A novel E4BP4 element drives circadian expression of mPeriod2

Period2 (Per2) is an essential component of the mammalian clock mechanism and robust circadian expression of Per2 is essential for the maintenance of circadian rhythms. Although recent studies have shown that the circadian E2 enhancer (a non-canonical E-box) accounts for most of the circadian transcriptional drive of mPer2, little is known about the other cis-elements of mPer2 oscillatory transcription. Here, we examined the contribution of E4BP4 to Per2 mRNA oscillation in the cell-autonomous clock. Knockdown experiments of E4BP4 in both Northern blots and real-time luciferase assays suggested that endogenous E4BP4 negatively regulates Per2 mRNA oscillation. Sequence analysis revealed two putative E4BP4-binding sites (termed A-site and B-site) on mammalian Per2 promoter regions. Luciferase assays with mutant constructs showed that a novel E4BP4-binding site (B-site) is responsible for E4BP4-mediated transcriptional repression of Per2. Furthermore, chromatin immunoprecipitation assays in vivo showed that the peak of E4BP4 binding to the B-site on the Per2 promoter almost matched the trough of Per2 mRNA expression. Importantly, real-time luciferase assays showed that the B-site in addition to the E2 enhancer is required for robust circadian expression of Per2 in the cell-autonomous clock. These findings indicated that E4BP4 is required for the negative regulation of mammalian circadian clocks

Predominant accumulation of a 3-hydroxy-γ-decalactone in the male rectal gland complex of the Japanese orange fly, Bactrocera tsuneonis

The Japanese orange fly, Bactrocera tsuneonis, infests various citrus crops. While male pheromone components accumulated in the rectal glands are well characterized for Bactrocera, but information regarding the chemical factors involved in the life cycles of B. tsuneonis remains scarce. Herein, several volatile chemicals including a γ-decalactone, (3R, 4R)-3-hydroxy-4-decanolide [(3R, 4R)-HD], were identified as major components, along with acetamide and spiroketals as minor components in the rectal gland complexes of male B. tsuneonis flies. The lactone (3R, 4R)-HD was also identified in female rectal gland complexes. The amount of this compound in mature males was significantly higher than those observed in females and immature males. The lactone (3R, 4R)-HD was detected in flies fed with sucrose only, indicating that this lactone is not derived from dietary sources during adulthood, but biosynthesized in vivo. The predominant accumulation of (3R, 4R)-HD in mature males also suggests a possible role in reproductive behavior

Bulk motion-independent analyses of water diffusion changes in the brain during the cardiac cycle

金沢大学医薬保健研究域保健学系We evaluated dynamic changes in water diffusion in the brain during the cardiac cycle by using cine diffusion MRI. On a 1.5-T MRI, ECG-triggered single-shot diffusion echo planar imaging was used with sensitivity encoding, halfscan, and rectangular field of view techniques for minimizing bulk motion effects such as brain pulsation, with a data-sampling window of 3 ms. The apparent diffusion coefficient (ADC) and fractional anisotropy (FA) in the white matter zone were determined in ten healthy volunteers and then compared with the intracranial volume change (ICVC) revealed by phase-contrast cine MRI during the cardiac cycle. In addition, a frequency analysis of these waveforms was performed. ADC and FA values changed significantly during the cardiac cycle, despite minimizing the effect of bulk motion, i.e., independent of bulk motion. The ADC was synchronized with the ICVC during the cardiac cycle. A significant positive correlation was noted among their amplitudes. Analysis of the dynamic change of water diffusion by use of cine diffusion MRI facilitates the assessment of intracranial conditions. © 2009 Japanese Society of Radiological Technology and Japan Society of Medical Physics

Application of the sliding window method and Mask-RCNN method to nuclear recognition in oral cytology

