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The production of red blood cells in vitro, which is useful for basic or clinical research, has been improved. Further optimization of culture protocols may facilitate erythroid differentiation from hematopoietic stem cells to red blood cells. However, the details of erythropoiesis, particularly regarding the behaviors of differentiation-related proteins, remain unclear. Here, we performed erythroid differentiation using two independent bone marrow- or cord blood-derived CD34+ cell sources and identified proteins showing reproducible differential expression in all groups. Notably, most of the proteins expressed at the early stage were downregulated during erythroid differentiation. However, seven proteins showed upregulated expression in both bone marrow cells and cord blood cells. These proteins included alpha-synuclein and selenium-binding protein 1, the roles of which have not been clarified in erythropoiesis. There is a possibility that these factors contribute to erythroid differentiation as they maintained a high expression level. These findings provide a foundation for further mechanistic studies on erythropoiesis.

α-Synuclein (αSyn), the major constituent of Lewy bodies and Lewy neurites, is generally expressed in presynapses and is involved in synaptic function. However, we previously demonstrated that some neurons, including those in the olfactory bulb, show high αSyn expression levels in the cell body under normal conditions. αSyn is also known to be important for adult neurogenesis. Thus, in present study, we examined the role of αSyn in juxtaglomerular neurons (JGNs) with high αSyn expression in the mouse olfactory bulb. Most αSyn-enriched JGNs expressed sex-determining region Y-box 2 (Sox2), which functions to maintain neural immature identity. Interestingly, in αSyn homozygous (-/-) knockout (KO) mice, Sox2-positive JGNs were significantly increased compared with heterozygous (+/-) KO mice. Following global brain ischemia using wild-type mice, there was also a significant decrease in Sox2-positive JGNs, and in the co-expression ratio of Sox2 in αSyn-enriched JGNs. By contrast, the co-expression ratio of neuronal nuclei (NeuN, mature neuronal marker) was significantly increased in αSyn-enriched JGNs. However, this ischemia-induced decrease of Sox2-positive JGNs was not observed in αSyn homozygous KO mice. Overall, these data suggest that αSyn functions to promote the maturation of immature JGNs in the mouse olfactory bulb.

Some lactic acid bacteria (LAB) are known to improve atopic dermatitis (AD) through the regulation and stimulation of the host immune system. In this study, we found that ingestion of yogurt containing Lactococcus lactis 11/19-B1 strain (L. lactis 11/19-B1) daily for 8 weeks significantly improved the severity scoring of atopic dermatitis (SCORAD) system score from 38.8 ± 14.4 to 24.2 ± 12.0 in children suffering from AD. We tried to identify which LAB species among the five species contained in the test yogurt contributed to the improvement in AD pathology using an AD mouse model induced by repeated application of 1-fluoro-2, 4-dinitrobenzene (DNFB). AD-like skin lesions on the dorsal skin and ear were most improved by L. lactis 11/19-B1 intake among the five LAB species. In addition, analysis of CD4+ T cell subsets in Peyer’s patches (PPs) and cervical lymph nodes (CLNs) indicated that the intake of L. lactis 11/19-B1 generally suppressed all subsets related to inflammation, i.e., Th1, Th2 and Th17, instead of activating the suppressive system, Treg, in the AD mouse model. Histological observations showed ingestion of L. lactis 11/19-B1 significantly suppressed severe inflammatory findings, such as inflammatory cell filtration, epidermal erosion and eosinophil infiltration. These results suggest that the immunomodulatory effects of L. lactis 11/19-B1 contribute to improvements in AD pathology.

Serotonin 2C receptor (5-HT2CR) belongs to the superfamily of seven transmembrane domain receptors coupled to G proteins (GPCR). It is broadly distributed in the CNS and its expression is relatively high in the limbic system including the amygdala, nucleus accumbens (NAc), hippocampus, and hypothalamus. Based on its expression patterns and numerous pharmacological studies, 5-HT2CR is thought to be involved in various brain functions including emotion, appetite, and motor behavior. Here, we review 5-HT2CR and its relationship with alcohol intake with a particular focus on the involvement of 5-HT2CR mRNA editing and its association with alcohol preference in mice. RNA editing is a post-transcriptional modification mechanism. In mammals, adenosine is converted to inosine by the deamination enzymes ADAR1 and ADAR2. 5-HT2CR is the only GPCR subjected to RNA editing within the coding region. It has five editing sites in exon 5 that encode the second intracellular loop. Consequently, three amino acids residues (I156, N158, and I160) of the unedited receptor (INI) may be altered to differently edited isoforms, resulting in a change of receptor activity such as 5-HT potency and G-protein coupling. 5-HT2CR in the NAc is involved in enhanced alcohol drinking after chronic alcohol exposure and alterations in 5-HT2CR mRNA editing is important in determining the alcohol preference using different strains of mice and genetically modified mice. RNA editing of this receptor may participate in the development of alcoholism.

