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The measurement protocol of solid particle number with the lower detection limit (D50) at 10 nm (SPN10) is planned to be implemented in European emission regulations by means of laboratory-grade measurement systems. Furthermore, SPN10 measurement as the real driving emissions (RDE) regulations is under development by defining appropriate technical specifications for the portable emissions measurement system (PEMS). It is under discussion to implement SPN10 limits as one of additional pollutants to the new European emissions regulations, so-called “Euro 7”. As the Consortium for ultra LOw Vehicle Emissions (CLOVE) has proposed, RDE testing by means of PEMS will be the primary means of emissions determination for certification purposes. Measurement equivalency between laboratory-grade emissions measurement systems and PEMS is still important due to the necessity of validation in laboratories before on-road testing by comparing determined emissions by both. The current draft technical requirements for SPN10 measurement in laboratory and on-board allow certain difference in detection efficiencies, especially around the D50 of 10 nm. However, good agreement in the efficiencies between the commercially available laboratory systems and PEMS was observed experimentally by using similar measurement principles based on condensation particle counters (CPCs). The results suggested that the difference in detection efficiencies for the applicable particle size range greater than 10 nm between the laboratory system and PEMS could be within ±10%. Moreover, it is found that the entire system detection efficiencies calibrated directly were equivalent to the calculated efficiencies by individual calibration coefficients of the volatile particle remover and the CPC defined by the regulations.

The European Union’s ambition to reach climate neutrality and a toxic-free environment by 2050 entails, among other things, cleaner road vehicles. The European Commission’s proposal for the next regulatory emissions standard, Euro 7, requires the measurement of pollutants currently not regulated on the road. In this study we compared a prototype portable emissions measurement system (PEMS) measuring CO2, CO, NO, NO2, N2O, NH3, CH4, and HCHO based on infrared laser absorption modulation (IRLAM), and two Fourier transform infrared (FTIR) spectrometers with laboratory grade analyzers. To this end, one Euro 6d Diesel, one Euro 6d gasoline, and one Euro 4 gasoline vehicle were tested at −7 °C and 23 °C with various driving cycles covering traffic conditions to highway dynamic driving. The results demonstrated that the differences among the instruments were small: ±1 mg/km for HCHO, N2O, and CH4, ±2.5 mg/km for NH3, ±10–15 mg/km for NOx, ±50 mg/km or ±15% for CO (whichever was larger), and ±10–15 g/km for CO2. These values corresponded to

北海道屈斜路カルデラとその周辺は，約40 kaから続いている後カルデラ期に活発な火山活動と地形変化が生じてきた。とくに約20～23 kaのアトサヌプリカルデラ形成時に噴出したアトサヌプリ軽石（Ap）とおよそ7.6 kaの摩周カルデラより噴出した摩周f（Ma-f）は噴火規模が大きく，火砕流台地を形成するとともに釧路川を閉塞し屈斜路湖の水位を上昇させた。湖水位の上昇と火砕流二次堆積物の供給によって屈斜路湖岸および釧路川河岸には複数の段丘が形成された。また，このような地形面形成と並行して屈斜路湖東岸のアトサヌプリ火山群周辺では激しい隆起運動が生じ，隆起速度は最大2 m/kyr程度以上に至る。そしてこの隆起運動に伴い断層群が形成され，一部はA級活断層に匹敵する変位速度に達している。

There is a clinical need for sensitive acute kidney injury (AKI) biomarkers that enable early therapeutic interventions and prediction of disease prognosis. In this study, we monitored interleukin (IL)-24 expressed in kidneys with severe AKI that progresses to atrophic kidney in a mouse model of ischemia-reperfusion injury (IRI). Therefore, we evaluated IL-24 as a potential biomarker not only for early diagnosis of AKI, but also for predicting progression to chronic kidney disease (CKD). Serum IL-24 was detected earlier than the elevation of serum creatinine levels and urinary IL-24 was detected as early as neutrophil gelatinase associated lipocalin (NGAL) in severe AKI (60 min of IRI). In addition, serum and urine IL-24 levels tended to increase in relation to ischemia duration. In such kidneys, vascular smooth muscle cells expressed IL-24 in response to the injury in the renal tubular epithelial cell and its target was the renal tubular epithelial cell itself. IL-24 may play a pivotal role in the communication between tubular epithelial cells and vascular smooth muscle cells and, in conclusion, IL-24 can be used as a sensitive biomarker for AKI.

本稿では，EPMA主成分化学組成による対比と現地調査結果に基づき，飯縄山西麓における，中期更新世以降の模式テフラ層序を明らかにした。その結果，広域テフラであるAT，DKP，およびAso-4を含めた9層の示標テフラ層を認定した。本研究で新称した中社スコリア層（CS）は，飯縄山の西麓～南西麓に広く分布する飯縄山起源のテフラである。その層位はATとDKPの間に存在し，この間の褐色ローム層の堆積速度を考慮すると，CSの噴出年代は約45～51 kaであると見積もられる。

Many countries worldwide have introduced a limit for solid particles larger than 23 nm for the type approval of vehicles before their circulation in the market. However, for some vehicles, in particular for port fuel injection engines (gasoline and gas engines) a high fraction of particles resides below 23 nm. For this reason, a methodology for counting solid particles larger than 10 nm was developed in the Particle Measurement Programme (PMP) group of the United Nations Economic Commission for Europe (UNECE). There are no studies assessing the reproducibility of the new methodology across different laboratories. In this study we compared the reproducibility of the new 10 nm methodology to the current 23 nm methodology. A light-duty gasoline direct injection vehicle and two reference solid particle number measurement systems were circulated in seven European and two Asian laboratories which were also measuring with their own systems fulfilling the current 23 nm methodology. The hot and cold start emission of the vehicle covered a range of 1 to 15 × 1012 #/km with the ratio of sub-23 nm particles to the >23 nm emissions being 10–50%. In most cases the differences between the three measurement systems were ±10%. In general, the reproducibility of the new methodology was at the same levels (around 14%) as with the current methodology (on average 17%).

