Search

Kenji Inoue,1 Junji Inoue,2 Shiho Kunimatsu-Sanuki,2 Norie Nozaki,3 Kosuke Shimizu,4 Kyoko Ishida,5 Goji Tomita1,5 1Inouye Eye Hospital, Chiyoda-Ku, Tokyo, Japan; 2Nishikasai Inouye Eye Hospital, Edogawa-Ku, Tokyo, Japan; 3Omiya Inouye Eye Clinic, Saitama-Shi, Saitama, Japan; 4Sapporo Inouye Eye Clinic, Sapporo-Shi, Sapporo, Japan; 5Department of Ophthalmology, Toho University Ohashi Medical Center, Meguro-ku, Tokyo, JapanCorrespondence: Kenji InoueInouye Eye Hospital, Chiyoda-Ku, Tokyo, JapanTel +81 03 3295 0911Fax +81 03 3295 0917Email 2inoue-k@inouye-eye.or.jpPurpose: To retrospectively evaluate the short-term efficacy of omidenepag isopropyl (EYBELIS 0.002%) by assessing its intraocular pressure (IOP)-lowering capability and safety in patients with normal-tension glaucoma (NTG).Patients and Methods: Fifty-four NTG patients (54 eyes) who were newly administrated with omidenepag isopropyl were enrolled in the study. The subjects comprised 22 men and 32 women, and the mean age of the subjects was 55.0 ±  14.1 years. The mean deviation value using the Humphrey visual field test program (30– 2 SITA Standard) was − 5.03 ±  3.38 dB. The following data were retrieved from the medical records and used for retrospective analyses: IOP at baseline 1– 2 months and 3– 4 months after administration. The frequency of non-responder patients who had less than 10% IOP reduction was evaluated. Patients were observed for adverse reactions and dropouts at each time point.Results: IOP at baseline, after 1– 2 months and after 3– 4 months was 15.7 ±  2.6 mmHg, 13.5 ±  2.3 mmHg, and 13.6 ±  2.4 mmHg, respectively. There was a significant decrease in IOP after administration (p< 0.0001). Eleven patients (22.4%) were non-responders. Adverse reactions occurred in 4 patients (7.4%), including conjunctival hyperemia in 3 patients (after 1 week, 2 weeks, and 1 month, respectively) and eye pain in 1 patient (after 1 month). Five patients (9.3%) dropped out of the study because of an adverse reaction in 3 patients, insufficient IOP reduction in 1 patient, and discontinuation of follow-up of 1 patient at our institution.Conclusion: After administration of omidenepag isopropyl, IOP in patients with NTG was significantly decreased. However, adverse reactions occurred in 7.4% of patients.Keywords: intraocular pressure, adverse reactions, EP2 receptor agonist, conjunctival hyperemia

Background: BRAK/CXCL14 is a chemokine expressed in many normal cells and tissues. Several papers have reported tissue distribution, but no study has examined the serum protein level of this chemokine. Methods: We evaluated serum levels of BRAK/CXCL14 by enzyme-linked immunosorbent assay, after determining the conditions necessary to increase the sensitivity and reproducibility of the method. Results: The average level of males was significantly higher than that of females. Two percent of the volunteers showed a value 2 S.D. higher than the average value without any obvious abnormality, but no person was lower than 1 S.D. of the average value. The serum BRAK/CXCL14 levels of individual subjects were constant. Conclusion: These data indicate that the serum BRAK/CXCL14 level is highly conserved and suggest that the levels are determined by the genetic background and/or lifestyle of the individual. [ABSTRACT FROM AUTHOR]

We investigated muscle strains that occurred in a team from the Japan Professional Football League. Numbers of muscle strains tended to rise in proportion to the increase in the number of matches played. Many muscle strains occurred by eccentric force. Proximal hamstring tendon injuries happened in hip flexion and knee extension position. From the MRI findings and the actual period of returning to training in each case, we learned to gauge the approximate period of returning to training. The cases of bleeding in the muscle or between the muscles or on the muscle fascia returned to training from several days to within 2 weeks. The cases of injury of the musculotendinous junction returned to training from 4 weeks to 3 months. The cases of injury of the aponeurosis of the semimembranous muscle returned to training from 4 to 10 weeks. And the cases of tendon rupture required at least 4 months after surgery. Prevention of muscle strains is becoming an increasingly important consideration for athletes. [ABSTRACT FROM AUTHOR]