α-Synuclein, the major component of Lewy bodies (LBs) and Lewy neurites (LNs), is expressed in presynapses under physiologically normal conditions and is involved in synaptic function. Abnormal intracellular aggregates of misfolded α-synuclein such as LBs and LNs are pathological hallmarks of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). According to previous studies using pathological models overexpressing α-synuclein, high expression of this protein in neurons is a critical risk factor for neurodegeneration. Therefore, it is important to know the endogenous expression levels of α-synuclein in each neuronal cell type. We previously reported differential expression profiles of α-synuclein in vitro and in vivo. In the wild-type mouse brain, particularly in vulnerable regions affected during the progression of idiopathic PD, α-synuclein is highly expressed in neuronal cell bodies of some early PD-affected regions, such as the olfactory bulb, the dorsal motor nucleus of the vagus, and the substantia nigra pars compacta. Synaptic expression of α-synuclein is mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory synapse marker protein. In contrast, α-synuclein expression in inhibitory synapses differs among brain regions. Recently accumulated evidence indicates the close relationship between differential expression profiles of α-synuclein and selective vulnerability of certain neuronal populations. Further studies on the regulation of α-synuclein expression will help to understand the mechanism of LB pathology and provide an innovative therapeutic strategy to prevent PD and DLB onset.

Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system, including the cerebral cortex, nucleus accumbens (NAc) and hypothalamus. We previously analyzed the behavior of transgenic mice exclusively expressing an unedited RNA isoform of the 5-HT2C receptor. These mice showed decreased NPY gene expression in the NAc and exhibited behavioral despair, suggesting that NAc NPY neurons may be involved in mood disorder; however, their role in this behavior remained unknown. Therefore, in the present study, we investigated the functional role of NAc NPY neurons in anxiety-like behavior by examining the impact of specific ablation or activation of NAc NPY neurons using NPY-Cre mice and Cre-dependent adeno-associated virus. Diphtheria toxin-mediated ablation of NAc NPY neurons significantly increased anxiety-like behavior in the open field and elevated plus maze tests, compared with before toxin treatment. Moreover, chemogenetic activation of NAc NPY neurons reduced anxiety-like behavior in both behavioral tests compared with control mice. These results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.

Background Serum bone alkaline phosphatase (ALP) is a marker of bone formation and metabolism. However, existing methods for measuring it have their limitations and their accuracy has not been determined. Methods We measured serum bone ALP activity in 127 patients with liver disease using 2 methods: electrophoresis and chemiluminescent enzyme immunoassay (CLEIA). The results of these 2 methods were compared and analyzed according to gender, age and several serum biochemical markers. Results When ALP3 (%; bone-type isozyme activity as a percentage of total ALP activity) values were high, the 2 methods showed good correlation. However, with a decrease in ALP3 (%) levels, the correlation coefficient (R) also decreased. Starting with ALP3 (%) 0.05). Five outliers displayed low ALP3 (%) activity levels. Furthermore, in regard to genders, there were significant differences in total cholesterol (TC), γ-glutamyltransferase (γ-GTP), ALP and ALP3 (%) levels (p Conclusions When serum ALP3 (%) levels were high in patients with liver disease, the accuracy of electrophoresis was comparable to that of CLEIA. However, the accuracy of electrophoresis needs to be evaluated with further when patient samples under certain conditions.

Serotonin 2C receptors (5-HT2 C Rs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT2 C R mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT2 C R INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT2 C R RNA editing, and its impairment may be linked to mood disorders.

The effect of long-term visible-light irradiation on the photo-induced deformation of amorphous carbon nitride (a-CN x ) films was investigated. a-CN x films were deposited on SiO 2 substrates (30 × 2 × 0.05 mm 3 ) using reactive radio frequency magnetron sputtering. Deformation of the a-CN x films was measured using continuous wave (CW) or pulsed monochromatic light with a wavelength of 470 nm. Pulsed light irradiation was applied for a total of 60 min with an on/off pulse period of 60 s, while CW light irradiation was performed for 120, 190, and 759 min with different light intensities so that the total photon flux remained constant. In all cases, the extent of photo-induced deformation of the a-CN x films before and after irradiation did not change. The chemical bonding states determined from X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy analyses indicated no significant changes after illumination. In addition, electron spin resonance (ESR) spectroscopy measurements indicated that there was no increase in the defect density after illumination. The long-term stability of a-CN x films is one of the main advantages for their use in light-driven microactuator systems.

