Impacts of dosing and drug withdrawal period on tacrolimus-based triple therapy in a non-human primate renal transplantation model.

Non-human primate (NHP) renal transplantation models are widely used vivo models for researching new immunosuppressive therapies including allograft tolerance strategies. To enroll animals into a tolerance study, an immunosuppressive regimen that efficiently establishes stable renal function in NHPs is needed. Here, we assessed the effect of triple therapy comprising 2.0 mg/kg tacrolimus, mycophenolate mofetil and a steroid and its success rate for achieving stable renal function. In addition, to predict the pathophysiological consequences of withdrawing immunosuppressants, an indispensable process after induction of tolerance, we also assessed changes in the stable renal state maintained by triple therapy after drug withdrawal. Six cynomolgus monkeys were used. The median survival time was >176 days over the dosing period and 45 days after drug withdrawal. The triple therapy successfully induced stable graft function without calcineurin inhibitor nephrotoxicity in three of six recipients, although adopting trough-dependent tacrolimus dose adjustment rather than a preset dose regimen could improve on the present strategy. Further, drug withdrawal led to deterioration of renal function, de novo donor specific antibody production and increased the memory/naïve T cell ratio within two weeks post drug withdrawal. We expect that these findings contribute to establish one of the choices for animal model for evaluating future tolerance therapy for renal transplantation.

Unique left pulmonary vein isolation in straight common trunk based on longitudinal conduction of left lateral ridge.

BACKGROUND: A left common pulmonary vein (LCPV) is the most common anatomical variation in the pulmonary vein (PV) and often influences strategies of PV isolation for atrial fibrillation (AF). Our objective was to elucidate the electrical properties of the specific shape of LCPV and to apply it to an ablation procedure. METHODS AND RESULTS: We investigated consecutive 12 out of 204 paroxysmal AF patients who had the shape of a straight common trunk in LCPV defined by the formation of a single conduit with parallel cranial and caudal walls after the coalescence of superior and inferior PVs on the distal side. The distance between the top of the bifurcation of LPVs and the level coinciding with the middle of the anterior wall of LCPV (left lateral ridge: LLR) was more than 10 mm in all the patients. The activation pattern of the LLR showed longitudinal conduction without outside connections. All the LCPV except one were successfully isolated without ablating the LLR (C-shape ablation). Only one patient had AF recurrence during the follow-up period. CONCLUSION: The LLR in LCPV with a straight common trunk has longitudinal conduction without outside connections, which permits the isolation of LCPV without ablating LLR.

Different characteristics of postoperative atrial tachyarrhythmias between congenital and non-congenital heart disease.

PURPOSE: The chance of encountering tachyarrhythmias has been increasing in adult congenital heart disease (CHD) patients with previous open-heart surgery, along with the improvement of their longevity. However, the characteristics of these arrhythmias remain to be elucidated. METHODS: We examined the characteristics of atrial tachyarrhythmias (ATs) in 26 consecutive CHD patients (M/F 17/9) referred for catheter ablation and compared them with 16 non-CHD patients with cardiac surgery (M/F 11/5). RESULTS: The CHD group was younger and had a longer period from cardiac surgery until the occurrence of ATs compared with the non-CHD group (44.8 ± 19.5 vs. 67.6 ± 12.5 years old, and 23.3 ± 13.2 vs. 6.3 ± 4.9 years, respectively, both P < 0.05). Multiple ATs were equally induced in both groups, 12 in CHD (46.1%) and 5 in non-CHD (31.3%). Although the prevalence of macro-reentrant ATs (cavo-tricuspid isthmus-dependent atrial flutter (AFL) or intra-atrial reentrant tachycardia (IART)) was comparable, the mechanisms were different between the 2 groups (AFL and IART), 34% and 27% in CHD and 71% and 24% in non-CHD, respectively. Furthermore, focal AT (FAT) was noted in 9 patients (34.6%) in CHD but none in non-CHD (P < 0.05). Electroanatomical mapping showed that the surface area and low-voltage area (LVA) of the right atrium were significantly larger in CHD than in non-CHD (197.1 ± 56.4 vs. 132.4 ± 41.2 cm2, and 40.8 ± 33.3 vs. 13.6 ± 9.0 cm2, respectively, both P < 0.05). Ten out of 14 FATs (71.4%) were highly associated with LVA, especially near the crista terminalis. CONCLUSIONS: The development of ATs in CHD patients could be associated with large atrial remodeling, resulting in complicated ATs.

Synthesis of Uracil-Iodonium(III) Salts for Practical Utilization as Nucleobase Synthetic Modules.