Photobrightening of photodarkened CdS- and CdS xSe1 − x-doped glasses was investigated using luminescence and electron spin resonance (ESR). Photobrightening depends on wavelength of laser light, and it is observed in all samples investigated, when wavelength of laser light is longer than that of absorption edge. Photobrightening depends on glass composition. [ABSTRACT FROM AUTHOR]

To prevent osteoporosis, which is expected to increase in incidence in this rapidly aging society, in recent years bone mineral density (BMD) has frequently been measured as a predisposition index. However, these measurements are made on different sites with different apparatus, and the results are independently studied by different institutions. In our present investigation, to establish the standard radius BMD as determined by dual-energy X-ray absorptiometry (DXA), we carried out a general population survey in 29 municipalities and prefectures on 11,252 locally residing females aged 15 to 83 years (mean, 35.61 +/- 12.85 years). Their YAM (young adult mean) BMD was estimated at 0.664 +/- 0.054 g/cm2, which was almost the same as the figure given in the 1996 version of the diagnostic criteria for primary osteoporosis. We further studied the relationships of BMD to age and physical factors known to be influential to BMD. It was found that BMD was correlated negatively to age and positively to body mass index (BMI). The average values we obtained for age and physique groups appeared to have provided reliable indices for the primary prevention of osteoporosis. [ABSTRACT FROM AUTHOR]

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Mammary gland tumors (MGT) are the most common tumors in sexually intact female dogs. The functional regulation of miRNAs, a type of noncoding RNAs (ncRNAs), in canine MGT has been extensively investigated. However, the expression of other ncRNAs, such as YRNAs and transfer RNA-derived fragments (tRFs) in canine MGT is unknown. We investigated ncRNAs other than miRNAs from our small RNA project (PRJNA716131) in different canine MGT histologic subtypes. This study included benign tumors (benign mixed tumor, complex adenoma) and malignant tumors (carcinoma in benign tumor and carcinoma with metastasis) samples. Aberrantly expressed ncRNAs were examined by comparisons among MGT subtypes. The relative expression trends were validated in canine MGT tissues, plasma, extracellular vesicles, and MGT cell lines using quantitative reverse transcription PCR. Three aberrantly expressed ncRNAs were identified by comparisons among MGT subtypes. YRNA and tRNA-Gly-GCC distinguished benign mixed tumor from other MGT histologic subtypes, while tRNA-Val differentiated complex adenoma, carcinoma in benign tumors, and carcinoma with metastasis. The ROC curve of the three ncRNAs showed they might be potential biomarkers to discriminate malignant from benign MGT. YRNA and tRFs expression levels were decreased in metastatic compared with primary canine MGT cell lines. To the best of our knowledge, this is the first investigation of YRNA and tRFs in canine MGT. The three identified ncRNAs may be biomarkers for differentiating MGT histologic subtypes. Suggested Reviewers: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporatio., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

An inter-hospital heart team conference based collaborative follow-up (FU) may facilitate outpatient cardiac rehabilitation (CR) programs, especially in hospitals without an outpatient CR center. Consecutive 145 patients with cardiovascular disease who received inpatient treatment at Yamagata University Hospital were divided into collaborative (n = 76) and same-hospital (n = 69) FU groups. In the collaborative FU group, patients received outpatient care at a university hospital and outpatient CR at different hospitals. In the same-hospital FU group, patients received outpatient care and outpatient CR at the same hospital other than the university hospital. The collaborative FU group held monthly 60-minute inter-hospital heart team conferences with CR specialists. No cardiovascular accidents occurred during the outpatient CR program in either group. Peak oxygen uptake VO 2 , anaerobic threshold, brain natriuretic peptide level, and left ventricular ejection fraction significantly improved in both groups. Kaplan-Meier analysis revealed no significant difference in prognosis between the collaborative and same-hospital FU groups (P = 0.246). Of the patients who had collaborative CR programs, 29 (38.2%) patients (37 consultations) were discussed at an inter-hospital heart team conference. Eighteen (48.6%) consultations were for issues related to continuing outpatient CR programs. Collaborative FU was as useful as same-hospital FU in terms of safety, efficacy, and prognosis in patients with cardiovascular disease. We conclude that regular inter-hospital heart team conferences are useful for facilitating collaboration among outpatient CR programs.