A 75 mm $^{2}$ low power 64 Gb MLC NAND flash memory capable of 30 MB/s program throughput and 533 MB/s data transfer rate at 1.8 V supply voltage is developed in 15 nm CMOS technology. 36% power reduction from 3.3 V design is achieved by a new pumping scheme. New low current peak features reduce a multi-die concurrent programming peak by 65% for 4-die case, and an erase verifying peak by 40%, respectively. Nanoscale transistors reducing bit-line discharge time by 70% is introduced to improve performance.

Immortalized erythroid progenitor cell lines, which exhibit potential for enucleated red blood cell (RBC) production, are expected to serve as an in vitro source of RBCs. These erythroid progenitor cell lines have previously been established from a variety of sources; however, large numbers of cell lines have not been established, characterized, and compared from a common cell source. In the present study, 37 cell lines were established from human bone marrow cells from a single donor. The time required for the establishment of each cell line varied greatly from 46 to 246 days. Of these lines, five were selected and their characteristics were analyzed. The cell lines established at the earliest time point showed better results in terms of both karyotype and differentiation potential than those established the latest. Moreover, obvious differences were noted even when cell lines were established at the earliest time point from the same source. These results suggest that it is important to select the best cell lines from ones established at the earliest time point for generating cell lines with low genomic abnormality and high differentiation ability. We have successfully generated an adult type of cell line with 50% cells carrying a normal karyotype and with 25% enucleation efficiency. These findings could be valuable in the development of an optimal method for establishing cell lines.

Heat shock factor 1 (HSF1) is a master regulator of heat shock response and controls transcription of heat shock proteins, including molecular chaperones. It is recently becoming clear that macroautophagy (hereafter autophagy) is regulated by the HSF1-mediated proteostasis network in cells exposed to stress conditions. HSF1 controls expression of several autophagy related genes and activates the autophagy receptor sequestosome1 (p62/SQSTM1). This chapter focuses on regulation of p62-associated formation of inclusions and selective autophagy clearance of harmful proteins by HSF1. The contributions of autophagy induction by HSF1 activation to lifespan extension are also discussed.

Epigenetics is the study of heritable changes in gene expression that are not produced by modification of gene nucleotide sequences. There is growing evidence that epigenetic changes are important in the etiology or progression of a wide range of diseases. Over the last decade in particular, the technologies available to study the mechanisms and consequences of epigenetic modifications have increased exponentially. The stimulus for this has been the rapid increase in our understanding and appreciation of the importance of epigenetic changes on phenotypes and in the etiology of diseases, allied to technological breakthroughs that have made it possible to undertake large-scale epigenomic studies. This chapter summarizes some of the contemporary methods used to study epigenetics and highlights new methods and strategies that have considerable potential for future epigenetic and epigenomic studies. Finally, we describe an epigenomic analysis strategy that focuses on chromosome band structures, the boundaries of which have been shown to be epimutation-sensitive genomic regions at the genome sequence level.

α-Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α-synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α-synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α-synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α-synuclein was highly expressed in the neuronal cell bodies of some early PD-affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α-synuclein was mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory presynaptic marker. In contrast, expression of α-synuclein in the GABAergic inhibitory synapses was different among brain regions. α-Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD-affected human brain regions express high levels of perikaryal α-synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α-synuclein are different in various brain regions.

The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.

The current study investigated whether ABO blood mistyping of human biological samples is induced by soil bacteria. A total of 380 bacterial strains were isolated from 50 discrete soil samples using human blood agar, and glycosidase activity evaluated for all strains using 4-nitropheny glycosides (4-nitrophenyl n-acetyl-α-D-galactosaminide, 4-nitrophenyl-α-D-galactopyranoside, 4-nitrophenyl-α-L-fucopyranoside) as substrates. Thirteen strains possessed α-galactosidase activity, and 16S rRNA sequence analysis revealed a close relatedness to the genus Bacillus. An indirect competitive enzyme-linked immunosorbent assay confirmed seven strains exhibited type B antigen degradation activity. These results demonstrated that 1.8% of the bacteria isolated from soil, were Bacillus sp., possessed galactosidase activity, and had the potential to cause ABO blood mistyping.