Iodonium(III) salts bearing uracil moieties have recently appeared in the literature, but their structural scope and utilization are limited because of their hygroscopic characteristics. In this study, we describe our detailed investigations for synthesizing a series of uracil iodonium(III) salts derived with various structural motifs and counterions. These new compounds have been utilized as attractive synthetic modules in constructing functionalized nucleobase and nucleosides.

Characteristics of ventricular tachycardia and long-term treatment outcome in patients with dilated cardiomyopathy complicated by lamin A/C gene mutations.

BACKGROUND: Dilated cardiomyopathy caused by lamin A/C gene (LMNA) mutation is complicated with atrioventricular (AV) conduction disturbances, malignant ventricular arrhythmias, and progressive severe heart failure. Radiofrequency catheter ablation (RFCA) of ventricular tachycardia (VT) has been reported to be challenging due to the high recurrence rate in patients with LMNA-related cardiomyopathy. However, electrophysiological and histopathological characteristics of VT substrate remain to be fully elucidated. METHODS AND RESULTS: We experienced 6 familial patients with LMNA-related cardiomyopathy in 3 pedigrees (6 males, 43.7±4.5 [SD] years). All patients had first VT attack at 50±6.6 [SD] years of age, and 4 underwent RFCA for incessant VT. Their electrocardiograms during VT showed similar QRS morphologies, characterized by an inferior axis, SR pattern in aVR, and QS pattern in aVL, suggesting the origin of the basal anterior ventricle. Indeed, the VTs had multiple exits around the basal anterior ventricular septum in all RFCA cases. Although we performed multiple RFCA procedures including epicardial ablation and surgical cryoablation, all cases experienced VT recurrences in 4.5±6.4 [SD] months after last procedure. All patients developed end-stage heart failure with frequent VT events, and died at 59.5±3.6 years of age (severe heart failure in 5 and lung disease in 1). In three autopsy cases with RFCA, fibrofatty degeneration was noted in the AV node. In addition, in the deep basal ventricular septum, inhomogenous fibrotic degenerated tissue was noted beyond the reach of RF lesions. CONCLUSIONS: These results demonstrate that patients with LMNA-related cardiomyopathy are characterized by VTs refractory to RFCA probably because of the deep intramural focus at the basal ventricular septum, resulting in poor prognosis with progressive severe heart failure despite all available optimized therapies. Thus, we should consider heart transplantation in their early 50s when several VT events begin to occur.

Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice.

It is uncertain which ß4-galactosyltransferase (ß4GalT; gene name, B4galt), ß4GalT-5 and/or ß4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout (B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Time Course and Factors Correlating With Ventricular Tachyarrhythmias After Introduction of Steroid Therapy in Cardiac Sarcoidosis.

BACKGROUND: The time course and factors correlating with ventricular tachyarrhythmias (VTs) after introduction of corticosteroid therapy in patients with cardiac sarcoidosis remain to be elucidated. METHODS AND RESULTS: We examined 68 consecutive patients with cardiac sarcoidosis in the Tohoku University Hospital from October 1998 to September 2014 (age: 57±11 years old; male:female 18:50) and evaluated VTs after initiation of steroid therapy. VTs were defined as documented ventricular tachycardia or ventricular fibrillation lasting for more than 30 seconds or resulting in cardiovascular collapse, or appropriate implantable cardioverter defibrillator therapy. During a mean follow-up of 5.5 years, 20 out of 68 patients (29%) experienced VTs after initiation of corticosteroid therapy, especially in the first 12 months in 14 patients (70%). A multivariable analysis revealed that positive gallium scintigraphy had a significant correlation with VTs (hazard ratio, 11.33; 95% confidence interval, 3.22-39.92; P<0.001), in addition to reduced left ventricular ejection fraction (hazard ratio, 0.94; 95% confidence interval, 0.90-0.97; P=0.001). Furthermore, electrical storm was noted in 10 patients (14.7%), 8 within the first 12 months of treatment, whereas the recurrence of electric storm was relatively less. CONCLUSIONS: These results indicate that VTs and electric storm frequently occur in the first 12 months after initiation of corticosteroid therapy, presumably because of inflammatory conditions, and that the positive gallium scintigraphy is a significant and independent predictor of VTs. The present findings may be useful to further improve the management of VTs in patients with cardiac sarcoidosis.

Effect of Respiratory Therapy on the Prognosis of Chronic Heart Failure Patients Complicated With Sleep-Disordered Breathing　- A Pilot Efficacy Trial.

