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The formation and the chemical characterization of single atoms of dubnium (Db, element 105), in the form of its volatile oxychloride, was investigated using the on‐line gas phase chromatography technique, in the temperature range 350–600 °C. Under the exactly same chemical conditions, comparative studies with the lighter homologues of Group 5 in the Periodic Table clearly indicate the volatility sequence being NbOCl3 > TaOCl3 ≥ DbOCl3. From the obtained experimental results, thermochemical data for DbOCl3 were derived. The present study delivers reliable experimental information for theoretical calculations on chemical properties of transactinides., Dubnium obeys Mendeleev's periodicity. Fifty years after the discovery of this element, its chemical behavior was experimentally revisited. From the gas chromatographic investigation of the volatile oxychloride compound, a trend of decreasing volatility along Group 5 elements was deduced, in agreement with the expectations by the laws established in the Periodic Table.

Impaired social facilitation was reported in autism spectrum disorder (ASD) children. However, behavioral analysis methods of social facilitation for ASD model have not been reported. We developed a novel breeding home cage for social facilitation. Voluntary exercise of more social C57BL/6 J mice was significantly increased in the presence of observer mouse compared to that in the absence of observer mouse. In contrast, the presence of observer mouse did not affect voluntary exercise of less social BALB/cCrSlc mice. These suggest that BALB/cCrSlc mice, a mouse model of ASD, exhibited impaired social facilitation. Our method would provide novel clues for ASD pathophysiology.

While designing deuteron accelerator neutron sources for radioisotope production, nuclear data for light elements such as Li, Be, and C have been systematically measured in the deuteron energy rang...

Objective Owing to variations in the exterior appearances of noncancerous diseases in the oral cavity, clinicians may have difficulty diagnosing oral squamous cell carcinoma (OSCC). Tissue biopsy is confirmatory, but invasive. Therefore, reliable tumor markers for OSCC are required. Here, exosomal Alix (exoAlix) levels were measured in serum/salivary samples from patients with OSCC and healthy controls (HCs). Methods Fifty-seven patients admitted to Nagoya University Hospital from 2017 through 2019 were enrolled, and serum samples (OSCC, n = 29; HC, n = 21) and/or saliva samples (OSCC, n = 23; HC, n = 20) were collected. Exosomal fractions were isolated using ultracentrifugation. ExoAlix levels were measured using enzyme-linked immunosorbent assay. Results Serum/salivary exoAlix levels were significantly higher in patients with OSCC than in HCs. Receiver operating characteristic analyses revealed that sensitivity, specificity, positive predictive value, and area under the curve were 0.345, 1.000, 1.000, and 0.685, respectively, for serum exoAlix and 0.348, 1.000, 1.000, and 0.712, respectively, for salivary exoAlix at optimal cut-off values (serum, 0.205; saliva, 0.193). All tested OSCC tissue sections (n = 21) were immuno-reactive for Alix. Conclusion Serum and salivary exoAlix were identified as potential diagnostic OSCC biomarkers. Serum exoAlix was suitable for prediction of therapeutic responses.

Clinical interest into the function of tuft cells in human intestine has increased in recent years. However, no quantitative study has examined intestinal tuft cells in pathological specimens from patients. This study quantified tuft cell density by using a recently identified marker, specific for tyrosine phosphorylation (pY1798) of girdin (also known as CCDC88A or GIV) in the duodenum of pediatric patients. Deidentified sections with pathological diagnosis of acute duodenitis, ulcer, or celiac disease, and age-matched normal control were analyzed under double-blind conditions. Immunostaining for pY1798-girdin demonstrated the distinct shape of tuft cells with and filopodia-like basolateral membrane structure and a small apical area, which densely expressed gamma-actin. As compared to normal tissues, the specimens diagnosed as celiac disease and duodenal ulcer had significantly fewer tuft cell numbers. In contrast, acute duodenitis showed varied population of tuft cells. The mucosa with severe inflammation showed lower tuft cell numbers than the specimens with none to mild inflammation. These results suggest that loss of tuft cells may be involved in prolonged inflammation in the duodenal mucosa and disrupted mucosal integrity. pY1798-girdin and gamma-actin are useful markers for investigating the distribution and morphologies of human intestinal tuft cells under healthy and pathological conditions.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E–based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

Charged Particle Activation Analysis (CPAA) utilizing an 8-MeV proton beam has been studied for determination of 35 long-lived radioactive nuclides. We accumulated the reaction cross section and nu...