Video-assisted thoracoscopic surgery (VATS) is the standard treatment for patients with spontaneous pneumothorax (SP). However, postoperative recurrence is not infrequent even with an absorbable covering sheet used to reinforce the visceral pleura. Recent reports suggest that intraoperative injection of a highly concentrated glucose solution into the thoracic cavity provides effective prophylaxis against postoperative SP recurrence. Since September 2015, we have been injecting 50 ml of 50 % glucose solution intraoperatively for pleural coating (GPC) around an absorbable sheet to prevent postoperative SP recurrence.We evaluated 340 patients who underwent VATS between February 2011 and June 2017(88 patients:GPC group, 252:non-GPC group), and we retrospectively analyzed the efficacy of GPC in preventing postoperative SP recurrence.One year postoperative recurrence rates of GPC and non-GPC groups were 9.0 and 17.9%,respectively. The log-rank test revealed GPC as a significant factor in preventing postoperative recurrence (p=0.020). No severe adverse events occurred in either group. Minor postoperative complications, viz., high blood sugar, high volume of chest tube drainage occurred in the GPC group.Application of GPC is beneficial in reducing postoperative recurrence of SP.

A 77-year-old man with myelodysplastic syndrome suffered from duodenal perforation after undergoing endoscopic submucosal dissection (ESD) for treatment of duodenal cancer. He presented with hemorrhagic shock, peritonitis and disseminated intravascular coagulation (DIC), and received transfusions of red blood cells (RBC), fresh frozen plasma (FFP), γ-globulin and albumin (Alb). One month after the last RBC transfusion, prolonged thrombocytopenia was observed, and platelet concentrate (PC) was transfused. However, immediately after starting PC transfusion, he developed dyspnea, hypotension and rash, and was thus diagnosed as being in anaphylactic shock. Analysis of the patient's serum revealed absence of haptoglobin (Hp) and the presence of anti-Hp antibody. Further studies, using PCR detected Hp

There is considerable current interest in the function of epigenetic mechanisms in neuroplasticity with regard to learning and memory formation and to a range of neural diseases. Previously, we described replication timing on human chromosome 21q in the THP-1 human cell line (2n = 46, XY) and showed that several genes associated with neural diseases, such as the neuronal glutamate receptor subunit GluR-5 (GRIK1) and amyloid precursor protein (APP), were located in regions where replication timing transitioned from early to late S phase. Here, we compared replication timing of all known human glutamate receptor genes (26 genes in total) and APP in 6 different human cell lines including human neuron-related cell lines. Replication timings were obtained by integrating our previously reported data with new data generated here and information from the online database ReplicationDomain. We found that many of the glutamate receptor genes were clearly located in replication timing transition zones in neural precursor cells, but this relationship was less clear in embryonic stem cells before neural differentiation; in the latter, the genes were often located in later replication timing zones that displayed DNA hypermethylation. Analysis of selected large glutamate receptor genes (>200 kb), and of APP, showed that their precise replication timing patterns differed among the cell lines. We propose that the transition zones of DNA replication timing are altered by epigenetic mechanisms, and that these changes may affect the neuroplasticity that is important to memory and learning, and may also have a role in the development of neural diseases.

The aim of the study was to evaluate the feasibility and compare the outcomes of single-incision thoracoscopic surgery using a chest wall pulley for lung excision (PulLE) vs. those of conventional video-assisted thoracic surgery (cVATS) in patients with primary spontaneous pneumothorax (PSP). Sixty-nine patients who underwent PulLE (n = 34) or cVATS (n = 35) between January 2009 and December 2013 were enrolled in this study. PulLE was performed as follows. After making a 17- to 25-mm single incision in the 6th intercostal space (6ICS) at the median axillary line, the visceral pleura near the bulla was sutured for traction. The parietal pleura at 3ICS was then sutured from the thoracic cavity to serve as the chest wall pulley and a traction thread was passed through the pulley. By manipulating the traction thread, it was possible to move the lesion to an arbitrary site for excision. The postoperative scar was nearly invisible. The operative time, duration of postoperative drainage, and postoperative hospital stay were equivalent for PulLE vs. cVATS. There was no significant difference in postoperative recurrence rates. PulLE has cosmetic benefits over cVATS and is easy to perform. We believe our novel procedure has the potential to become the standard operative treatment for PSP.

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H2O2 used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens.

A reversible photo-induced deformation was found in amorphous carbon nitride (a-CN x ) thin films prepared by reactive radio frequency magnetron sputtering method. The a-CN x films were deposited on a rectangular shaped ultrathin Si substrate at different temperatures in the range of room temperature (RT) to 600 °C. A deflection of a-CN x /Si bilayer system was measured using optical cantilever technique with laser light. The bending signal indicates contraction of the film under illumination. The deflection increased with increasing the intrinsic stress of a-CN x films. An increase the ratio of deflection to the intrinsic stress corresponds to an expansion of optical band gap. As a result of Raman spectra, the photo-induced deformation was found to be inhibited with increasing sp 2 cluster size.