BACKGROUND: Sleep-disordered breathing (SDB) has been reported to influence mortality and occurrence of ventricular tachyarrhythmia in patients with chronic heart failure (CHF). It remains to be elucidated, however, whether respiratory therapy (RT) can affect the occurrence of fatal ventricular tachyarrhythmia in CHF patients with SDB. METHODS AND RESULTS: We prospectively examined whether the severity of SDB was associated with fatal cardiac events in CHF patients and, if so, whether RT for SDB improved prognosis. We enrolled 95 patients with stable CHF, in whom SDB was examined on overnight polygraphy. The severity of SDB was quantified using the apnea-hypopnea index (AHI). All patients with AHI ≥10 (n=42) at initial evaluation were recommended to have RT, such as home oxygen therapy and continuous positive airway pressure, and 24 agreed to this. During the follow-up period of 29±17 months, 8 ventricular tachyarrhythmias occurred and 14 of the 95 patients died. On multivariate proportional hazard analysis AHI ≥5 was a risk factor for fatal arrhythmic events (P=0.026). Although RT significantly reduced AHI, it did not significantly reduce the event rates, but 4 patients with AHI <5 on RT had no fatal arrhythmic events or death. CONCLUSIONS: SDB is an independent prognostic factor and thus an important therapeutic target in CHF patients.

Current status of primary prevention of sudden cardiac death with implantable cardioverter defibrillator in patients with chronic heart failure--a report from the CHART-2 Study.

BACKGROUND: The current status of primary prevention of sudden cardiac death (SCD) with implantable cardioverter defibrillator (ICD) in patients with heart failure with reduced ejection fraction remains to be fully elucidated in Japan. METHODS AND RESULTS: In the chronic heart failure (CHF) cohort study, the CHART-2 Study, we enrolled 2,778 consecutive patients with NYHA class II-III. According to the Japanese Circulation Society guideline of prophylactic ICD, we divided them into 3 groups: group A, class I indication; B, class IIa; and C, no indication. During the (median) 3.2-year follow-up, 79 fatal arrhythmic events (FAE), defined as composite of sudden cardiac/arrhythmic death, ventricular tachycardia/fibrillation and appropriate ICD therapy, occurred. In the groups A, B and C, the prevalence of FAE was 16.1%, 8.9% and 1.9%, respectively; the use of prophylactic ICD among those with FAE, however, was only 44%, 9% and 6%, respectively. In the groups A and B combined, chronic atrial fibrillation (cAF) and left ventricular end-diastolic dimension (LVDd) ≥ 65 mm were independent predictors of FAE, and, when combined, their prognostic impact was highly significant (hazard ratio, 7.01; P<0.001). CONCLUSIONS: Primary prevention of SCD with ICD in CHF patients is validated but is still underused in Japan, and the combination of cAF and LVDd ≥ 65 mm may be a useful indication of prophylactic ICD implantation.

Characteristics of heart failure associated with the Great East Japan Earthquake.

BACKGROUND: On March 11, 2011, the Tohoku district was struck by the most powerful known earthquake to hit Japan. Although stress-induced heart diseases rise after strong psychosocial stress, little is known about the characteristics of heart failure (HF) caused by psychosocial stress related to earthquakes. METHODS: We examined patients admitted to our hospital for HF during a three-week period between March 11 and March 31, 2011 (Disaster group) and compared them to patients during the corresponding period of 2010 (Non-Disaster group). RESULTS: The number of patients was larger in the Disaster group (n=30, 18 men, 12 women; mean age 77.3±9.8 years) than in the Non-Disaster group (n=16, 8 men, 8 women; mean age 77.3±11.6 years). A total of 14 of 30 patients (46.7%) in the Disaster group did not have past history of admission for HF, compared to 2 patients (12.5%) in the Non-Disaster group (p=0.02). The number of patients with hypertension was larger in the Disaster group than in the Non-Disaster group (53.3% vs. 37.5%, p=0.04). The number of patients with atrial fibrillation was also larger in the Disaster group than in the Non-Disaster group (56.7% vs. 25.0%, p=0.03). Left ventricular systolic ejection fraction (EF) did not differ between the Disaster and Non-Disaster groups (45.2±17.8% vs. 45.6±14.0%, p=0.46), however, the proportion of patients whose EF was more than 45% were significantly higher in the Disaster group more than in the Non-Disaster group (56.7% vs. 43.8%, p=0.04). The in-hospital mortality rate for patients in the Disaster group was higher than in the Non-Disaster group (20.0% vs. 6.3%, p=0.04). CONCLUSION: The incidence and in-hospital mortality rate of HF increased after the Great East Japan Earthquake, suggesting that psychosocial stress brought on by such a disaster could lead to the development of HF with preserved EF more than that with reduced EF.