This study aimed to investigate blood flow dynamics in the bilateral prefrontal cortex during silent and oral reading using near-infrared spectroscopy (NIRS). The subjects were 40 right-handed university students (20.5±1.8 years old, 20 men and 20 women). After completing the NIRS measurements, the subjects were asked to rate their level of proficiency in silent and oral reading, using a 5-point Likert scale. During oral reading, the left lateral prefrontal cortex (Broca's area) was significantly more active than the right side. During silent reading, prefrontal cortex activity was lower than that during oral reading, and there was no significant difference between both sides of the brain. A significant negative correlation was found between the change in oxy-hemoglobin (oxy-Hb) concentration in the left and right lateral prefrontal cortex during silent reading and silent reading speed. In addition, students with lower self-reported reading proficiency had significantly greater changes in oxy-Hb concentrations in the left and right lateral prefrontal cortex during silent/oral reading than did students with higher self-reported reading proficiency. Reading task assessment using NIRS may be useful for identifying language lateralization and Broca's area. The results demonstrate that NIRS is useful for assessing effortful reading and may be used to diagnose developmental dyslexia in children. J. Med. Invest. 71 : 92-101, February, 2024.

We examined effects of a visual search task (VST) in virtual reality (VR) with a moving background on spatial cognition and standing balance in left hemiparetic strokes. The VST with background deviation was allocated to Case A. In Case B, the VST without the deviation was performed. As a results, in Case A, the reaction time of VST was shortened in the paretic space and ability of weight-shift to the paretic side was improved. In conclusion, the VST in the VR with a spatial manipulation may improve spatial cognition and standing balance in left hemiparetic strokes.

Despite the rapid progress of organic electronics, developing high-performance n-type organic semiconductors is still challenging. Donor-acceptor (D-A) type conjugated structures have been an effective molecular design strategy to achieve chemically-stable semiconductors and the appropriate choice of the acceptor units determines the electronic properties and device performances. We have now synthesized two types of A 1 -D-A 2 -D-A 1 type conjugated molecules, namely, NDI-BTT-NDI and NDI-TBZT-NDI, with different central acceptor units. In order to investigate the effects of the central acceptor units on the charge-transporting properties, organic field-effect transistors (OFETs) were fabricated. NDI-TBZT-NDI had shallower HOMO and deeper LUMO levels than NDI-BTT-NDI. Hence, the facilitated charge injection resulted in ambipolar transistor performances with the optimized hole and electron mobilities of 0.00134 and 0.151 cm 2  V -1  s -1 , respectively. In contrast, NDI-BTT-NDI displayed only an n-channel OFET performance with the electron mobility of 0.0288 cm 2  V -1  s -1 . In addition, the device based on NDI-TBZT-NDI showed a superior air stability to that based on NDI-BTT-NDI. The difference in these OFET performances was reasonably explained by the contact resistance and film morphology. Overall, this study demonstrated that the TBZ acceptor is a promising building block to create n-type organic semiconductors., (© 2022 The Authors. Chemistry - An Asian Journal published by Wiley-VCH GmbH.)

Purpose: To retrospectively evaluate the 1-year efficacy and safety of single-agent of omidenepag isopropyl in patients with normal-tension glaucoma (NTG). Methods: One hundred patients (100 eyes) newly administered omidenepag isopropyl were enrolled. Intraocular pressure (IOP) was compared at baseline and 3, 6, 9, and 12 months after administration. The mean deviation values at baseline and 12 months measured using the Humphrey visual field test (30-2 Swedish Interactive Threshold Algorithm standard) were compared. Adverse reactions and dropouts were observed. Results: IOP significantly decreased from 15.5 ± 2.7 mmHg at baseline to 13.3 ± 2.5 mmHg after 3 months, 13.7 ± 2.3 mmHg after 6 months, 13.9 ± 2.4 mmHg after 9 months, and 13.7 ± 2.3 mmHg after 12 months ( P  < 0.0001). There was no significant difference in the mean deviation values at baseline (-3.66 ± 3.49 dB) and after 12 months (-3.41 ± 3.80 dB). Adverse reactions occurred in 9 patients (9.0%): conjunctival hyperemia ( n  = 6), eye pain ( n  = 1), iritis ( n  = 1), and blepharitis ( n  = 1). Twenty-one patients (21.0%) discontinued administration because of changes in medication ( n  = 7), interruption of visits ( n  = 5), adverse reactions ( n  = 4), and others. Conclusions: After administering omidenepag isopropyl, the IOP in patients with NTG decreased within 1 year, visual fields were maintained, and safety was satisfactory. Omidenepag isopropyl can be used as the first-line medication for patients with NTG.