The human genome is composed of large-scale compartmentalized structures resulting from variations in the amount of guanine and cytosine residues (GC%) and in the timing of DNA replication. These compartmentalized structures are related to the light- and dark-staining bands along chromosomes after the appropriate staining. Here we describe our current understanding of the biological importance of the boundaries between these light and dark bands (the so-called R/G boundaries). These R/G boundaries were identified following integration of information obtained from analyses of chromosome bands and genome sequences. This review also discusses the potential medical significance of these chromosomal regions for conditions related to genomic instability, such as cancer and neural disease. We propose that R/G-chromosomal boundaries, which correspond to regions showing a switch in replication timing from early to late S phase (early/late-switch regions) and of transition in GC%, have an extremely low number of replication origins and more non-B-form DNA structures than other genomic regions. Further, we suggest that genes located at R/G boundaries and which contain such DNA sequences have an increased risk of genetic instability and of being associated with human diseases. Finally, we propose strategies for genome and epigenome analyses based on R/G boundaries.

Central ghrelin is required for the rewarding properties of drug abuse. We investigated whether alcohol affects ghrelinergic, dopaminergic, and serotoninergic neurons and growth hormone secretagogue receptor 1A (GHS-R1A) levels in the reward system of the brain. Alcohol-naive C57BL/6J mice received 2g/kg ethanol (EtOH) intraperitoneally (i.p.). Plasma ghrelin levels decreased between 1 and 4h. We investigated the effects of EtOH administration on plasma ghrelin levels in two different animal models at 1, 3, and 10months of age. Plasma ghrelin levels decreased following the EtOH treatment in 1- and 3-month-old short-term (1-day) alcohol vapor-exposed (STA) mice. In contrast, EtOH administration increased plasma ghrelin levels in 1- and 3-month-old long-term (20-day) alcohol vapor-exposed (LTA) mice. In vivo ghrelin release in the lateral hypothalamus (LH) increased in STA and LTA mice after the i.p. administration of EtOH. EtOH increased in vivo dopamine (DA), but not serotonin (5-HT) release in the LH of STA mice, and increased in vivo DA and 5-HT release in the LH of LTA mice. GHS-R1A mRNA expression and GHS-R1A protein levels in the LH were increased in LTA mice. The number of GHS-R1A-immunoreactive cells was greater in the LH and amygdala of LTA mice. These results support the neurobiological correlation between the development of drinking behavior and activation of ghrelinergic and serotonergic neurons in the LH. The activation of ghrelinergic systems in the amygdala may also induce an increase in 5-HT release in the LH during long-term alcohol intake.

The superlattice films, which consist of amorphous silicon (a-Si) and amorphous gold (Au), were prepared by ultra-high vacuum evaporation system. The first layer was grown a-Si with a thickness of 4.2 nm and the second layer was grown Au with a thickness of 0.8 nm. Thermal annealing was performed at 473, 673, and 873 K, respectively. The structural properties of the films were investigated using transmission electron microscope (TEM), X-ray diffraction (XRD), and Raman scattering spectroscopy. The electrical property was assessed by the temperature dependence of electrical conductivity. A crystallization of Si and a forming of Au nanoparticles were observed in all of the annealing films. The crystalline volume fraction reached 70% by annealing time for 15 min. An average diameter of the Au nanoparticles embedded in Si matrix also increased with increasing the annealing temperature. At annealing temperature above 873 K, Au atoms migrated toward the film surface. It was observed that the electrical conductivity changed in several temperatures.

Dye-doped deoxyribonucleic acids (DNA)–tetradecyltrimethylammonium (TTA) films have been prepared. Rhodamine 6G, known as laser dyes, can be spontaneously doped by immersing the DNA–TTA film in rhodamine 6G-acetonitrile solutions. It is surmised that rhodamine 6G monomers and dimers diffuse within the hydrophobic TTA sites, and then monomers presumably intercalate between adjacent base pairs of DNA. Optical absorption spectra reveal that rhodamine 6G molecules in the sample undergo an unusual transformation from the dimer state to the monomer state with the elapse of time. Rhidamine 6G molecules doped in DNA–TTA show enhanced photostability and concentration quenching than those in PMMA. The environment, conformation and chemical stability of rhodamine 6G are different between DNA–TTA and PMMA, and are presumably modified by the intercalation.