An isothermal gas-chromatographic (IGC) device has been developed and tested for on-line gas phase studies of volatile oxychlorides of short-lived group-5 transition metals. Radioisotopes of niobium and tantalum, produced in nuclear fusion evaporation reactions, are directly flushed into the IGC setup by an inert gas-jet. Oxychloride compounds are formed by the addition of SOCl2 and O2. Parameters influencing the formation and transport of NbOCl3 and TaOCl3 are discussed. For nuclides with half-lives (t1/2) of about 30 s, an overall efficiency of 7% is obtained, rendering the IGC setup suitable for the chemical exploration of 262Db (t1/2 = 34 s).

Chondroitin sulfate (CS) and its isomeric variant, dermatan sulfate (DS), are complex glycosaminoglycans (GAGs) which are ubiquitous components of the extracellular matrix in various tissues including the brain. CS and/or DS are known to bind to a variety of growth factors and regulate many cellular events such as proliferation and differentiation. Although the biological activities of CS and/or DS towards neural stem/progenitor cells (NSPCs) have been well investigated, the CS and/or DS of hematopoietic stem cells (HSCs) have not been fully characterized. Here, we analyzed GAGs on mononuclear cells of rat umbilical cord blood cells (UCB-MNCs). CS was detected in vascular intima and media of rat umbilical cord at embryonic day 19 (E19) by immunohistochemistry. The stem-cell-enriched-UCBCs (SCE-UCBCs), which were expanded from rat UCB-MNCs, expressed CS. CS chains are composed of repeating disaccharide units, which are classified into several types such as O-, A-, B-, C-, D-, and E-unit according to the number and positions of sulfation. A disaccharide composition analysis revealed that CS and/or DS were abundant in rat UCB-MNCs as well as in their expanded SCE-UCBCs, while the amount of heparan sulfate (HS) was less. The degree of sulfation of CS/DS was relatively low and the major component in UCB-MNCs and SCE-UCBCs was the A-unit. A colony-forming cell assay revealed that the percentage of colony-forming cells decreased in culture with CS degradation enzyme. The CS and/or DS of UCBCs may be involved in biological activities such as stem cell proliferation and/or differentiation.

In 2014 the first synthesis of a transactinide carbonyl complex – seaborgium hexacarbonyl – was reported. This was achieved in gas-phase chemical experiments in a beam-free environment behind the recoil separator GARIS. Extending this work to heavier elements requires more efficient techniques to synthesize carbonyl complexes as production rates of transactinide elements drop with increasing atomic number. A novel approach was thus conceived, which retains the benefit of a beam-free environment but avoids the physical preseparation step. The latter reduces the yields for products of asymmetric reactions such as those used for the synthesis of suitable isotopes of Sg, Bh, Hs and Mt. For this a series of experiments with accelerator-produced radioisotopes of the lighter homologues W, Re and Os was carried out at the tandem accelerator of JAEA Tokai, Japan. A newly developed double-chamber system, which allows for a decoupled recoil ion thermalization and chemical complex formation, was used, which avoids the low-efficiency physical preseparation step. Here, we demonstrate the feasibility of this newly developed method using accelerator-produced short-lived radioisotopes of the 5d homologues of the early transactinides.

The formation of NbOCl3 and TaOCl3 and their adsorption behavior on quartz surfaces was explored by applying an isothermal gas-chromatographic method. Trace amounts of short-lived Nb and Ta isotopes were used. Adsorption enthalpy values (ΔHads) at zero surface coverage of –ΔHads(NbOCl3) = 102 ± 4 kJ/mol and –ΔHads(TaOCl3) = 128 ± 5 kJ/mol were determined by analyzing the chromatographic behavior of the Nb and Ta complexes with a Monte-Carlo simulation method based on an adsorption–desorption kinetic model. By applying an empirical correlation, the experimental ΔHads values were successively related to the macroscopic standard sublimation enthalpy, ΔH°subl, as a measure of the volatility of each substance. The inferred sublimation enthalpies are in agreement with tabulated thermochemical values. Thus, the linear empirical correlation between ΔHads and ΔH°subl for metal-oxychlorides was updated with the inclusion of the present data. According to the predicted ΔH°subl(DbOCl3), a ΔHads(DbOCl3) value of 135 ± 2 kJ/mol was extrapolated. The future accomplishment of comparative studies with DbOCl3 under the same experimental conditions will provide valuable information on the volatility trend in Group-5 elements, together with an indication on the magnitude of relativistic effects on the electronic structure of dubnium.