This study was conducted to elucidate the involvement of the PINK1-Parkin pathway in ethanol-induced mitophagy among Sertoli cells (SCs). In the research, adult rats were given intraperitoneal injections of ethanol (5 gm/kg) and sacrificed at various time periods within 24 h. Transmission electron microscopy was applied to reveal enhanced mitochondrial damage in SCs of the ethanol-treated rats (ETRs) in association with a significant increase in numbers of mitophagic vacuoles (mitophagosomes and autolysosomes) in contrast to very low levels in a control group treated with phosphate-buffered saline (PBS). This enhancement was ultra-structurally verified via observation of trapped mitochondria within LC3-labeled membranes, upregulation of LC3 protein levels, colocalization of LC3 and cytochrome c, and reduced expression of mitochondrial proteins. Importantly, Parkin expression was found to be upregulated in ETR SCs, specifically in mitochondria and mitophagosomes in addition to colocalization with PINK1 and pan-cathepsin, indicating augmented mitophagy. Transcription factor EB (TFEB, a transcription factor for autophagy and mitophagy proteins) was also found to be upregulated in nuclei of ETR SCs and associated with enhanced expression of iNOS. Enhanced Parkin-related mitophagy in ETR SCs may be a protective mechanism with therapeutic implications. To the authors&rsquo, knowledge, this is the first report demonstrating the ultrastructural characteristics and molecular mechanisms of Parkin-related mitophagy in ETR SCs.

Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) © 2016 Japanese Teratology Society.

Gully erosion has important on and off site effects. Therefore, several studies have been conducted over the past decades to quantify gully headcut retreat (GHR) in different environments. Although these led to important site-specific and regional insights, the overall importance of this erosion process or the factors that control it at a global scale remain poorly understood. This study aims to bridge this gap by reviewing research on GHR and conducting a meta-analysis of measured GHR rates worldwide. Through an extensive literature review, GHR rates for 933 individual and actively retreating gullies have been compiled from more than 70 study areas worldwide (comprising a total measuring period of > 19 600 years). Each GHR rate was measured through repeated field surveys and/or analyses of aerial photographs over a period of at least one year (maximum: 97 years, median: 17 years). The data show a very large variability, both in terms of gully dimensions (cross-sectional areas ranging between 0.11 and 816 m 2 with a median of 4 m 2 ) and volumetric GHR rates (ranging between 0.002 and 47 430 m 3 year − 1 with a median of 2.2 m 3 year − 1 ). Linear GHR rates vary between 0.01 and 135 m year − 1 (median: 0.89 m year − 1 ), while areal GHR rates vary between 0.01 and 3628 m 2 year − 1 (median: 3.12 m 2 year − 1 ). An empirical relationship allows estimating volumetric retreat rates from areal retreat rates with acceptable uncertainties. By means of statistical analyses for a subset of 724 gullies with a known contributing area, we explored the factors most relevant in explaining the observed 7 orders of magnitudes of variation in volumetric GHR rates. Results show that measured GHR rates are significantly correlated to the runoff contributing area of the gully (r 2 = 0.15) and the rainy day normal (RDN; i.e. the long-term average annual rainfall depth divided by the average number of rainy days; r 2 = 0.47). Other factors (e.g. land use or soil type) showed no significant correlation with the observed GHR rates. This may be attributed to the uncertainties associated with accurately quantifying these factors. In addition, available time series data demonstrate that GHR rates are subject to very large year-to-year variations. As a result, average GHR rates measured over short ( 100%) uncertainties. We integrated our findings into a weighted regression model that simulates the volumetric retreat rate of a gully headcut as a function of upstream drainage area and RDN. When weighing each GHR observation proportional to its measuring period, this model explains 68% of the observed variance in GHR rates at a global scale. For 76% of the monitored gullies, the simulated GHR values deviate less than one order of magnitude from their corresponding observed value. Our model clearly indicates that GHR rates are very sensitive to rainfall intensity. Since these intensities are expected to increase in most areas as a result of climate change, our results suggest that gully erosion worldwide will become more intense and widespread in the following decades. Finally, we discuss research topics that will help to address these challenges.