This paper describes temperature-dependent time-resolved photoluminescence of two-dimensional organic–inorganic layered perovskite compounds, (C6H5C2H4NH3)2PbX4 (X = Br and I) prepared by solution-based self-assembly. (C6H5C2H4NH3)2PbX4 shows fluorescence from free-exciton even at room temperature. At low temperatures below 90 K, the Stokes-shifted fluorescence from bound excitons appears in (C6H5C2H4NH3)2PbI4. In contrast, (C6H5C2H4NH3)2PbBr4 exhibits Stokes-shifted phosphorescence from the triplet excited state to the ground state. The local structure between the organic and inorganic layers plays a prominent role in the formation of triplet excitons.

Dopamine and serotonin (5-HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol-drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol-exposed mice, the expression of 5-HT(2C) receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5-HT(2C) receptor significantly increased in the ACC. The expression of 5-HT(7) receptor mRNA increased in the ACC and DRN. Contents of 5-HT decreased in the ACC shell (ACC(S) ) and DRN of the alcohol-exposed mice. The basal extracellular releases of dopamine (DA) and 5-HT in the ACC(S) increased more in the alcohol-exposed mice than in alcohol-naïve mice. The magnitude of the alcohol-induced ACC(S) DA and 5-HT release in the alcohol-exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC(S) of the 5-HT(2C) receptor antagonist, SB-242084, suppressed voluntary alcohol-drinking behavior in the alcohol-exposed mice. But the i.p. administration of the 5-HT(7) receptor antagonist, SB-258719, did not have significant effects on alcohol-drinking behavior in the alcohol-exposed mice. The effects of the 5-HT(2C) receptor antagonist were not observed in the air-exposed control mice. These results suggest that adaptations of the 5-HT system, especially the upregulation of 5-HT(2C) receptors in the ACC(S) , are involved in the development of enhanced voluntary alcohol-drinking behavior.

Mutations in the human NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), but the pathogenesis of CADASIL has remained unclear. Recently, endocytosis of the Notch ectodermal domain into ligand-expressing cells, called transendocytosis, has come to be considered critical for Notch activation. We hypothesized that the mutant NOTCH3 protein, particularly the ectodermal domain of NOTCH3 (N3ECD), may be refractory to degradation on the cell surface due to impaired transendocytosis. We established a co-culture system in which HEK293 cells stably expressing one copy of tetracycline-regulated NOTCH3 were cultured with NOTCH3 ligand Jagged1 (Jag1)-expressing HEK293 cells. We obtained three main results: first, the C185R mutant N3ECD on the cell surface was degraded significantly more slowly than the wild N3ECD when NOTCH3 cells were co-cultured with Jag1-expressing cells. Second, both the wild-type and mutant NOTCH3-expressing cells increased HES1 expression on co-culture with ligand-expressing cells. Third, vesicles containing N3ECD were observed in Jag1-expressing cells. Vesicles of mutant N3ECD within the Jag1-expressing cells were significantly less in number than in the case of wild-type N3ECD. These results indicated that the process of degradation of mutant N3ECD on the cell surface is disturbed due to impairment of transendocytosis. Such disturbance on the surface of vascular smooth muscle cells may contribute to the pathogenesis of CADASIL.

症例は60歳代，男性．8年前に肝細胞癌に対し肝部分切除術が施行された．平成23年2月の精査で肝S4/8とS6に再発が認められたため手術予定となった．糖尿病のコントロールが悪くHbA1cが8％台と長期間高値であったため術前にdi-peptidyl peptidase-IV（以下DPP-4）阻害薬内服によりコントロールを行った．DPP-4阻害薬内服前の腫瘍最大径はダイナミックCTでS4/8が5.0 cm，S6が2.5 cmであったが，内服開始3週間後のダイナミックCTでは腫瘍最大径はS4/8が2.5 cm，S6が2.0 cmと縮小した．AFPとPIVKA-IIはDPP-4阻害薬内服後に著明に低下した．病理組織学検査で肝細胞癌内に著明なリンパ球浸潤を認め，免疫組織化学染色で浸潤リンパ球はCD8陽性T細胞であった．免疫応答が強く関与していると考えられる肝細胞癌自然退縮の1例を経験した．

This paper describes the synthesis and characterization of organic–inorganic layered perovskite compounds, (CnH2n+1NH3)2PbI4 (n=4, 5, 7, 8 and 9). The effect of the number of carbon atoms on luminescence properties has been examined. Thin films of microcrystalline (CnH2n+1NH3)2PbI4 fabricated by spin-coating are highly oriented, with the c-axis perpendicular to the substrate surface. Temperature-dependent optical absorption spectra reveal that (CnH2n+1NH3)2PbI4 films (n=4, 7, 8 and 9) show the structural phase transitions. The excitonic structures of (CnH2n+1NH3)2PbI4 vary with the number of carbon atoms of the alkyl chain length. At low temperatures below 100 K, the lowest-energy free-exciton band of (CnH2n+1NH3)2PbI4 (n=7, 8 and 9) split into three fine-structure levels. In contrast to (CnH2n+1NH3)2PbBr4 films, (CnH2n+1NH3)2PbI4 (n=7, 8 and 9) shows no triplet exciton emission, but it shows the Stokes-shifted emission from bound excitons.