Background: We aimed to develop an artificial intelligence (AI)-assisted oral cytology method, similar to cervical cytology. We focused on the detection of cell nuclei because the ratio of cell nuclei to cytoplasm increases with increasing cell malignancy. As an initial step in the development of AI-assisted cytology, we investigated two methods for the automatic detection of cell nuclei in blue-stained cells in cytopreparation images.Methods: We evaluated the usefulness of the sliding window method (SWM) and mask region-based convolutional neural network (Mask-RCNN) in identifying the cell nuclei in oral cytopreparation images. Thirty cases of liquid-based oral cytology were analyzed. First, we performed the SWM by dividing each image into 96 × 96 pixels. Overall, 591 images with or without blue-stained cell nuclei were prepared as the training data and 197 as the test data (total: 1,576 images). Next, we performed the Mask-RCNN by preparing 130 images of Class II and III lesions and creating mask images showing cell regions based on these images.Results: Using the SWM method, the highest detection rate for blue-stained cells in the evaluation group was 0.9314. For Mask-RCNN, 37 cell nuclei were identified, and 1 cell nucleus was identified as a non-nucleus after 40 epochs (error rate:0.027).Conclusions: Mask-RCNN is more accurate than SWM in identifying the cell nuclei. If the blue-stained cell nuclei can be correctly identified automatically, the entire cell morphology can be grasped faster, and the diagnostic performance of cytology can be improved

Flecainide reduces ventricular arrhythmias via a mechanism that differs from that of β-blockers in catecholaminergic polymorphic ventricular tachycardia

AbstractBackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by episodic ventricular tachycardia induced by adrenergic stress. Although β-blockers are used as first-line therapy, their therapeutic effects are largely incomplete. Flecainide has recently been shown to modify the molecular defects in CPVT. The aim of this study was to investigate the effects of flecainide as an add-on to conventional therapy on exercise-induced ventricular arrhythmia and compare them with those of conventional therapy alone.MethodsThe study included 5 CPVT patients with a mutation in RYR2. They experienced episodic arrhythmic events despite conventional β-blocker therapy and were therefore given flecainide in addition. The effects of the addition of flecainide therapy on ventricular arrhythmia during exercise testing were compared with those of conventional therapy alone.ResultsBoth β-blockers alone and with additional flecainide increased the maximal workload attained at the onset of ventricular arrhythmia; however, only flecainide increased the sinus rate at the onset of ventricular arrhythmias. Furthermore, flecainide increased the exercise capacity by preventing exercise-induced arrhythmias. During a follow-up period of 17±2 months, 1 patient experienced recurrent arrhythmic episodes that were associated with noncompliance. All patients reported improvements in their ability to perform the activities of daily living.ConclusionFlecainide effectively reduced ventricular arrhythmias via a mechanism that differs from that of β-blockers in genotype-positive patients with CPVT. The specific effects of flecainide may be critical in the improvement noted in the patients' ability to perform daily activities

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination

Recently, it was reported that 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-β-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-β-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-β-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-β-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-β-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner

Postoperative supraventricular tachycardia and polymorphic ventricular tachycardia due to a novel SCN5A variant: a case report of a rare comorbidity that is difficult to diagnose.

Background:Loss-of-function mutations of human cardiac sodium channel gene SCN5A induce a wide range of arrhythmic disorders. Mutation carriers with co-existing conditions such as congenital heart diseases and histories of cardiac surgeries, could develop complex arrhythmic events that are difficult to diagnose.Case presentation:A 41-year-old Japanese male with a history of a surgical closure of an ASD presented impairment of consciousness by wide QRS tachycardia. Because the patient\u27s baseline ECG in sinus rhythm showed similar QRS axis with right bundle brunch block morphology, we suspected supraventricular tachycardia (SVT). During hospitalization, the patient developed polymorphic ventricular tachycardia that was induced by bradycardia. In an electrophysiological study, the SVT was identified as right atrial incisional tachycardia circulating around the scar in the right atrium. The genetic analysis revealed a heterozygous SCN5A c.4037-4038 del TC, p. L1346HfsX38 variant. We diagnosed this patient as having progressive cardiac conduction disorder (PCCD) and polymorphic VT caused by the mutation. Incisional tachycardia with wide QRS morphology was a by-standing comorbidity related to the history of cardiac surgery which could miss lead the diagnosis. The patient\u27s SVT was eliminated by radiofrequency catheter ablation. An implantable cardioverter defibrillator (ICD) was implanted for the secondary prevention of polymorphic VT. Cardiac pace-making therapy by the ICD to avoid bradycardia effectively suppressed the patient\u27s arrhythmic events.Conclusions:We treated a patient with a sodium channel gene variant. Co-existing SVT originated by a scar in the right atrium made the diagnosis extremely difficult. A multilateral diagnostic approach using an ECG analysis, an electrophysiological study, and genetic screening enabled effective combination therapy comprised of catheter ablation and an ICD