Objective: Heart failure (HF) is a common and highly morbid cardiovascular disorder. Oxidative stress worsens HF, and uric acid (UA) is a useful oxidative stress marker. The novel anti-hyperuricemic drug febuxostat is a potent non-purine selective xanthine oxidase inhibitor. The present study examined the UA-lowering and prognostic effects of febuxostat in patients with HF compared with conventional allopurinol., Methods: This multicenter, randomized trial included 263 patients with chronic HF who were randomly assigned to two groups and received allopurinol or febuxostat (UA >7.0 mg/dL). All patients were followed up for 3 years after enrollment., Results: There were no significant differences in baseline clinical characteristics between the two groups. The UA level was significantly decreased after 3 years of drug administration compared with the baseline in both groups. Urine levels of the oxidative stress marker 8-hydroxy-2'-deoxyguanosine were lower in the febuxostat group than in the allopurinol group (11.0 ± 9.6 vs. 22.9 ± 15.9 ng/mL), and the rate of patients free from hospitalization due to worsening HF tended to be higher in the febuxostat group than in the allopurinol group (89.0% vs. 83.0%)., Conclusions: Febuxostat is potentially more effective than allopurinol for treating patients with chronic HF and hyperuricemia.This study was registered in the University Hospital Medical Information Network Clinical Trials Registry (https://www.umin.ac.jp/ctr/; ID: 000009817).

Background and Aim: Although tenofovir alafenamide (TAF), as well as entecavir (ETV), is widely used as first-line treatment for patients with chronic hepatitis B, there are only a few studies comparing sequential therapy from ETV to TAF and continuous ETV monotherapy in patients with maintained virologic response to ETV., Methods: In a retrospective multicenter study, we investigated the efficacy and safety of sequential therapy from ETV to TAF (ETV-TAF group) and compared them with continuous ETV monotherapy (ETV group), using propensity score matching, in chronic hepatitis B patients., Results: From 442 patients, we analyzed 142 patients from each group comprising 71 patients matched for several data, including age, HBV genotype, hepatitis B envelope antigen, cirrhosis, alanine aminotransferase, platelet count, prior ETV monotherapy period, and hepatitis B surface antigen (HBsAg) change during prior ETV monotherapy. In the ETV-TAF group, HBsAg levels significantly decreased from baseline to 48 weeks after switching to TAF (-0.02 log IU/mL, P = 0.038). HBcrAg levels also significantly decreased after switching to TAF (-0.1 log IU/mL, P = 0.004). However, there were no significant differences in the reduction of HBsAg and HBcrAg levels between the ETV-TAF and ETV groups. There was no significant difference in the change of estimated glomerular filtration rate levels from baseline to 48 weeks between the two groups., Conclusions: The present study indicated that the efficacy, especially of the HBsAg-reducing action, and safety of sequential therapy from ETV to TAF were similar to those of continuous ETV monotherapy among chronic hepatitis B patients with maintained virologic response to ETV., (© 2020 The Authors. JGH Open published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Post-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications., Competing Interests: Competing interestsN.N. is a founder of Mab Institute Inc., (© 2020. Published by The Company of Biologists Ltd.)

1,3-Diaza-2-oxophenoxazine ("phenoxazine"), a tricyclic cytosine analogue, can strongly bind to guanine moieties and improve π-π stacking effects with adjacent bases in a duplex. Phenoxazine has been widely used for improving duplex-forming abilities. In this study, we have investigated whether phenoxazine and its analogue, 1,3,9-triaza-2-oxophenoxazine (9-TAP), could improve triplex-forming abilities. A triplex-forming oligonucleotide (TFO) incorporating a phenoxazine component was found to show considerably decreased binding affinity with homopurine/homopyrimidine double-stranded DNA, so the phenoxazine system was considered not to function as either a protonated cytosine or thymine analogue. Alternatively, a 9-TAP-containing artificial nucleobase developed by us earlier as a new phenoxazine analogue functioned as a thymine analogue with respect to AT base pairs in a parallel triplex DNA motif. The fluorescence of the 9-TAP moiety was maintained even in triplex (9-TAP:AT) formation, so 9-TAP might be useful as an imaging tool for various oligonucleotide nanotechnologies requiring triplex formation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.