Online gas-solid adsorption studies with single atom quantities of Hg, Tl, and Pb on SiO$_{2}$ and Au surfaces were carried out using short-lived radioisotopes with half-lives in the range of 4-49 s. This is a model study to measure adsorption enthalpies of superheavy elements Cn, Nh, and Fl. The short-lived isotopes were produced and separated by the gas-filled recoil separator TASCA at GSI. The products were stopped in He gas, and flushed into gas chromatography columns made of Si detectors whose surfaces were covered by SiO$_{2}$ or Au. The short-lived Tl and Pb were successfully measured by the Si detectors with the SiO$_{2}$ surface at room temperature. On the other hand, the Hg did not adsorb on the SiO$_{2}$ surface, but adsorbed on the Au surface. The results demonstrated that the adsorption properties of short-lived Hg, Tl, and Pb could be studied with this setup, and that this method is applicable to the experiment for Cn, Nh, and Fl., 半減期4$\sim$49秒の短寿命同位体を用いて、水銀(Hg), タリウム(Tl), 鉛(Pb)の単一原子レベルでのSiO$_{2}$及びAu表面への固体吸着挙動をオンライン気相実験により調べた。超重元素Cn, Nh, Flの吸着エンタルピー測定のためのモデル実験として実施した。短寿命同位体はドイツ重イオン研究所の反跳核分離装置TASCAを用いて生成・分離した。生成核はヘリウムガス中に捕集された後、SiO$_{2}$あるいはAuで表面を覆われたSi検出器で作製されたガスクロマトグラフィーカラムへと導入した。短寿命Tl及びPb同位体は室温においてSiO$_{2}$表面に吸着し、Si検出器で測定することに成功した。一方、HgはSiO$_{2}$表面には吸着せず、Au表面に吸着した。この結果より、本実験のセットアップを用いることで短寿命Hg, Tl, Pb同位体の吸着特性を調べることができると証明され、この手法がCn, Nh, Flの実験にも適用できることが確認された。

We report the first ionization potentials (IP1) of the heavy actinides, fermium (Fm, atomic number Z = 100), mendelevium (Md, Z = 101), nobelium (No, Z = 102), and lawrencium (Lr, Z = 103), determined using a method based on a surface ionization process coupled to an online mass separation technique in an atom-at-a-time regime. The measured IP1 values agree well with those predicted by state-of-the-art relativistic calculations performed alongside the present measurements. Similar to the well-established behavior for the lanthanides, the IP1 values of the heavy actinides up to No increase with filling up the 5f orbital, while that of Lr is the lowest among the actinides. These results clearly demonstrate that the 5f orbital is fully filled at No with the [Rn]5f(14)7s(2) configuration and that Lr has a weakly bound electron outside the No core. In analogy to the lanthanide series, the present results unequivocally verify that the actinide series ends with Lr.