// Kohei Taniguchi 1, 2 , Miku Sakai 1 , Nobuhiko Sugito 1 , Minami Kumazaki 1 , Haruka Shinohara 1 , Nami Yamada 1 , Tatsushi Nakayama 1 , Hiroshi Ueda 1 , Yoshihito Nakagawa 3 , Yuko Ito 4 , Manabu Futamura 5 , Bunji Uno 1 , Yoshinori Otsuki 4 , Kazuhiro Yoshida 5 , Kazuhisa Uchiyama 2 , Yukihiro Akao 1 1 United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, Japan 2 Department of General and Gastroenterological Surgery, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan 3 Department of Gastroenterology, Fujita Health University, School of Medicine, Kutsukake-cho, Toyoake, Aichi, 470-1192, Japan 4 Department of Anatomy and Cell Biology, Division of Life Sciences, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan 5 Department of Oncological Surgery, Gifu University School of Medicine, Gifu 501-1193, Japan Correspondence to: Kohei Taniguchi, e-mail: sur144@osaka-med.ac.jp Keywords: miR-1, miR-133, Warburg effect, PTBP1, PKM Received: September 28, 2015 Accepted: February 05, 2016 Published: March 09, 2016 ABSTRACT It is known that pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has essential roles in the Warburg effect and that expression of cancer-dominant PKM2 is increased by polypyrimidine tract-binding protein 1 ( PTBP1 ), which is a splicer of the PKM gene. In other words, PKM2 acts as a promoter of the Warburg effect. Previously, we demonstrated that the Warburg effect was partially established by down-regulation of several microRNAs (miRs) that bind to PTBP1 and that ectopic expression of these miRs suppressed the Warburg effect. In this study, we investigated the functions of miR-1 and -133b, which are well known as muscle-specific miRs, from the viewpoint of the Warburg effect in colorectal tumors. The expression levels of miR-1 and -133b were relatively high in colon tissue except muscle and very frequently down-regulated in 75 clinical colorectal tumors samples, even in adenomas, compared with those of the adjacent normal tissue samples. The ectopic expression of these miRs induced growth suppression and autophagic cell death through the switching of PKM isoform expression from PKM2 to PKM1 by silencing PTBP1 expression both in vitro and in vivo . Also, we showed that the resultant increase in the intracellular level of reactive oxygen species (ROS) was involved in this mechanism. Furthermore, PTBP1 was highly expressed in most of the 30 clinical colorectal tumor samples examined, even in adenomas. Our results suggested that PTBP1 and PTBP1-associated miR-1 and -133b are crucial molecules for the maintenance of the Warburg effect in colorectal tumors.

In clinical medicine, indomethacin (IND, a non‐steroidal anti‐inflammatory drug) is used variously in the treatment of severe osteoarthritis, rheumatoid arthritis, gouty arthritis or ankylosing spondylitis. A common complication found alongside the therapeutic characteristics is gastric mucosal damage. This complication is mediated through apoptosis and autophagy of the gastrointestinal mucosal epithelium. Apoptosis and autophagy are critical homeostatic pathways catalysed by caspases downstream of the gastrointestinal mucosal epithelial injury. Both act through molecular signalling pathways characterized by the initiation, mediation, execution and regulation of the cell regulatory cycle. In this study we hypothesized that dysregulated apoptosis and autophagy are associated with IND‐induced gastric damage. We examined the spectra of in vivo experimental gastric ulcers in male Sprague‐Dawley rats through gastric gavage of IND. Following an 18‐hour fast, IND was administered to experimental rats. They were sacrificed at 3‐, 6‐ and 12‐hour intervals. Parietal cells (H(+), K(+)‐ATPase β‐subunit assay) and apoptosis (TUNEL assay) were determined. The expression of apoptosis‐signalling caspase (caspases 3, 8, 9 and 12), DNA damage (anti–phospho‐histone H2A.X) and autophagy (MAP‐LC3, LAMP‐1 and cathepsin B)‐related molecules in gastric mucosal cells was examined. The administration of IND was associated with gastric mucosal erosions and ulcerations mainly involving the gastric parietal cells (PCs) of the isthmic and upper neck regions and a time‐dependent gradual increase in the number of apoptotic PCs with the induction of both apoptotic (upregulation of caspases 3 and 8) cell death and autophagic (MAP‐LC3‐II, LAMP‐1 and cathepsin B) cell death. Autophagy induced by fasting and IND 3 hours initially prompted the degradation of caspase 8. After 6 and 12 hours, damping down of autophagic activity occurred, resulting in the upregulation of active caspase 8 and its nuclear translocation. In conclusion we report that IND can induce time‐dependent apoptotic and autophagic cell death of PCs. Our study provides the first indication of the interactions between these two homeostatic pathways in this context.

Keloid is a fibro-proliferative skin disorder with tumor-like behavior and dependence on anaerobic glycolysis (the Warburg effect), but its exact pathogenesis is unknown. Although autophagy is widely accepted as a lysosomal pathway for cell survival and cellular homeostasis (specifically upon exposure to stressors such as hypoxia), very few studies have investigated the involvement of autophagy and related glycolytic effectors in keloidogenesis. Here the authors examined the expression and cellular localization of autophagy proteins (LC3, pan-cathepsin), glycolytic markers (LDH, MCT1, MCT4) and the transcription factor HIF isoforms in human keloid samples using immunohistochemical analysis and double-labeling immunofluorescence methods. Based on HE staining and expression of CD31, keloids were compartmentalized into hypoxic central and normoxic marginal zones. Vimentin-expressing fibroblasts in the central zone exhibited greater autophagy than their marginal-zone counterparts, as evidenced by increased LC3 puncta formation and co-localization with lysosomal pan-cathepsin. LDH (a lactate stimulator), MCT4 (a lactate exporter) and HIF-1 α expression levels were also higher in central-zone fibroblasts. Conversely, HIF-2 α expression was upregulated in fibroblasts and endothelial cells of the peripheral zone, while MCT1 was expressed in both zones. Taken together, these observations suggest that upregulation of autophagy and glycolysis markers in keloid hypoxic-zone fibroblasts may indicate a prosurvival mechanism allowing the extrusion of lactate to marginal-zone fibroblasts via metabolic coupling. The authors believe this is the first report on differential expression of autophagic and glycolytic markers in keloid-zone fibroblasts. The study results indicate that autophagy inhibitors and MCT4 blockers may have therapeutic implications in keloid treatment.

