material Bi4Br4 is a higher-order topological insulator. Our demonstration that various topological states can be selected by stacking chains differently, combined with the advantages of van der Waals materials, offers a playground for engineering topologically non-trivial edge states towards future spintronics applications.

Pulmonary vascular resistance and compliance in individuals with trisomy 18.

Individuals with trisomy 18 (T18) usually have congenital heart disease, often with pulmonary hypertension, which is associated with poor outcomes. This study aimed to explore the characteristics of pulmonary circulation including pulmonary vascular resistance (Rp) and compliance (Cp) among them. We retrospectively reviewed cardiac catheterization data in subjects with T18, trisomy 21 (T21), and without chromosomal anomaly (control group) who were referred due to heart failure associated with ventricular septal defect between 2000 and 2020. Pulmonary hemodynamic parameters including Rp and Cp were compared between these groups. We studied 20 subjects with T18, 88 subjects with T21, and 240 control subjects. There was no significant difference in age (T18: 4.6 [3.0-6. 9] vs. T21: 2.8 [1.9-4.0] vs. control: 2.9 [1.6-3.2] months, p = 0.06) and mean pulmonary arterial pressure (T18: 41 [33-49] vs. T21: 35 [30-41] vs. control: 36 [28-43] mmHg, p = 0.121) between the groups. The pulmonary to systemic blood flow ratio (Qp/Qs) (p = 0.983), Rp (p = 0.449), and Cp (p = 0.195) did not differ between T18 and control groups. However, Qp/Qs and Cp in T18 group were significantly greater than that in T21 group (T18: Qp/Qs: 3.4 [2.3-5.2] vs. T: 21 2.3 [1.7-3.7], p = 0.001. Cp: 3.5 [2.3-5.5] vs. 2.3 [1.6-3.1] mmHg/mL/m2 , p = 0.007), while Rp was identical between the groups (T18: 2.0 [1.6-3.3] vs. T21: 2.3 [1.7-3.7], p = 0.386). The pulmonary circulation in T18 subjects differed from that observed in T21 subjects, and identical to that observed in control subjects. Pulmonary hypertension is expected to be normalized after reasonable corrective surgery in T18 patients with congenital heart disease.

Coronary arterial dominance in patients with congenital heart disease.

Coronary arterial dominance is concerned in the management of ischemic heart disease. In particular, right coronary arterial dominance is having a risk for three-vessel coronary artery disease. Thus, this study aimed to explore coronary arterial dominance in patients with congenital heart disease. The study involved 250 patients, of which 105 patients were with tetralogy of Fallot (TOF), 100 patients with ventricular septal defect (VSD), and 45 patients with Kawasaki disease (KD). We retrospectively reviewed their ascending aortography to determine their coronary arterial dominance, Z-scores of coronary artery diameter, and the ascending aortic curvature, which pertained to the angle between the aortic annulus plane and ascending aortic plane. We identified relevant factors that contribute to having right coronary arterial dominance. Age and weight of the 250 subjects were 2.9 (1.0-8.7) months and 7.7 (5.0-9.4) kg, respectively. The Z-scores of right coronary and anterior descending arteries significantly differed among patients with TOF, VSD, and KD (P < 0.001, P = 0.001). However, there were no significant differences in the Z-scores of left main trunk and circumflex arteries. Right coronary arterial dominance occurred in 89%, 49%, and 61% in patients with TOF, VSD, and KD, respectively (P < 0.001). The presence of TOF was the most powerful predictor for right coronary arterial dominance (odds ratio: 10.31, 95% confidence interval: 4.11-27.2, P < 0.001). We found the robust relationship between right coronary arterial dominance and TOF. Patients with TOF may have an increased risk for the development of coronary artery disease during adulthood.

Distensibility of the ductus arteriosus in neonates and young infants undergoing transcatheter closure.

Device migration is one of serious complications in neonates and infants undergoing transcatheter closure of the patent ductus arteriosus (PDA). We hypothesize that neonates and young infants possess the distensibility of the ductus, which may be related to device migration. We retrospectively reviewed angiographic findings in 41 neonates and infants who underwent transcatheter closure of PDA. We measured diameters of the ductus at the pulmonary (PA) side, the center, and the aortic (AO) side before PDA closure, and the device center diameter after device closure. The distensibility index was defined as the ratio of the device center diameter after device deployment to the diameter at the center of the ductus before PDA closure. Age and weight at the procedure were 168 (117-260) days and 5.3 (4.3-6.9) kg, respectively. Thirty-seven subjects accomplished the successful device closure, and four subjects were declined because of the device instability or migration. Implanted devices included Amplatzer Duct Occluders in 33 subjects and Amplatzer Vascular Plug-2 in 8 subjects. The PDA diameters at PA side, at the center, AO side, and the device center diameter were 3.2 (2.2-4.3) mm, 4.7 (3.6-5.7) mm, 7.7 (6.3-9.4) mm, and 5.8 (4.2-6.9) mm, respectively. The PDA diameter before closure was not correlated age and weight. The distensibility index was 1.28 (1.06-1.64), which was significantly correlated to age (r = - 0.49, P = 0.001) and weight (r = - 0.53, P < 0.001). Infants with the younger age and the lower weight have the more distensible PDA, which may be a risk for device migration.