We have studied extraction behavior of group-6 elements Mo and W to search for suitable conditions for an on-line extraction experiment of their heavier homolog, seaborgium (Sg). Batch-wise extraction of carrier-free radiotracers 93mMo and 177,179,181W were carried out from 0.10 – 8.6 M H2SO4 and 1.0 × 10−4 − 5.0 M HF/1.0 M HCl into toluene with a quaternary ammonium compound, Aliquat336. Anionic sulfate complexes of Mo and W with charge − 2 were extracted with Aliquat336 from H2SO4 solutions with concentrations of [H2SO4] > 2 M. In HF/1.0 M HCl, oxyfluoro complexes of Mo and W with charge − 1 were interpreted to be formed and extracted with Aliquat336. From these results, favorable conditions for the extraction of Sg are discussed.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed in several types of human cancers, particularly squamous cell carcinomas. We previously reported that CD109-deficient mice exhibit epidermal hyperplasia and chronic skin inflammation. Although we found that CD109 regulates differentiation of keratinocytes in vivo, the function of CD109 in tumorigenesis remains unknown. In this study, we investigated the role of CD109 in skin tumorigenesis using a two-stage carcinogenesis model in CD109-deficient mice with chronic skin inflammation. Immunohistochemical analysis revealed a higher level of TGF-β protein expression in the dermis of CD109-deficient mice than in that of wild-type mice. Additionally, immunofluorescence analysis showed that Smad2 phosphorylation and Nrf2 expression were enhanced in primary keratinocytes from CD109-deficient mice compared with in those from wild-type mice. Although no significant difference was found in conversion rates from papilloma to carcinoma between wild-type and CD109-deficient mice in the carcinogenesis model, we observed fewer and smaller papillomas in CD109-deficient mice than in wild-type mice. Apoptosis and DNA damage marker levels were significantly reduced in CD109-deficient skin compared with in wild-type skin at 24 h after 7, 12-dimethylbenz (α) anthracene treatment. Furthermore, mutation-specific PCR revealed that the mutation frequency of the H-ras gene was less in CD109-deficient skin than in wild-type skin in this model. These results suggest that CD109 deficiency suppresses skin tumorigenesis by enhancing TGF-β/Smad/Nrf2 pathway activity and decreasing the mutation frequency of the H-ras gene.

// Shotaro Ando 1 , Jun-ichi Kawada 1 , Takahiro Watanabe 2 , Michio Suzuki 1 , Yoshitaka Sato 2 , Yuka Torii 1 , Masato Asai 3 , Fumi Goshima 2 , Takayuki Murata 2 , Norio Shimizu 4 , Yoshinori Ito 1 , Hiroshi Kimura 2 1 Departments of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 2 Departments of Virology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan 3 Departments of Pathology, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8550, Japan 4 Center of Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan Correspondence to: Jun-ichi Kawada, email: kawadaj@med.nagoya-u.ac.jp Keywords: tofacitinib, EBV, lymphoma, cell-cycle arrest Received: June 13, 2016 Accepted: October 03, 2016 Published: October 08, 2016 ABSTRACT Epstein-Barr virus (EBV) infects not only B cells, but also T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoma. These lymphoid malignancies are refractory to conventional chemotherapy. We examined the activation of the JAK3/STAT5 pathway in EBV-positive and -negative B, T and NK cell lines and in cell samples from patients with EBV-associated T cell lymphoma. We then evaluated the antitumor effects of the selective JAK3 inhibitor, tofacitinib, against these cell lines in vitro and in a murine xenograft model. We found that all EBV-positive T and NK cell lines and patient samples tested displayed activation of the JAK3/STAT5 pathway. Treatment of these cell lines with tofacitinib reduced the levels of phospho-STAT5, suppressed proliferation, induced G1 cell-cycle arrest and decreased EBV LMP1 and EBNA1 expression. An EBV-negative NK cell line was also sensitive to tofacitinib, whereas an EBV-infected NK cell line was more sensitive to tofacitinib than its parental line. Tofacitinib significantly inhibited the growth of established tumors in NOG mice. These findings suggest that tofacitinib may represent a useful therapeutic agent for patients with EBV-associated T and NK cell lymphoma.

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, whereas extra-hypothalamic CRH has a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma adrenocorticotropic hormone, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open-field, elevated plus maze, holeboard, light-dark box and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Neurodevelopmental disorders, including intellectual disability and autism spectrum disorder, are often caused by de novo autosomal dominant mutations. While mouse models are frequently used to investigate these disorders, the genetic background sometimes affects the appearance or severity of mutant phenotypes. In a previous report, we developed a system to produce de novo heterozygous mutant mice using the Cre-LoxP system without the need to maintain the heterozygous mutant line itself (Takagi et al. 2015). To further verify the applicability of the de novo mutation system in sperm, we used this system to produce a mouse model for Rubinstein-Taybi syndrome, using a Cbp heterozygous mutant, which has been reported to be difficult to maintain on a C57BL/6 background. Here, we show that de novo Cbp- loss-of-function heterozygous mutant mice with a C57BL/6 background, present with a clear craniofacial phenotype and reduced locomotor activity in the open field test, which was not observed in the loss-of-function of Cbp heterozygous mutant line mice with a mixed genetic background, but was observed in the dominant negative Cbp heterozygous mutant line with a mixed genetic background. Meanwhile, the de novo heterozygous Cbp mutant mice still showed great variability in survival rates despite their inbred background. These results further confirmed that the de novo mutation system used in germ cells is effective for stable production and analysis of an autosomal dominant disorder mouse model, which is often difficult to maintain as a mutant mouse line.