Surface plasmon resonance (SPR) has been extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. Practical procedures, however, for measurement of the protein-DNA association in crude nuclear extract are yet to be defined.Crude nuclear extract was obtained from MCF-7 cells or recombinant estrogen receptor alpha (ERα) was used for analysis. To suppress signal from non-specific bindings in SPR assay using Biacore, DNA fragments with minimal protein binding activity were identified in a database for transcription factors and included in the study.It is known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams exhibit exponential tendency. Based on statistical analysis, the dissociation phase between the ERα complex from crude nuclear extract and DNA oligonucleotides could be fitted exponentially. Following extrapolation of the dissociation phase, theoretical amount of bound antibodies could be estimated and compared for significant difference.Our procedures made SPR technique such as Biacore a practical technique for measurement of protein-DNA associations in crude nuclear extract with reproducible and reliable results.

Background High-grade chondrosarcoma, which has a high incidence of local recurrence and pulmonary metastasis despite surgical resection, is associated with poor prognosis. Therefore, new and effective adjuvant therapies are urgently required for this disease. Gamma-aminobutyric acid (GABA), which acts as a neurotrophic factor during nervous system development, is related to the proliferation and migration of certain cancer cells. The GABAergic system, which is composed of GABA, the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), and GABA receptors, has an important function in nerve growth and development of neural crest. Therefore, the GABAergic system may play important functional roles in the proliferation of chondrosarcoma cells, which are derived from neural crest cells. We examined the anti-tumor effects of the GABAergic system on a chondrosarcoma cell line. Methods We evaluated the underlying mechanisms of the anti-tumor effects of the GABAergic system, such as the involvement of different signaling pathways, apoptosis, and cell cycle arrest, in the high-grade chondrosarcoma cell line OUMS-27. In addition, we performed whole-cell patch-clamp recordings for Ca2+ currents and evaluated the changes in intracellular Ca2+ concentration via Ca2+ channels, which are related to the GABAB receptor in high-grade chondrosarcoma cells. Results The GABAB receptor antagonist CGP had anti-tumor effects on high-grade chondrosarcoma cells in a dose-dependent manner. The activities of caspase 3 and caspase 9 were significantly elevated in CGP-treated cells compared to in untreated cells. The activity of caspase 8 did not differ significantly between untreated cells and CGP-treated cells. However, caspase 8 tended to be up-regulated in CGP-treated cells. The GABAB receptor antagonist exhibited anti-tumor effects at the G1/S cell cycle checkpoint and induced apoptosis via dual inhibition of the PI3/Akt/mTOR and MAPK signaling pathways. Furthermore, the changes in intracellular Ca2+ via GABAB receptor-related Ca2+ channels inhibited the proliferation of high-grade chondrosarcoma cells by inducing and modulating apoptotic pathways. Conclusions The GABAB receptor antagonist may improve the prognosis of high-grade chondrosarcoma by exerting anti-tumor effects via different signaling pathways, apoptosis, cell cycle arrest, and Ca2+ channels in high-grade chondrosarcoma cells.

Delta-like 3 (DLL3) is a member of the Delta/Serrate/Lag-2 family of ligands for the Notch receptor and plays a role in Notch signaling. We have previously revealed that the expression of DLL3 is silenced by aberrant DNA methylation and that overexpression of DLL3 in the HuH2 hepatocellular carcinoma (HCC) cell line induced apoptosis. In the present study, we first confirmed the methylation of DLL3 in HuH2 cells and analyzed the methylation status of the DLL3 promoter region by bisulfite sequencing. Furthermore, we investigated whether other epigenetic modifications, such as histone acetylation and histone methylation, affected the expression of DLL3. Treatment with the DNA methylation inhibitor, 5-azadeoxycytidine (5-Aza-dC) slightly reactivated DLL3 mRNA expression and bisulfite sequencing revealed that CpG sites in the DLL3 promoter region of the HuH2 cells were densely-methylated. In addition, a significant increase in the expression of DLL3 was observed when the cells were treated with 5-Aza-dC in combination with the histone deacetylase inhibitor trichostatin A. However, an inhibitor of the dimethylation of histone H3 lysine 9 (H3K9me2) or the trimethylation of histone H3 lysine 27 (H3K27me3), modifications that are associated with gene silencing, had no effect on DLL3 reactivation. In combination with the findings from our previous study, these results indicated that DLL3 expression was silenced in HCC cells by DNA methylation and was more readily affected by histone acetylation than histone methylation (H3K9me2 or H3K27me3).

Biacore is a system that is extensively used to characterize the interactions between molecules in terms of their binding specificity, affinity, and kinetics. The practical procedures, however, for measurement of protein-DNA association in crude nuclear extract are yet to be defined. In the present study, DNA fragments with the least protein binding activity were identified in database for transcription factors and included in Biacore assay as control, so that the signals from non-specific binding were markedly suppressed. It was known that when analytes were purified transcription factors, the dissociation curves in Biacore sensorgrams showed exponential tendency. Further analysis showed that the interaction between ERα complex from crude nuclear extract and DNA oligos could be fitted to mono- or bi-exponential functions. Discrimination between orders of exponential function was based on the results of several statistical analyses with an average score of more than 95%. As exponential characteristics allow extrapolation of the dissociation, theoretical amount of bound anti-ERα antibodies could thus be evaluated statistically. Our procedures made Biacore a practical technique like Supershift Assay to measure protein-DNA association in crude nuclear extract with reproducible and reliable results.