A nonproteinogenic amino acid, ß-tyrosine, accumulates in young rice leaves via long-distance phloem transport from mature leaves.

Oryza sativa L. ssp. japonica cv. Nipponbare produces a nonproteinogenic amino acid (3R)-ß-tyrosine from l-tyrosine by tyrosine aminomutase (OsTAM1). However, physiological and ecological function(s) of ß-tyrosine have remained obscure. Often an improved understanding of metabolite localization and transport can aid in design of experiments to test physiological functions. In the current study, we investigated the distribution pattern of ß-tyrosine in rice seedlings and found that ß-tyrosine is most abundant in the youngest leaves. Based upon observations of high TAM1 activity in mature leaves, we hypothesized that ß-tyrosine is transported from mature leaves to young leaves. Patterns of predominant mature synthesis and young leaf accumulation were supported by stable isotope studies using labeled ß-tyrosine and the removal of mature leaves. Stem exudate analyses was also consistent with ß-tyrosine transport through phloem. Thus, we identify young leaves as a key target in efforts to understand the biological function(s) of ß-tyrosine in rice.

Fetal cardiac capillary hemangioma resulting in tachyarrhythmia.

We present the case of a fetus with cardiac capillary hemangioma in the right atrial cavity. The tumor showed dramatic growth between the 28th and 32nd week of gestation and resulted in tachyarrhythmia. The patient was born at the 33 weeks of gestation weighing 2430 g via urgent cesarean section because the rapidly growing cardiac tumor caused incessant tachyarrhythmia, pericardial effusion, and fetal circulatory incompetence. Coronary angiography revealed that the right coronary artery drained into the tumor. Due to hemodynamic deterioration, the patient underwent subtotal resection of the tumor on the 2nd day after birth. Histopathological examination revealed an undifferentiated capillary hemangioma. The patient was discharged at the age of 86 days, as the tachyarrhythmia and hemodynamic incompetence had subsided; however, bradycardia and intermittent atrioventricular conduction disturbance gradually developed. Capillary hemangioma, a rare primary cardiac space-occupying tumor in children, can invade the conduction system.

Ventricular Repolarization Dispersion is a Potential Risk for the Development of Life-Threatening Arrhythmia in Children with Hypertrophic Cardiomyopathy.

The risk stratification and early interventions are necessary in patients with hypertrophic cardiomyopathy (HCM), as life-threatening arrhythmia (LTA) is a leading cause of death. This study aimed to explore whether an interval between the peak of the T wave to the end terminal of the T wave (Tp-e), which represents ventricular repolarization dispersion, could predict the risk for LTA in children with HCM. We analyzed electrocardiography at the first and last visits in children (aged < 15 years) with HCM, and compared Tp-e interval and the ratio of Tp-e interval to QT interval (Tp-e/QT) between children with and without LTA. We studied 25 children with HCM. During the follow-up of 85 (38-146) months, there were 7 children with LTA. The 5-year sudden cardiac death (SCD) risk was 1.4 (1.1-2.5) %, which suggested that our cohort consisted of patients at a lower risk for SCD. Age was significantly older in children with LTA compared to those without it (12.5 vs.1.0 years, P = 0.037), although sex, the presence of family history and symptoms at diagnosis, the maximum left ventricular wall thickness Z-score did not differ between the groups. At the last electrocardiography before LTA, corrected Tp-e interval and Tp-e/QT ratio were significantly greater in patients with LTA compared to those in patients without LTA (corrected Tp-e: 103 vs. 78 ms, P = 0.020; Tp-e/QT: 0.28 vs. 0.22, P = 0.046). Corrected Tp-e and Tp-e/QT ratio cutoff values of 91 ms and 0.28 yielded as the predictors for LTA with sensitivity of 85% and 72%, specificity of 71% and 89%, respectively. Prolonged absolute and corrected Tp-e intervals and an increase in the Tp-e/QT ratio can be useful predictors for LTA in children with HCM. We offer temporal assessments of ventricular repolarization dispersion to stratify the risk for the development of LTA/SCD among children with HCM.

Long-Term Outcomes After an Individualized Strategy in Patients with Pulmonary Atresia and Intact Ventricular Septum.