Metal-mediated base pairs (MMBPs) formed by natural or artificial nucleobases have recently been developed. The metal ions can be aligned linearly in a duplex by MMBP formation. The development of a three- or more-metal-coordinated MMBPs has the potential to improve the conductivity and enable the design of metal ion architectures in a duplex. This study aimed to develop artificial self-bases coordinated by three linearly aligned Ag I ions within an MMBP. Thus, artificial nucleic acids with a 1,3,9-triaza-2-oxophenoxazine (9-TAP) nucleobase were designed and synthesized. In a DNA/DNA duplex, self-base pairs of 9-TAP could form highly stable MMBPs with three Ag I ions. Nine equivalents of Ag I led to the formation of three consecutive 9-TAP self-base pairs with extremely high stability. The complex structures of 9-TAP MMBPs were determined by using electrospray ionization mass spectrometry and UV titration experiments. Highly stable self-9-TAP MMBPs with three Ag I ions are expected to be applicable to new DNA nanotechnologies., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The 2'-O,4'-C-methylene-bridged or locked nucleic acid (2',4'-BNA/LNA), with an N-type sugar conformation, effectively improves duplex-forming ability. 2',4'-BNA/LNA is widely used to improve gene knockdown in nucleic acid based therapies and is used in gene diagnosis. Metal-mediated base pairs (MMBPs), such as thymine (T)-Hg II -T and cytosine (C)-Ag I -C have been developed and used as attractive tools in DNA nanotechnology studies. This study aimed to investigate the application of 2',4'-BNA/LNA in the field of MMBPs. 2',4'-BNA/LNA with 5-methylcytosine stabilized the MMBP of C with Ag I ions. Moreover, the 2',4'-BNA/LNA sugar significantly improved the duplex-forming ability of the DNA/DNA complex, relative to that by the unmodified sugar. These results suggest that the sugar conformation is important for improving the stability of duplex-containing MMBPs. The results indicate that 2',4'-BNA/LNA can be applied not only to nucleic acid based therapies, but also to MMBP technologies., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A growing population and urbanization is a challenge for finite natural resources. Without strict regulation to recycle, recover and reuse resources, waste is discarded with no value. Every year throughout the world, more than twenty-five million vehicles turn into end-of-life vehicles (ELV) and most of their valuable resources end up in landfill sites. This research analyses the effect of regulation on ELV innovation for additional recovery of resources in Japan and EU nations using patent data as a proxy. The analysis determines the statistical difference in patent activity before and after regulations were enacted in the case studies. The relevant data on ELV technologies was gathered for the period 1985-2013. The study suggests that in general environmental regulation in Japan drove innovation and reveals that environmental policy in Japan was more effective in enabling innovation compared to EU nations. Specifically, the results from these developed countries can be used by the rapidly growing developing countries in automobile manufacturing like China for amendment of their ELV regulation accordingly., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

DNA double-strand breaks (DSBs) are repaired by either the homology-directed repair (HDR) or the non-homologous end-joining (NHEJ) pathway. RIF1 (RAP1-interacting factor homolog) was recently shown to stimulate NHEJ through an interaction with 53BP1 (p53-binding protein 1) phosphorylated at S/TQ sites, but the molecular mechanism underlying pathway choice remains unclear. Here, we show that SCAI (suppressor of cancer cell invasion) binds to 53BP1 phosphorylated at S/TP sites and facilitates HDR. Upon DNA damage, RIF1 immediately accumulates at damage sites and then gradually dissociates from 53BP1 and is subsequently replaced with SCAI. Depletion of SCAI reduces both the accumulation of HDR factors, including BRCA1 (breast cancer susceptibility gene 1), at damage sites and the efficiency of HDR, as detected by a reporter assay system. These data suggest that SCAI inhibits RIF1 function to allow BRCA1-mediated repair, which possibly includes alt-NHEJ and resection-dependent NHEJ in G1, as well as HDR in S/G2., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies.