Obesity is characterized by massive adipose tissue accumulation and is associated with psychiatric disorders and cognitive impairment in human and animal models. However, it is unclear whether high-fat diet (HFD)-induced obesity presents a risk of psychiatric disorders and cognitive impairment. To examine this question, we conducted systematic behavioral analyses in C57BL/6J mice (male, 8-week-old) fed an HFD for 7 weeks. C57BL/6J mice fed an HFD showed significantly increased body weight, hyperlocomotion in the open-field test (OFT) and Y-maze test (YMZT), and impaired sucrose preference in the sucrose consumption test, compared to mice fed a normal diet. Neither body weight nor body weight gain was associated with any of the behavioral traits we examined. Working memory, as assessed by the YMZT, and anxiety-like behavior, as assessed by the elevated plus maze test (EPMT), were significantly correlated with mice fed an HFD, although these behavioral traits did not affect the entire group. These results suggest that HFD-induced obesity does not induce neuropsychiatric symptoms in C57BL/6J mice. Rather, HFD improved working memory in C57BL/6J mice with less anxiety, indicating that an HFD might be beneficial under limited conditions. Correlation analysis of individual traits is a useful tool to determine those conditions.

An isothermal vacuum chromatography setup for superheavy element chemistry studies was developed and tested online at the one-atom-at-a-time level. As a model system, the adsorption behavior of thallium on quartz was chosen with respect to a future chemical characterization of its superheavy homologue, element 113 (E113, Z = 113), using the described setup. Short-lived 184Tl (t1/2 = 10.1(5) s) was produced in the reaction 152Gd(35Cl, 3n)184Tl and delivered as a mass-separated ion beam to the chemistry experiment: A subsurface implantation and a subsequent fast thermal release from a metal matrix was followed by isothermal vacuum chromatography as the chemical separation stage. Single atomic species passing this chromatographic separation were finally identified by time- and energy-resolved event-by-event α-spectroscopy using a diamond-based solid-state detector. The derived adsorption enthalpy of −ΔHadsSiO2(Tl) = 158 ± 3 kJ·mol–1 significantly exceeds available data but correlates well with the adsorption...

To control the Karman vortex street formed behind a thin airfoil at a certain incidence, a control cylinder was placed at the suction side apart from the airfoil in the downstream region. Both smoke visualization and hot-wire measurements confirmed that the Karman vortex street was completely suppressed. The mechanism for suppression of the vortex street was examined with a hot-wire survey.

In gastric cancer, the non‐canonical Wnt signaling pathway is activated by Wnt5a, which has a critical role in disease outcome. Previous studies have shown that Wnt5a mediates the expression of the extracellular matrix protein laminin γ2 through Rac and JNK activation to promote gastric cancer progression. However, the mechanism of this regulatory pathway has not been completely addressed. The scaffold protein Dvl is a major component of the Wnt signaling pathway. Here, we show that Dvl‐associating protein with a high frequency of leucine residues (Daple) mediates Wnt5a‐induced laminin γ2 expression. Immunohistochemical analysis showed marked expression of Daple in advanced clinical stages of gastric cancer, where it highly correlated with Wnt5a/b and laminin γ2 expression, the depth of wall invasion, and the frequency of lymph node metastasis. In cultured cancer cells, Daple depletion led to the suppression of Wnt5a‐induced Rac and JNK activation, laminin γ2 expression, and cell migration and invasion. Accordingly, Daple depletion also suppressed liver metastasis in a mouse xenograft model of gastric cancer. These results suggest that the non‐canonical Wnt signaling pathway contributes to gastric cancer progression at least in part via Daple, which provides a new therapeutic opportunity for the treatment of the disease.