The human DEAD/H-box RNA helicase gene DDX6 is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein has been shown to cause malignant transformation. DDX6 has a variety of functions such as translation initiation, pre-mRNA splicing, ribosome assembly, and more. However, details of the regulatory mechanism of DDX6 and functions of DDX6 in cancer cells are largely unknown. On the other hand, the Warburg effect is a well-known feature of cancer cells. Pyruvate kinase in muscle (PKM), which is a rate-limiting glycolytic enzyme, has 2 isoforms, PKM1 and PKM2. It has been frequently reported that PKM2 is a tumor-specific isoform and promotes the Warburg effect. However, the functions of the PKM1 gene have been hardly mentioned. Here, we showed that DDX6 was overexpressed in colorectal cancer specimens and regulated by microRNA (miR)-124 in colon cancer cells. Also, a DDX6/c-Myc/PTB1 positive feedback circuit regulated by miR-124 was shown to be established and to contribute to maintenance of the Warburg effect. Moreover, we showed that knockdown of DDX6 induced mainly apoptosis through an imbalance of PKM gene expression, especially causing down-regulation of PKM1 in colon cancer cells. These results suggest that miR-124 is a fine tuner of the Warburg effect and that DDX6 is one of the key molecules in Warburg effect-related miR-124 targeting various genes.

Altered levels and functions of microRNAs (miRs) have been associated with carcinogenesis. In this study, we investigated the role of miR-124 in colorectal adenoma (CRA) and cancer (CRC). The expression levels of miR-124 were decreased in CRA (81.8%) and CRC (57.6%) in 55 clinical samples. The ectopic expression of miR-124 induced apoptosis and autophagy in colon cancer cells. Also, miR-124 targeted polypyrimidine tract-binding protein 1 (PTB1), which is a splicer of pyruvate kinase muscles 1 and 2 (PKM1 and PKM2) and induced the switching of PKM isoform expression from PKM2 to PKM1. Also, siR-PTB1 induced drastic apoptosis in colon cancer cells. Furthermore, we found that the ectopic expression of miR-124 enhanced oxidative stress and the miR-124/PTB1/PKM1/PKM2 axis constituted a feedback cascade. Finally, we showed that intratumor injection of miR-124 and siR-PTB1 induced a tumor-suppressive effect in xenografted mice. The axis was established by both in vitro and in vivo experiments to function in human colorectal cancer cells. These findings suggest that miR-124 acts as a tumor-suppressor and a modulator of energy metabolism through a PTB1/PKM1/PKM2 feedback cascade in human colorectal tumor cells.

The retroesophageal right subclavian artery may be associated with multiple neurovascular variations, which may impact various cervicothoracic surgeries. During the dissection of 27 cadavers, the authors detected a retroesophageal right subclavian artery in old man arising from distal aortic arch, crossed ventrally by left recurrent laryngeal nerve and dorsally by thoracic duct. On the right side, the aberrant subclavian artery was associated with nonrecurrent laryngeal nerve crossing a large tortuous inferior thyroid artery at multiple intersection points. Importantly, the nonrecurrent laryngeal nerve gave rise to extra laryngeal branch passing over Zuckerkandl’s tubercle of thyroid gland before its termination into the larynx. The relationships of the aberrant subclavian artery in the present case to thoracic duct, recurrent and nonrecurrent laryngeal nerves may have clinical relevance to various operations such as thyroidectomy, esophagectomy and surgical correction of the aberrant vessel. Therefore, these relationships should be well known by surgeons for prevention of iatrogenic damage of essential neurovascular structures during various surgeries.

The purpose is to evaluate quantified kidney echogenicity as a biomarker for the early diagnosis of acute kidney injury (AKI) and predicting progression to chronic kidney disease (CKD) in a mouse model of ischemia–reperfusion injury (IRI). Two separate protocols of murine models of IRI were used: (1) 10, 30, and 40 min of bilateral ischemia duration and (2) 45 and 60 min of unilateral ischemia duration. Renal echogenicity was measured with ultrasound and compared with serum creatinine or urine neutrophil gelatinase-associated lipocalin (NGAL) at various timepoints after IRI. In mice subjected to 10, 30, and 40 min of bilateral ischemia, renal echogenicity increased about 2 h after IRI for all ischemia times, earlier than serum creatinine or urine NGAL. In those subjected to 45 and 60 min of unilateral ischemia, 60 min of unilateral ischemia, which represents atrophic changes 28 days after IRI, resulted in a sustained high level of echogenicity and was significantly different 24 h after IRI, while 45 min of unilateral ischemia resulted in trivial levels of histological damage 28 days after IRI. Renal echogenicity might have the potential to be a biomarker for the early diagnosis of AKI and the prognosis of CKD.

Background/Aims: Chymase inhibition has been shown to attenuate matrix metalloproteinase (MMP)-9 and tumor necrosis factor (TNF)-α, both of which are associated with the pathogenesis of acute liver failure (ALF). This study investigated the effects of the chymase inhibitor TY-51469 on lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced ALF in hamsters. Methods: TY-51469 (10 or 30 mg/kg) or placebo was administered 1 h before the LPS (160 µg/kg)/GalN (400 mg/kg) injection. Results: Hepatic chymase activity was significantly increased after the LPS/GalN injection, but the significant increase was dose-dependently and significantly attenuated by treatment with TY-51469. Significant increases in hepatic MMP-9 activity and TNF-α concentration were observed after the LPS/GalN injection, but these increases were also attenuated by treatment with TY-51469. Plasma aspartate aminotransferase and alanine aminotransferase activities were significantly increased after LPS/GalN injection in the placebo-treated group, but the increases were significantly attenuated in the TY-51469-treated group. The area of hepatic necrotic after LPS/GalN injection was significantly reduced by treatment with TY-51469. Treatment with TY-51469 resulted in significant reductions in the hepatic malondialdehyde concentration, mast cell numbers, and gene expressions of interleukin-1β and myeloperoxidase. Discussion: Chymase inhibition could be a useful strategy to attenuate LPS/GalN-induced ALF in hamsters.