Glycopeptide identification using liquid-chromatography-compatible hot electron capture dissociation in a radio-frequency-quadrupole ion trap.

We developed a liquid chromatography (LC) compatible electron capture dissociation (ECD) mass spectrometer for glycoproteomics, with which ECD and hot ECD (HECD) experiments can be flexibly switched by quickly changing the electron energy without further tuning of the mass spectrometer. Desialylated glycopeptides were dissociated well in both ECD and HECD experiments. For sialylated glycopeptides, on the other hand, ECD with electron energy higher than 4 eV showed significantly higher sequence coverage than that with an electron energy of 0.2 eV. A nano LC system was coupled to our ECD mass spectrometer to investigate N-linked glycopeptides from lysylendopeptidase (Lys-C) digests of human transferrin. ECD spectra at multiple electron energies of 0.2, 5.0, and 9.0 eV were obtained for each targeting precursor ion in a single LC injection. Glycopeptides with a sialylated bi-, tri-, or tetra-antennary complex N-glycan were identified with high sequence coverage by HECD. Glycopeptides with tri- or tetra-antennary N-glycans have seldom been analyzed by ECD or ETD before this report. We also found that a preferential dissociation of nonreducing termini of glycans in glycopeptides by ECD and HECD.

Elucidating the sequence of intact bioactive peptides by using electron capture dissociation and hot electron capture dissociation in a linear radio-frequency quadrupole ion trap.

RATIONALE: Electron capture dissociation (ECD) is useful tool for sequencing of peptides and proteins with post-translational modifications. To increase the sequence coverage for peptides and proteins, it is important to develop ECD device with high fragmentation efficiency. METHODS: Sequence analysis of intact undigested bioactive peptides (3000-5000 Da) was performed by use of electron capture dissociation (rf-ECD) and collision-induced dissociation (CID) in a linear radio-frequency quadrupole ion trap that was coupled to a time-of-flight mass spectrometer. We applied rf-ECD, hot rf-ECD (rf-ECD with high electron energy), and CID for intact bioactive peptide ions of various charge states and evaluated the sequence coverage of their fragment spectra. RESULTS: Hot rf-ECD produced a higher number of c- and z-type fragment ions of modified peptide ions as electron energy increased in lower charged peptide ions, and sequence coverage greater than 80% was obtained compared with the CID case (40-80%). CONCLUSIONS: The result indicates that intact bioactive modified peptides (Ghrelin, ANP) were correctly identified by use of hot rf-ECD.

An efficient approach for the characterization of mucin-type glycopeptides: the effect of O-glycosylation on the conformation of synthetic mucin peptides.

Despite the growing importance of mucin core O-glycosylation in many biological processes including the protection of epithelial cell surfaces, the immune response, cell adhesion, inflammation, and tumorigenesis/metastasis, the regulation mechanism and conformational significance of the multiple introduction of α-GalNAc residues by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs) remains unclear. Here we report an efficient approach by combining MS and NMR spectroscopy that allows for the identification of O-glycosylation site(s) and the effect of O-glycosylation on the peptide backbone structures during enzymatic mucin domain assembly by using an isoform UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T2 (ppGalNAcT2) in vitro. An electron-capture dissociation device in a linear radio-frequency quadrupole ion trap (RFQ-ECD) combined with a time-of-flight (TOF) mass spectrometer was employed for the identification of Thr/Ser residues occupied by α-GalNAc branching among multiple and potential O-glycosylation sites in the tandem repeats of human mucin glycoproteins MUC4 (Thr-Ser-Ser-Ala-Ser-Thr-Gly-His-Ala-Thr-Pro-Leu-Pro-Val-Thr-Asp) and MUC5AC (Pro-Thr-Thr-Val-Gly-Ser-Thr-Thr-Val-Gly). In the present study, O-glycosylation was initiated specifically at Thr10 in naked MUC4 peptide and additional introduction of α-GalNAc proceeded preferentially but randomly at three other Thr residues to afford densely glycosylated MUC4 containing six α-GalNAc residues at Thr1, Ser2, Ser5, Thr6, Thr10, and Thr15. On the contrary, O-glycosylation of naked MUC5AC peptide occurred predominantly at consecutive Thr residues and led to MUC5AC with four α-GalNAc residues at Thr2, Thr3, Thr7, and Thr8. The solution structures determined by NMR spectroscopic studies elicited that the preferential introduction of α-GalNAc at Thr10 of MUC4 stabilizes specifically a ß-like extended backbone structure at this area, whereas other synthetic models with a single α-GalNAc residue at Thr1, Thr6, or Thr15 did not exhibit any converged three-dimensional structure at the proximal peptide moiety. Such conformational impact on the underlying peptides was proved to be remarkable in the glycosylation at the consecutive Thr residues of MUC5AC.