A multicenter randomized controlled trial to evaluate the efficacy and safety of nelfinavir in patients with mild COVID-19

Nelfinavir, an orally administered inhibitor of human immunodeficiency virus protease, inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. We conducted a randomized controlled trial to evaluate the clinical efficacy and safety of nelfinavir in patients with SARS-CoV-2 infection. We included unvaccinated asymptomatic or mildly symptomatic adult patients who tested positive for SARS-CoV-2 infection within 3 days before enrollment. The patients were randomly assigned (1:1) to receive oral nelfinavir (750 mg; thrice daily for 14 days) combined with standard-of-care or standard-of-care alone. The primary endpoint was the time to viral clearance, confirmed using quantitative reverse-transcription PCR by assessors blinded to the assigned treatment. A total of 123 patients (63 in the nelfinavir group and 60 in the control group) were included. The median time to viral clearance was 8.0 (95% confidence interval [CI], 7.0 to 12.0) days in the nelfinavir group and 8.0 (95% CI, 7.0 to 10.0) days in the control group, with no significant difference between the treatment groups (hazard ratio, 0.815; 95% CI, 0.563 to 1.182; P = 0.1870). Adverse events were reported in 47 (74.6%) and 20 (33.3%) patients in the nelfinavir and control groups, respectively. The most common adverse event in the nelfinavir group was diarrhea (49.2%). Nelfinavir did not reduce the time to viral clearance in this setting. Our findings indicate that nelfinavir should not be recommended in asymptomatic or mildly symptomatic patients infected with SARS-CoV-2. The study is registered with the Japan Registry of Clinical Trials (jRCT2071200023). IMPORTANCE The anti-HIV drug nelfinavir suppresses the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. However, its efficacy in patients with COVID-19 has not been studied. We conducted a multicenter, randomized controlled trial to evaluate the efficacy and safety of orally administered nelfinavir in patients with asymptomatic or mildly symptomatic COVID-19. Compared to standard-of-care alone, nelfinavir (750 mg, thrice daily) did not reduce the time to viral clearance, viral load, or the time to resolution of symptoms. More patients had adverse events in the nelfinavir group than in the control group (74.6% [47/63 patients] versus 33.3% [20/60 patients]). Our clinical study provides evidence that nelfinavir, despite its antiviral effects on SARS-CoV-2 in vitro, should not be recommended for the treatment of patients with COVID-19 having no or mild symptoms

Percutaneous coronary intervention using new-generation drug-eluting stents versus coronary arterial bypass grafting in stable patients with multi-vessel coronary artery disease: From the CREDO-Kyoto PCI/CABG registry Cohort-3

AIMS: There is a scarcity of studies comparing percutaneous coronary intervention (PCI) using new-generation drug-eluting stents (DES) with coronary artery bypass grafting (CABG) in patients with multi-vessel coronary artery disease. METHODS AND RESULTS: The CREDO-Kyoto PCI/CABG registry Cohort-3 enrolled 14927 consecutive patients who underwent first coronary revascularization with PCI or isolated CABG between January 2011 and December 2013. The current study population consisted of 2464 patients who underwent multi-vessel coronary revascularization including revascularization of left anterior descending coronary artery (LAD) either with PCI using new-generation DES (N = 1565), or with CABG (N = 899). Patients in the PCI group were older and more often had severe frailty, but had less complex coronary anatomy, and less complete revascularization than those in the CABG group. Cumulative 5-year incidence of a composite of all-cause death, myocardial infarction or stroke was not significantly different between the 2 groups (25.0% versus 21.5%, P = 0.15). However, after adjusting confounders, the excess risk of PCI relative to CABG turned to be significant for the composite endpoint (HR 1.27, 95%CI 1.04-1.55, P = 0.02). PCI as compared with CABG was associated with comparable adjusted risk for all-cause death (HR 1.22, 95%CI 0.96-1.55, P = 0.11), and stroke (HR 1.17, 95%CI 0.79-1.73, P = 0.44), but with excess adjusted risk for myocardial infarction (HR 1.58, 95%CI 1.05-2.39, P = 0.03), and any coronary revascularization (HR 2.66, 95%CI 2.06-3.43, P<0.0001). CONCLUSIONS: In this observational study, PCI with new-generation DES as compared with CABG was associated with excess long-term risk for major cardiovascular events in patients who underwent multi-vessel coronary revascularization including LAD