Tingible body macrophages (TBMs) play essential roles in the phagocytosis of apoptotic lymphocytes, specifically under exposure to various stressors. Although excessive ethanol consumption may enhance thymocyte apoptosis, reports investigating the autophagic response of the thymus to ethanol toxicity are still lacking. We investigated apoptosis and autophagy in thymi of an animal model of binge ethanol exposure. Adult male Wistar rats were injected intraperitoneally either with 5 g/kg ethanol or phosphate buffer saline (for the control group) and sacrificed 0, 3, 6 and 24 hours after injection. Light and transmission electron microscopy (TEM) studies revealed enhanced formation of TBMs phagocytosing many apoptotic thymocytes in the thymic cortex of the ethanol-treated rats (ETRs), and this formation was particularly marked at 24 h. The macrophages showed signs of activation under TEM and immunofluorescence double labeling with RM4 (a macrophage marker) and iNOS. Additionally, in comparison to the control group, autophagy was enhanced in ETR thymic TBMs as evidenced ultrastructurally by accumulation of autophagic vacuoles, immunohistochemical increases in LC3 puncta, Western blot analysis of the latter protein, and colocalization of LC3 and RM4 in immunofluorescence double labeling. Immunoelectron microscopy also revealed LC3-labeled autophagic vacuoles and apoptotic cell phagosomes in ETR TBMs, suggesting the possibility of LC3-related phagocytosis. This was confirmed by enhanced colocalization of LC3 with lysosomal cathepsins in double labeling. These results indicate that enhanced autophagy in ETR thymic TBMs is not only a cytoprotective mechanism but could also be involved in the clearance of apoptotic thymocytes, thus preventing autoimmune reactions and suppressing inflammatory response.

In a recent study, we reported that acute ethanol exposure enhanced autophagy in Sertoli cells (SCs) of adult rats. However, further research is needed to clarify the specific spermatogenic stage exhibiting the highest autophagic response, the mechanisms behind such specificity, and the related relevance to sperm. This brief report provides results indicating that stages VII–VIII (androgen-dependent or spermiation stages) of the spermatogenic cycle exhibited more marked autophagic response in acute-ethanol treated rats (ETRs) than other stages based on suppression of androgen receptor (AR), analysis of microtubule-associated protein 1 light chain 3 (LC3) (an autophagosomal marker) immunostaining in SCs, double labeling of LC3 and lysosomal proteins and electron microscopy. Ultrastructural observations and TUNEL method revealed a notable presence of phagocytosed apoptotic germ cells and retained sperm in SCs of ETRs at these specific stages—a finding rarely observed in control testes. In addition, PTEN-induced putative kinase 1 ( PINK1) (a sensor of mitochondrial damage and mitophagy) and giant lipid droplets were found to have accumulated in SCs of ETRs at same stages. Our data show novel findings indicating that stages VII–VIII of the spermatogenic cycle exhibit high levels of autophagy, specifically under stress conditions, as expressed by the term autophagic stages. This stage-specific upregulation of autophagy in SCs may be related to AR suppression, mitochondrial damage, lipid accumulation, and phagocytosis of apoptotic cells. The phenomenon may be an essential part of ensuring the viability of SCs and supporting germ cells in toxic environments.

A growing body of evidence based on in vitro studies indicates that mitophagy (selective autophagic clearance of damaged mitochondria) is a prosurvival mechanism associated with cellular exposure to various mitochondrial stressors. Very recently, a limited number of publications on animal-based models of alcoholic fatty liver diseases have reported that Parkin-mediated mitophagy may mitigate hepatocyte apoptosis, improve mitochondrial quality and suppress steatosis (lipid accumulation). From this perspective, the authors focus on the mechanisms of Parkin mitochondrial translocation (a key consideration in mitophagy activation) and therapeutic implications of mitophagy in liver disease. DNA repair and other functions of Parkin beyond mitophagy are also briefly discussed. The paper additionally shows original data from the authors’ current research indicating enhanced hepatic mitophagy in ethanol-treated rats (ETRs), which is associated with Parkin mitochondrial translocation triggered by oxidative mitochondrial DNA damage. Natural or pharmaceutical products that may trigger Parkin mitochondrial translocation in hepatocytes and/or suppress repressors of such translocation could be a potential therapeutic target in alcoholic and non-alcoholic fatty liver disease

Mitophagy is a cytoprotective mechanism against mitochondrial damaging agents. Studies demonstrating morphological evidence for the involvement of the PINK1-Parkin pathway in the hepatocyte mitophagic response to ethanol toxicity, and potential links to apoptosis and mitochondrial alterations such as spheroid formation are still lacking. We addressed these unresolved issues using a rat model of binge alcohol exposure. Adult rats were injected with ethanol (5g/kg) and liver samples were taken at 0, 3, 6, and 24 hours after ethanol administration and processed for light and electron microscopic studies. Ethanol induced a low level of hepatocyte apoptosis, peaking at 3 h and decreasing significantly by 24 h. In contrast, there was enhanced formation of mitophagic vacuoles in the majority of normal hepatocytes of ethanol-treated rats (ETRs), which peaked at 6 h and was maintained up to 24 h based on electron microscopy and TUNEL/LC3 double labelling. Moreover, enhanced mitophagy in ETR hepatocytes was confirmed by increased LC3 puncta formation, and co-localization of Parkin and LC3 with mitochondrial and lysosomal markers. Immunoelectron microscopy demonstrated the localization of PINK1 and Parkin to damaged mitochondria of ETR hepatocytes, which was consistent with co-localization of Parkin with 8-OHdG, a marker of oxidative mitochondrial DNA damage. Furthermore, electron microscopy showed enhanced formation of mitochondrial spheroids in ETR hepatocytes. These data are the first direct morphological evidence linking PINK1-Parkin pathway activation to the enhanced mitophagic response of hepatocytes to ethanol toxicity. Ethanol-induced hepatic mitophagy may be a prosurvival mechanism, which may have therapeutic implications.