NBD-based green fluorescent ligands for typing of thymine-related SNPs by using an abasic site-containing probe DNA.

The binding behavior of green fluorescent ligands, derivatives of 7-nitrobenzo-2-oxa-1,3-diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N(1)-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)propane-1,3-diamine (NBD-NH(2)) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52 x 10(6) M(-1). From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD-NH(2) is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe-pteridine).

N1 component reflects difference of terminal chords in three-chord sequences.

We understand from experience that musical contexts are formed when chords are combined according to the rules of harmony. In this study, the N1 component of the auditory-evoked potential was measured using comparable three-chord sequences; these sequences were constructed as a consecutive task (cf. C-C-C vs. Cm-Cm-Cm; control) and a cadence task (cf. C-G-C vs. C-G-Cm). In the cadence task, compared with cadences ending with a major chord (anticipated chord), those ending with a minor chord (unanticipated chord) showed a significantly larger amplitude of N1 waves. These components of auditory-evoked potentials reflect the effect of chord progression in musical perception and suggest that the musical context is recognized at least 100 ms after a chord is played.

Fluorescence emission detection of single-nucleotide polymorphisms by a naphthyridine-benzofurazan conjugate.

We have successfully developed a class of ligand that exhibits a fluorescence-enhancement upon binding to pyrimidine bases opposite an AP site in DNA duplexes. The present ligand, Naph-c3-DBD, in which DBD (7-N,N-dimethylaminosulfonylbenzo-2-oxa-1,3-diazole) is connected to 2-amino-7-methyl-1,8-naphthyridine by a propylene linker, is capable of selectively binding to pyrimidine bases over purine bases, and the binding event is accompanied by a significant enhancement of emission due to the DBD moiety (emission maximum at 597 nm). The response of the ligand is almost specific to pyrimidine bases, making it possible to detect pyrimidine/purine transversion.

Fast multiple electron capture dissociation in a linear radio frequency quadrupole ion trap.

We developed a fast electron capture dissociation (ECD) device using a linear radio frequency-quadrupole (RFQ) ion trap. The device dissociated peptides and proteins using a focused electron beam with an intensity of 0.5 microA and a diameter of 1 mm. The electron capture rate was 13%/ms for doubly charged peptides, and the total amount of ECD products was identical to the theoretical limit, i.e., 50% of incident precursor ions were observed as maximum ECD products by electron irradiation of 7 ms in a pulse counting detection scheme. Coupling this ECD device to a time-of-flight mass spectrometer, we applied multiple ECD. Protonated ubiquitin precursor ions with a charge state of 10 were repeatedly cleaved by ECD, i.e., charge-reduced species and their highly charged fragments were cleaved again and again, creating lower charged products, leaving only singly to triply charged states among the final products. Meanwhile with the amount of electron irradiated, lower charged products increased. Applying an electron beam for 8 ms, we obtained 96% of the total sequence coverage using a 40 fmol sample except at three proline sites. This fast ECD device should be widely applicable to proteomics including post-translational modification analysis and top-down analysis.

Synthesis, stabilities, and redox behavior of mono-, di-, and tetracations composed of di(1-azulenyl)methylium units connected to a benzene ring by phenyl- and 2-thienylacetylene spacers. A concept of a cyanine-cyanine hybrid as a stabilized electrochromic system.

This paper describes the preparation of two tetracations 4a(4+) and 4b(4+) composed of di(1-azulenyl)methylium units based on a new structural principle of a cyanine-cyanine hybrid for the design of electrochromic materials with two color changes. Di- and monocations 5a(2+), 5b(2+) and 6a+, 6b+ composed of di(1-azulenyl)methylium units were also prepared for the purpose of comparison. The pKR+ values of the tetracations are rather high despite their tetracationic structure, although the stability of these cations decreases with the increase of the number of the existing cation units. The cyclic voltammetry (CV) of these cations revealed the presumed multielectron redox properties. However, the tetracations did not exhibit the idealized electrochemical behavior, in which subsequent two-electron reduction was presumed as the cyanine-cyanine hybrid, probably due to the less effective electrochemical interaction among the positive charges. The scope of the creation of the novel polyelectrochromic materials taking the new structural principle is demonstrated by these examples.