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 °C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding 'tuft.' Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling–dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation–induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism., Author summary The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master regulator of cell growth, which senses several extracellular signals, such as growth factors and nutrient levels, to coordinate cell metabolism. The activation of mTORC1 by amino acids requires many proteins such as Rag GTPase, GATOR, and Ragulator. However, how cells negatively regulate amino acid signaling remains largely unknown. In this study, we revealed that an endocytosis-related protein called Girdin negatively regulates amino acid–induced mTORC1 activation via the formation of a complex with 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters. We show that Girdin/4F2h complex formation requires growth factor-induced Girdin phosphorylation and amino acid–induced 4F2hc ubiquitination. We also find that the complex promotes the internalization of 4F2hc from the plasma membrane to the lysosomes. The subsequent decrease of 4F2hc in the cell surface results in a lower cytoplasmic glutamine and leucine content, which then down-regulates amino acid–induced mTORC1 activation. These findings uncover the mechanism underlying negative regulation of mTORC1 signaling, which may play a role in tightly regulated cell growth and metabolism.

In order to study the nucleus–nucleus interactions for syntheses of superheavy nuclei, we measured excitation functions for the quasielastic scattering of 48Ca+208Pb, 50Ti+208Pb, and 48Ca+248Cm using the gas-filled-type recoil ion separator GARIS. The quasielastic scattering events were clearly separated from deep-inelastic events by using GARIS and its focal plan detectors, except for high-incident-energy points. The quasielastic barrier distributions were successfully extracted for these systems, and compared with coupled-channels calculations. The results of the calculations indicate that vibrational and rotational excitations of the colliding nuclei, as well as neutron transfers before contact, strongly affect the structure of the barrier distribution. For the reactions of 48Ca+208Pb and 50Ti+208Pb, a local maximum of the barrier distribution occurred at the same energy as the peak of the 2n evaporation cross section of the system. On the other hand, for the hot fusion reaction of 48Ca+248Cm, the 4n e...

The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.

Rapid in situ formation of metal carbonyl complexes with short-lived nuclides has been demonstrated to be feasible with recoiling ions formed in nuclear fusion and fission reactions. These carbonyl complexes are highly volatile and can be transported rapidly in a gas-stream to counting or chemistry devices. This method was already successfully applied in the chemical investigation of the superheavy element seaborgium (Z = 106) and appears promising for various fields of nuclear research. In this article, we give an overview on the current status of metal carbonyl complex studies with short-lived d-element isotopes.

Fetal brain injury is often related to prenatal inflammation; however, there is a lack of effective therapy. Recently, molecular hydrogen (H2), a specific antioxidant to hydroxyl radical and peroxynitrite, has been reported to have anti-inflammatory properties. The aim of this study was to investigate whether maternal H2 administration could protect the fetal brain against inflammation. Pregnant C3H/HeN mice received an intraperitoneal injection of lipopolysaccharide (LPS) on gestational day 15.5 and were provided with H2 water for 24 h prior to LPS injection. Pup brain samples were collected on gestational day 16.5, and the levels of apoptosis and oxidative damage were evaluated using immunohistochemistry. Interleukin-6 (IL-6) levels were examined using real-time PCR. The levels of apoptosis and oxidative damage, as well as the levels of IL-6 mRNA, increased significantly when the mother was injected with LPS than that in the control group. However, these levels were significantly reduced when H2 was administered prior to the LPS-injection. Our results suggest that LPS-induced apoptosis, oxidative damage and inflammation in the fetal brain were ameliorated by maternal H2 administration. Antenatal H2 administration might protect the premature brain against maternal inflammation.

Adult Cebpb KO mice incisors present amelogenin-positive epithelium pearls, enamel and dentin allopathic hyperplasia, fewer Sox2-positive cells in labial cervical loop epitheliums, and reduced Sox2 expression in enamel epithelial stem cells. Thus, Cebpb acts upstream of Sox2 to regulate stemness. In this study, Cebpb KO mice demonstrated cementum-like hard tissue in dental pulp, loss of polarity by ameloblasts, enamel matrix in ameloblastic layer, and increased expression of epithelial-mesenchymal transition (EMT) markers in a Cebpb knockdown mouse enamel epithelial stem cell line. Runx2 knockdown in the cell line presented a similar expression pattern. Therefore, the EMT enabled disengaged odontogenic epithelial stem cells to develop supernumerary teeth. Cebpb and Runx2 knockdown in the cell line revealed higher Biglycan and Decorin expression, and Decorin-positive staining in the periapical region, indicating their involvement in supernumerary tooth formation. Cebpb and Runx2 acted synergistically and played an important role in the formation of supernumerary teeth in adult incisors.