Although it has been shown that the cyclic transition of endometrium is genetically controlled, we still do not know the specific genes involved. It has been suggested that the differences in estrogen-induced cell proliferation and apoptosis are involved in the different phenotypes between mammary gland and endometrium. In the glandular cells of human endometrium, the expression of Bcl-2 is increased at proliferative phases but not at late secretory through menstrual phases. The disappearance of Bcl-2 expression in glandular cells at late secretory phase is consistent with the appearance of apoptotic cells at the same phase. The proliferative phase-specific expression of Bcl-2 in glandular cells is regulated by the binding of C-Jun to its motifs in the promoter region. Furthermore, the menstrual cyclic expression of Bcl-2 is regulated either by the interaction of ERα with C-Jun that binds to its motifs in the Bcl-2 gene or by direct binding of ERα to ERE in the C-Jun promoter. The discovery of new mechanisms downstream of ERα will help pave the way for further understanding of human endometrial transition and function.

Chronic alcohol intake is associated with testicular damage, hypogonadism and infertility. Previously, we reported enhancement of testicular germ cell apoptosis in ethanol-treated rats (ETR) associated with upregulation of apoptosis related genes. Fas, FasL, p53, and activation of caspases. Here, we investigated the involvement of inducible nitric oxide synthase (iNOS) which is a major mediator of oxidative stress in ethanol-induced germ cell apoptosis. Moreover, we explored the various germ cell types undergoing apoptotic DNA fragmentation and their relationship to phagocytosing Sertoli cells. Adult Wistar rats were fed either 5% ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 12 weeks. iNOS expression in control and ETR was investigated using immunohistochemistry. DNA fragmentation in various germ cells of ETR was detected using TUNEL method under both light and electron microscopy. Damaged spermatids with apoptotic features were observed by transmission electron microsc...

Gene expression profiling can be of benefit in identifying critical factors in the process of disease initiation and development. However, in endometriosis it has proven difficult to identify common genes between DNA microarray studies, presumably because of tissue homogeneity in lesions and diversity in the patients' conditions. We attempted DNA microarray analysis in a mouse model for endometriosis with stable lesions and a homogeneous genetic background. Data extracted from the mouse model was then evaluated in human tissues. Mice of the ddY strain underwent surgery to remove the left side of the uterine horn, and the uterine tissue was then minced into small segments and auto-transplanted onto the left peritoneum. After 8 weeks, most of the uterine grafts were enlarged and had regenerated lumens. Comparison of the intensity of mRNA expression between grafts and normal uteri showed that genes encoding immune regulators (e.g. CXCL10) and metabolic factors (e.g. calbindin D-28K) were highly up-regulated in the grafts. Strongly inhibited genes in the grafts included prostaglandin-related factors [e.g. prostaglandin E receptor 3 (subtype EP3) and prostaglandin I2 synthase]. Variation in some candidate factors detected in the mouse model was observed by immunohistochemical studies in human adenomyosis tissues. The gene list in the present study is available for re-evaluation of past studies and provides new candidate factors potentially involved in the pathogenesis of endometriosis.

Gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the brain, is also located in many peripheral nonneuronal tissues. The glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mouse is a useful model for studying the distribution of GABAergic cells in many tissues and organs. The lungs of these mice contain cells with an intense GFP signal exclusively in the airway epithelium. We aimed to characterize the GFP-positive cells and to clarify their relationship with the GABAergic system. We identified the GFP-positive cells as pulmonary neuroendocrine cells (PNECs) by immunohistochemistry for the protein gene product 9.5 and calcitonin gene-related peptide and by ultrastructural analysis. Immunohistochemistry for GADs and GABA revealed GAD65/67 and GABA in GFP-positive PNECs. Reverse transcription-polymerase chain reaction analyses revealed mRNAs encoding the GABA(B) receptor subunits necessary for the assembly of functional receptors, R1 and R2, in the lung. GABA(B) receptor subunit R1 and R2 proteins were expressed in many airway epithelial cells including alveolar epithelial cells other than GFP-positive PNECs. The present findings demonstrated that PNECs in the airway epithelium have a GABA production system and indicated that GABA plays functional roles in airway epithelial cells through GABA(B) receptors.

Unique boreal forest, consisting mainly of sparse larch (Larix cajanderii), creeping pine (Pinus pumila) and birch forest (Betula ermanii), occurs above the lower elevational limit of discontinuous permafrost of about 500 m above sea level in the area around Esso, central Kamchatka (∼56°N). Permafrost is present beneath surfaces covered by Sphagnum or creeping pine, but is absent beneath birch forest. Thermal conductivities in mid-September are low in Sphagnum (0.07–0.19 W/(m·K)) and in the thick (13–20 cm) litter layer beneath creeping pine-covered areas (0.06–0.09 W/(m·K)). Seasonal changes in the subsurface ground temperatures beneath creeping pine show that the surface litter prevents ground temperature increases at depth in summer, resulting in a thin active layer (several decimetres). The surface organic layer appears to be the most important factor controlling the presence or absence of permafrost in this area. Copyright © 2008 John Wiley & Sons, Ltd.