In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Post-translational histone modifications are highly correlated with transcriptional activity, but the relative timing of these marks and their dynamic interplay during gene regulation remains controversial. To shed light on this problem and clarify the connections between histone modifications and transcription, we demonstrate how FabLEM (Fab-based Live Endogenous Modification labeling) can be used to simultaneously track histone H3 Lysine 9 acetylation (H3K9ac) together with RNA polymerase II Serine 2 and Serine 5 phosphorylation (RNAP2 Ser2ph/Ser5ph) in single living cells and their progeny. We provide a detailed description of the FabLEM methodology, including helpful tips for preparing and loading fluorescently conjugated antigen binding fragments (Fab) into cells for optimal results. We also introduce simple procedures for analyzing and visualizing FabLEM data, including color-coded scatterplots to track correlations between modifications through the cell cycle and temporal cross-correlation analysis to dissect modification dynamics. Using these methods, we find significant correlations that span cell generations, with a relatively strong correlation between H3K9ac and Ser5ph that appears to peak a few hours before mitosis and may reflect the bookmarking of genes for efficient re-initiation following mitosis. The techniques we have developed are broadly applicable and should help clarify how histone modifications dynamically contribute to gene regulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.

Inhibin-βA (INHBA), a ligand belonging to the transforming growth factor-β superfamily, is associated with cell proliferation in cancer. We studied the relations of INHBA gene expression to clinicopathological factors and outcomes in 168 patients with gastric cancer who underwent curative surgery. Relative INHBA gene expression was measured in surgical specimens of cancer tissue and adjacent normal mucosa by quantitative real-time, reverse-transcription polymerase chain reaction. INHBA expression levels were significantly higher in cancer tissue than in adjacent normal mucosa and were related to TNM stage and venous invasion. High INHBA gene expression was associated with significantly poorer 5-year overall survival than was low expression. On multivariate analysis, INHBA gene expression was an independent prognostic factor. Overexpression of the INHBA gene is considered a useful independent predictor of outcomes in patients with gastric cancer after curative surgery.

The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.

The purpose of this study was to investigate the roles of neuronal nitric oxide synthase (nNOS), Ca(2+)/calmodulin (CaM)-dependent protein kinases (CaMKs), and protein kinase C (PKC) in nicotine-induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) activation. Treatment with nicotine stimulated ERK1/2 and p38 MAPK phosphorylation in the PC12 cells expressing nNOS (NPC12 cells) as compared with that in control PC12 cells. An inhibitor of L-type voltage-sensitive Ca(2+) channel suppressed the nicotine-induced phosphorylation of p38 MAPK. The inhibition of CaMK-kinase, the upstream activator of CaMKI and CaMKIV, did not inhibit the enhanced their phosphorylation. ERK1/2 phosphorylation was attenuated by inhibitors of p38 MAPK, PKC, and MAPK-kinase 1/2, indicating the involvement of these protein kinases upstream of ERK1/2. Furthermore, we found that nNOS expression enhances the nicotine-induced increase in the intracellular concentration of Ca(2+), using the Ca(2+)-sensitive fluorescent probe Fura2. These data suggest that NO promotes nicotine-triggered Ca(2+) transient in PC12 cells to activate possibly CaMKII, leading to sequential phosphorylation of p38 MAPK and ERK1/2., (2013 Elsevier Inc. All rights reserved)

Human inactive X chromosome (Xi) forms a compact structure called the Barr body, which is enriched in repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3) and Lys27 (H3K27me3). These two histone marks are distributed in distinct domains, and X-inactive specific transcript (XIST) preferentially colocalizes with H3K27me3 domains. Here we show that Xi compaction requires HBiX1, a heterochromatin protein 1 (HP1)-binding protein, and structural maintenance of chromosomes hinge domain-containing protein 1 (SMCHD1), both of which are enriched throughout the Xi chromosome. HBiX1 localization to H3K9me3 and XIST-associated H3K27me3 (XIST-H3K27me3) domains was mediated through interactions with HP1 and SMCHD1, respectively. Furthermore, HBiX1 was required for SMCHD1 localization to H3K9me3 domains. Depletion of HBiX1 or SMCHD1, but not Polycomb repressive complex 2 (PRC2), resulted in Xi decompaction, similarly to XIST depletion. Thus, the molecular network involving HBiX1 and SMCHD1 links the H3K9me3 and XIST-H3K27me3 domains to organize the compact Xi structure.

Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.