Some dental patients have histories of adverse reactions to local anesthesia. The aim of the present study was to investigate the frequency of allergy to local anesthetics of dental patients who had histories of adverse reactions to local anesthesia based on the results of allergy tests in our institute over a period of 5 years. We investigated the past medical records of dental patients retrospectively, and twenty patients were studied. Three of the 20 showed a positive or false-positive reaction in the intracutaneous test, and one patient showed a false-positive reaction in the challenge test. Our results suggest that the frequency of allergy to local anesthetics is low even if patients have histories of adverse reactions to local anesthesia. However, allergy tests of local anesthetics should be performed in patients in whom it is uncertain whether they are allergic.

Single layered amorphous carbon nitride (a-CNx) films and a multilayered a-CNx film were prepared by reactive radio frequency magnetron sputtering of a graphite target and nitrogen gas. This paper describes the optical, electrical and opto-electrical properties of the a-CNx films. The optical band-gap of the single layered films increased with increasing nitrogen concentration, which was controlled through the deposition temperature. The photo-sensitivity values, a ratio of photo- and dark-conductivities, ranged from 2.2 to 6.0. In the multilayered film consisting of four a-CNx layers deposited at different temperatures, the photo-sensitivity of the multilayered film was over 1.2 times as compared with that of the single layered films.

Solid phase synthesis of the cyclic depsipeptides of callipeltin B analogues and evaluation of cytotoxicity of synthetic peptides are described. We attempted to synthesize cyclic depsipeptides via the esterification or the amide bond formation for cyclization steps. In the assay of synthetic peptides, the linear peptide (10) exhibited modest cytotoxicity against HeLa cells.

乳び胸の発症原因は胸部外科手術後，外傷，胸部悪性腫瘍などが知られているが，腸間膜腫瘍に合併するものは，これまで報告されていない．今回，われわれは腸間膜腫瘍手術後に乳び胸が消失した興味深い病態を経験したので報告する．症例は78歳，女性．平成16年4月に左側腹部痛が出現し，この時の腹部CTで，腸間膜に石灰化を伴う腫瘤を指摘．外来経過観察されていたが，平成19年9月より増大傾向を示し，平成21年10月のCTでは小腸の浮腫，腹水出現のほか，下痢，栄養状態悪化も認めるため，当科入院となった．入院時に右胸水貯留があり胸腔穿刺を行ったところ乳び胸水であった．腫瘍切除を試みたが，上腸間膜動静脈を巻き込み，周囲との強い癒着があったため生検に止め，浮腫をきたした部分のみ小腸切除した．術後に胸水の貯留はなく，下痢，栄養状態も改善し良好な経過であった．乳び胸の発生に腹腔内腫瘍の関与が推察され，念頭に置くべきと考えられた．

Relaxin-3 (RLN3) is a neuropeptide belonging to the insulin-relaxin superfamily. RLN3-expressing neurons are predominantly located in the dorsal pons known as the nucleus incertus, and project their axons to the forebrain including the hypothalamus. RLN3 has been suggested to be involved in the stress response. In the present study, we investigated the hypothalamic action of RLN3 in the stress-response system by intracerebroventricular (icv) administration of RLN3. Compared with saline icv injection, 1 nmol icv RLN3 injection induced c-Fos expression in the paraventricular nucleus of the hypothalamus (PVN) at 1 h after administration. Some RLN3-induced c-Fos-positive cells in the PVN were also corticotropin-releasing factor (CRF)-expressing neurons. CRF and c-fos mRNA levels in the PVN were increased at 2 h after RLN3 administration. Plasma adrenocorticotropic hormone (ACTH) levels were also increased after RLN3 administration. These results suggest that RLN3 is able to stimulate the hypothalamopituitary CRF-ACTH system during the acute response.