Nuclear factor-kappaB (NF kappaB) plays a pivotal role in cancer progression. In this study, we developed a decoy cis-element oligo-deoxyribonucleic acid against NF kappaB-binding site (NF kappaB-decoy), which effectively inhibits NF kappaB activity, and tested the effect of combined therapy comprising local transfection of NF kappaB-decoy into the liver and transportal injection of paclitaxel on cancer growth and metastasis using an orthotopic murine model of colon cancer liver metastasis. For NF kappaB-decoy transfection, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison. We examined the influence of NF kappaB-decoy transfer on susceptibility to paclitaxel in cancer cells and the mechanism involved using several in vitro analysis systems. We then studied the in vivo effect of combined NF kappaB-decoy transfer and paclitaxel in preventing cancer progression using a murine model of liver metastasis created by splenic injection of a human colon cancer cell line, HT29. In vitro experiments, including MTT-assay, fluorescence-activated cell sorter and cDNA array analysis, revealed that NF kappaB-decoy transfer significantly increased the susceptibility of cancer cells to paclitaxel, and that decreased expression of anti-apoptotic genes along with increased expression of genes relevant to the apoptosis-promotor may be involved. In vivo experiments showed that local transfection of NF kappaB-decoy into the liver followed by portal injection of paclitaxel effectively induced cancer cell apoptosis in the liver metastasis, and significantly prolonged animal survival compared to controls, without notable side effects. In conclusion, a combination of local NF kappaB-decoy transfer into the liver and transportal injection of paclitaxel may be a safe and effective new therapy for liver metastasis.

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein-Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.

Danazol, which has been used as a medicine for endometriosis, has a valid effect in pretreatment of patients receiving in vitro fertilization and embryo transfer, although its reproductive mechanism remains unclear. BALB/c mice were subcutaneously injected with danazol for 2 weeks. Blood and uteri were collected and cytokines were assayed. Following danazol treatment, an increase in pregnancy ratio was evident that was accompanied by up-regulation in serum macrophage-colony stimulating factor (M-CSF). RT-PCR analysis revealed that expression of M-CSF and Ly49, a phenotypic marker of natural killer (NK) cells, was up-regulated in the uteri of the danazol-treated mice. In immunohistochemical analysis, M-CSF and Ly49, together with alpha5 integrin, were clearly detected in the endometrium of the danazol-treated mice with very similar pattern of localization. These results suggest that danazol has an effect to promote pregnancy that induces recruitment of NK cells and a concomitant increase in the expression of M-CSF and alpha5 integrin in the uterus.

Homeobox genes play important roles in craniofacial morphogenesis. However, the characteristics of the transcription factor Hoxc during palate formation remain unclear. We examined the immunolocalization patterns of Hoxc5, Hoxc4, and Hoxc6 in palatogenesis of cleft palate (Eh/Eh) mice. On the other hand, mutations in the FGF/FGFR pathway are exclusively associated with syndromic forms of cleft palate. We also examined the immunolocalization of Fgfr1 and Erk1/2 to clarify their relationships with Hoxc in palatogenesis. Some palatal epithelial cells showed Hoxc5 labeling, while almost no labeling of mesenchymal cells was observed in +/+ mice. As palate formation progressed in +/+ mice, Hoxc5, Hoxc4, and Hoxc6 were observed in medial epithelial seam cells. Hoxc5 and Hoxc6 were detected in the oral epithelium. The palatal mesenchyme also showed intense staining for Fgfr1 and Erk1/2 with progression of palate formation. In contrast, the palatal shelves of Eh/Eh mice exhibited impaired horizontal growth and failed to fuse, resulting in a cleft. Hoxc5 was observed in a few epithelial cells and diffusely in the mesenchyme of Eh/Eh palatal shelves. No or little labeling of Fgfr1 and Erk1/2 was detected in the cleft palate of Eh/Eh mice. These findings suggest that Hoxc genes are involved in palatogenesis. Furthermore, there may be the differences in the localization pattern between Hoxc5, Hoxc4, and Hoxc6. Additionally, Hoxc distribution in palatal cells during palate development may be correlated with FGF signaling. (228/250 words) © 2016 Japanese Teratology Society.

The effect of recombinant human soluble thrombomodulin (TM-α) on acute liver failure (ALF) is unclear, and we elucidated the effect of TM-α in lipopolysaccharide (LPS)/d-galactosamine (GalN)-induced ALF in mice. Placebo (saline) or TM-α (100 mg/kg) was administered 1 h after LPS/GalN administration. Survival rates were evaluated for 24 h after LPS/GalN administration. Plasma and liver samples were evaluated 1, 3, and 7 h after LPS/GalN administration. Survival rates were significantly higher in the TM-α-treated group than in the placebo group. A significant augmentation of plasma high-mobility group box 1 protein (HMGB1) was observed 7 h after LPS/GalN administration. In the TM-α-treated mice, plasma HMGB1 was significantly lower than in the placebo group. A significant augmentation of hepatic nuclear factor (NF)-κB p65 was observed in the placebo-treated group, whereas a significant reduction, relative to placebo, was observed in the TM-α-treated group. Hepatic expression of tumor necrosis factor (TNF)-α and myeloperoxidase were significantly increased in the placebo group, and were similarly significantly attenuated in the TM-α-treated group. TM-α treatment also produced a significant attenuation of liver neutrophil accumulation after LPS/GalN administration. Thus, TM-α may become a useful treatment strategy for reducing the symptoms of ALF via the attenuation of LPS/GalN-induced HMGB1 levels.