Summary Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation-status–specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double-fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic-prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs.

In clathrin-mediated endocytosis (CME), specificity and selectivity for cargoes are thought to be tightly regulated by cargo-specific adaptors for distinct cellular functions. Here, we show that the actin-binding protein girdin is a regulator of cargo-selective CME. Girdin interacts with dynamin 2, a GTPase that excises endocytic vesicles from the plasma membrane, and functions as its GTPase-activating protein. Interestingly, girdin depletion leads to the defect in clathrin-coated pit formation in the center of cells. Also, we find that girdin differentially interacts with some cargoes, which competitively prevents girdin from interacting with dynamin 2 and confers the cargo selectivity for CME. Therefore, girdin regulates transferrin and E-cadherin endocytosis in the center of cells and their subsequent polarized intracellular localization, but has no effect on integrin and epidermal growth factor receptor endocytosis that occurs at the cell periphery. Our results reveal that girdin regulates selective CME via a mechanism involving dynamin 2, but not by operating as a cargo-specific adaptor.

Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV7 in epithelial ovarian cancer (EOC) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma (CCC) cells. Expression of REV7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in CCCs (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II–IV) EOC as assessed using Kaplan–Meier curves and log–rank tests. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate molecular target in CCC management.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275 ) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278 ) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth.

REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene expression, and carcinogenesis. In vitro studies show that REV7 interacts with several proteins and regulates their function. It has been reported that human REV7 is highly expressed in the adult testis by Northern blot analysis. However, the significance of REV7 in mammalian development has not been elucidated. Here, we present analyses of REV7-deficient (Rev7(-/-)) mice to clarify the significance of Rev7 in mouse development. In WT mice (Rev7(+/+)), Rev7 expression was ubiquitously observed in the embryo and confined to germ cells in the testes after birth. Rev7(-/-) mice exhibited growth retardation and a partial embryonic lethal phenotype. Mice that survived to adulthood were infertile in both sexes and showed germ cell aplasia in the testes and ovaries. Analyses of Rev7(-/-) embryos revealed that primordial germ cells (PGCs) were present at embryonic day 8.5 (E8.5). However, progressive loss of PGCs was observed during migration, and PGCs were absent in the genital ridges at E13.5. An increase of apoptotic cells was detected not only among PGCs but also in the forebrain of the Rev7(-/-) embryo, whereas cell proliferation was unaffected. Moreover, DNA damage accumulation and increased levels of histone methylation were detected in Rev7(-/-) embryos, and expression of Oct4 and Nanog was deregulated by REV7 deficiency at E8.5. These findings indicate that Rev7 is essential for PGC maintenance by prevention of apoptotic cell death in the mouse.

Resistance to platinum- and taxane-based chemotherapy is a major cause of treatment failure in ovarian cancer. Thus, it is necessary to develop a predictive marker and molecular target for overcoming drug resistance in ovarian cancer treatment. In a previous report, using an in vitro model, we found that the RET finger protein (RFP) (also known as tripartite motif-containing protein 27, TRIM27) confers cancer cell resistance to anticancer drugs. However, the significance of RFP expression in cancer patients remains elusive. In this study, we showed that RFP was expressed in 62% of ovarian cancer patients and its positivity significantly correlated with drug resistance. Consistent with clinical data, depletion of RFP by RNA interference (RNAi) in ovarian cancer cell lines, SKOV3 and HEY, significantly increased carboplatin- or paclitaxel-induced apoptosis and resulted in reduced anticancer drug resistance. In a nude mouse tumor xenograft model, inoculated RFP-knockdown ovarian cancer cells exhibited lower carboplatin resistance than control cells. These findings suggest that RFP could be a predictive marker for chemoresistance in ovarian cancer patients and also a candidate for a molecular-targeted agent.