This retrospective cohort study aimed to explore the long-term outcomes of an individualized strategy in patients with pulmonary atresia and intact ventricular septum (PAIVS). We analyzed survival and reintervention rates and identified risk factors for outcomes in patients with PAIVS treated based on individual right heart structures between 1979 and 2019. Ninety-five patients were included in this study. The z-scores of the pulmonary annulus, tricuspid annulus, and right ventricular end-diastolic volume were - 3.30 (- 15.15 to 1.83), - 0.70 (- 4.65 to 2.33), and - 1.51 (- 6.35 to 1.18), respectively. Right ventricular-dependent coronary circulation occurred in 15% of the patients. Among the 63 patients attempting biventricular strategy at first, 55 patients achieved biventricular circulation, 3 patients had one-and-a-half circulation, and 4 patients died perioperatively. Among the 33 patients attempting univentricular strategy at first, 10 patients died before the completion of Fontan operation, 17 patients (48%) accomplished Fontan operation, and 5 patients waited for Fontan operation. In one patient, conversion to biventricular circulation occurred. During the follow-up period of 720 person-years, the 20-year survival rate was significantly higher in patients with biventricular circulation than in those patients with univentricular circulation (93% vs. 67%, P < 0.001). Freedom from reintervention rates at 20 years was significantly lower in patients with biventricular circulation than in those patients with univentricular circulation (29% vs. 72%, P < 0.001). The pulmonary annulus z-score was an independent risk factor for reintervention in patients with biventricular circulation. Patients with biventricular circulation had an acceptable survival rate, but a high reintervention rate. Meanwhile, patients with univentricular circulation had high mortality before the completion of Fontan operation, although the reintervention rate was relatively low.

Localised laccase activity modulates distribution of lignin polymers in gymnosperm compression wood.

The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression-wood-specific S2 L cell wall layer with lignins highly enriched with p-hydroxyphenyl (H)-type units derived from H-type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)-type units from G-type monolignol with a trace amount of H-type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H-type and G-type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa). We performed a series of chemical-probe-aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases. Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H-type and G-type monolignols were precisely localised to distinct cell wall layers in which H-type and G-type lignin units were preferentially produced during the development of compression wood tracheids. We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements.

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3ß3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three ß-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two ß-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αß-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of ß and the loosening/tightening motions at the αß-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

MAIN CONCLUSION: Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly. ABBREVIATIONS: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II.

High-Frequency Heating Extraction Method for Sensitive Drug Analysis in Human Nails.

Background: A simple, sensitive, and rapid extraction method based on high-frequency (H-F) heating was developed for drug analysis in human nails. Methods: A human nail was placed in a glass tube with an extraction solvent (methanol and 0.1% formic acid; 7:3, v/v), and a ferromagnetic alloy (pyrofoil) was wrapped in a spiral around the glass tube. Then, the glass tube was placed in a Curie point pyrolyzer, and a H-F alternating voltage (600 kHz) was applied. The sample and extraction solvent were heated at the Curie temperature for 3 min. Different Curie temperatures were applied by changing the pyrofoil (160 °C, 170 °C, 220 °C, and 255 °C). Results: The caffeine in the nail was effectively and rapidly extracted into the extraction solvent with the pyrofoil at 220 °C. The peak area obtained for the caffeine using liquid chromatography mass spectrometry (LC-MS/MS) was five times that of what was obtained after conventional ultrasonic irradiation extraction. Because the extraction uses high-pressure and high-temperature conditions in a test tube, the drugs that were strongly incorporated in nails could be extracted into the solvent. The amount of caffeine extracted was independent of the size of the pieces in the sample. Conclusions: Therefore, the sensitive determination of target drugs in nails is possible with rapid (20 min, including H-F extraction for 3 min) and simple sample preparation. The developed method was applied to a nail from a patient with hypertension.

Immunocytochemical detection of rhamnogalacturonan II on forming cell plates in cultured tobacco cells.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.

Involvement of a proton-coupled organic cation antiporter in the blood-brain barrier transport of amantadine.

The blood-to-brain transport of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, has been shown previously to participate in the cationic drug-sensitive transport system across the mouse blood-brain barrier (BBB). The purpose of the present study was to characterize the influx transport system by means of both an in situ mouse brain perfusion technique and in vitro studies using rat immortalized brain capillary endothelial cells (GPNT). The observed concentration-dependent initial uptake rate of [(3) H]amantadine suggested the involvement of a carrier-mediated transport mechanism. The normal uptake at physiological pH 7.4 was decreased by 72.9% in acidic perfusate, while it was increased by 35.3% in alkaline perfusate. These results suggest that pH-dependent transport is regulated by utilizing an oppositely directed proton gradient as a driving force. In addition, the [(3) H]amantadine uptake was moderately inhibited by the adamantane structural analogs (rimantadine and memantine) and other cationic drugs (pyrilamine, clonidine, nicotine, etc.), but not by substrates or inhibitors of the well-characterized organic cation transporters (tetraethylammonium, l-carnitine and choline). A similar inhibition pattern was observed between the in vivo studies and the in vitro experiments. These results indicate that the influx transport for amantadine across the BBB involves a proton-coupled organic cation antiporter. Copyright © 2016 John Wiley & Sons, Ltd.