Dye-doped deoxyribonucleic acids (DNA)–tetradecyltrimethylammonium (TTA) films have been prepared. Acridine orange, known as a DNA-binding molecule, can be spontaneously doped by immersing the DNA–TTA film in an acetonitrile solution of the dye. Dye-doped samples exhibit two characteristic absorption bands corresponding to the dye monomer and aggregate, respectively. With the elapse of time after immersion, dye molecules undergo an unusual transformation from the aggregate state to the monomer state, and photoluminescence intensity also increases. Dye molecules in the sample exhibit a pronounced enhancement in their photoluminescence intensity than those in PMMA. The photoluminescence intensity of the samples strongly correlates to both of the dye concentration and monomer/(monomer + aggregates) ratio. Not only the hydrophobic interaction but also the electrostatic force between DNA and dyes play important roles in the formation of the dye-doped samples. It is surmised that monomers and aggregates disperse within the hydrophobic TTA sites in the early stage, and then a part of monomers presumably intercalate between adjacent base pairs of DNA with the elapse of time.

This paper describes the synthesis and characterization of self-assembled organic–inorganic layered perovskite compounds, (C6H5-CnH2n-NH3)2PbBr4 (n=1–4). the effect of the number of carbon atoms of the alkyl chain length (n) on optical properties has been studied. (C6H5-CnH2n-NH3)2PbBr4 films fabricated by spin-coating are microcrystalline form, single phase and oriented with the c-axis. Crystallinity, the maximum PL intensity and the lifetime of exciton emissions varied with the number of carbon atoms. the lowest-energy exciton splits into a few fine-structure levels at low temperatures. Time-resolved photoluminescence spectra reveal that (C6H5-CnH2n-NH3)2PbBr4 shows both singlet and triplet excitons. with decreasing temperature, triplet exciton emissions become dominant for (C6H5-CnH2n-NH3)2PbBr4 (n=1–3), while (C6H5-C4H8-NH3)2PbBr4 shows mainly singlet exciton emissions. The intersystem crossing from excited singlet state to triplet state plays an important role in the relaxation process of excitons.

Thin films of microcrystalline (CnH2n + 1NH3)2PbBr4 (n = 4, 5, 7 and 12) have been prepared by a modified spin-coating method, and the effect of the number of carbon atoms of the alkyl chain length (n) on optical properties has been investigated. Absorption spectra reveal that (CnH2n + 1NH3)2PbBr4 films show stable excitons with a binding energy of a few hundred meV. The excitonic structure of (CnH2n + 1NH3)2PbBr4 varies with the number of carbon atoms. The lowest-energy exciton splits into a few fine-structure levels at low temperature. (CnH2n + 1NH3)2PbBr4 films (n = 5, 7 and 12) show not only singlet excitons but also triplet excitons at low temperature, while (C4H9NH3)2PbBr4 films show only singlet excitons. The intersystem crossing from excited singlet state to triplet state plays an important role in the relaxation process of excitons.

Columnar structured amorphous carbon nitride films were deposited by reactive radio frequency magnetron sputtering from a graphite target in glow discharge plasma of nitrogen. The columnar structure was observed in all specimens of the amorphous carbon nitride (a-CNx) films deposited on substrates of crystalline Si and quartz glass within substrate temperatures between RT and 853 K in nitrogen gas pressure of 0.12 to 0.8 Torr. The average diameter of the columns obtained from SEM was found to be about 40 nm. The results of XRD and FE-TEM confirmed the columns were amorphous materials. However, the electrical properties reflected clearly the heterogeneous structure of a-CNx films, i.e., the resistivity in the vertical to a-CNx columns was lower as compared with that in the horizontal direction (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The transmission of genetic information relies on a coordinated network of cell cycle controls. Abnormalities in this network can result in genomic instability and lead to the transformation of normal cells into cancer cells. Chromosomal DNA replication is not only central to cellular division but also plays a crucial role in the maintenance of genomic integrity. DNA replication errors increase genetic instability, and may be a causative factor in diseases such as cancer and neuronal disorders. Replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict, often tissue-specific, temporal program. The genetic program that controls activation of replication origins in mammalian cells has still not been elucidated. There is evidence that specification of replication sites and timing of replication are dynamic processes that are regulated by tissue-specific and developmental cues and that are responsive to epigenetic modifications. Here, we focus on the spatiotemporal regulation of DNA replication in the human genome. There is growing evidence that chromosome band patterns and epigenetic transformation of chromatin influence the timing of replication. On the basis of this evidence, we propose that the chromatin regions showing switches in replication timing from early to late in S phase are correlated with chromosome band boundaries. These chromatin regions generally display transitions in GC contents and include more non-B-form DNA structures than other genomic regions. We also examine here the effect of changes in replication timing on genomic stability and the possible role of replication timing in the etiology of diseases such as cancer. Replication timing assays are one of many promising techniques under investigation that may in future allow much earlier cancer detection than is possible today.