Disinhibitory involvement of the anterior cingulate cortex in the descending antinociceptive effect induced by electroacupuncture simulation in rats

The present study was conducted to clarify the role of the anterior cingulate cortex(ACCX) in acupuncture analgesia. Experiments were performed on 35 female Wistar albino rats weighing about 300 g. Single unit recordings were made from ACCXneurons with a tungsten microelectrode. Descending ACCX neurons were identified byantidromic activation from electrical shocks applied to the ventral part of the ipsilateral PAG through a concentric needle electrode. Cathodal electroacupuncture stimulation of Ho-Ku (0.1 ms in duration, 45 Hz) for 15 min was done by inserting stainless steel needles bilaterally. An anodal silver-plate electrode (30 mm x 30 mm) was placed on the center of the abdomen. Naloxone (1.0 mg/kg, i.v.) was used to test whether changes of ACCX activities were induced by the endogenous opioid system or not. Data were collected from a total of 73 ACCX neurons. Forty-seven neurons had descending projection to the PAG, and 26 had no projections to the PAG. A majority of descending ACCX neurons were inhibited by electroacupuncture stimulation. By contrast, non-projection ACCX neurons were mainly unaffected by electroacupuncture. Naloxone did not reverse acupuncture effects on the changes of ACCX neuronal activities. Acupuncture stimulation had predominantly inhibitory effects on the activities of descending ACCX neurons. Since the functional connection between ACCX and 3/21 PAG is inhibitory, electroacupuncture caused disinhibition of PAG neurons, whose activity is closely related to descending antinociception to the spinal cord. Thisdisinhibitory effect elicited by acupuncture stimulation is thought to play a significant role in acupuncture analgesia

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.National Institutes of Health (U.S.) (Program Project Grant P01-CA026731)National Institutes of Health (U.S.) (R33-CA112151)National Institute of Environmental Health Sciences (P30-ES002109)Singapore-MIT Alliance for Research and Technology CenterNational Institutes of Health (U.S.) (P41-EB015871)National Cancer Institute (U.S.) (P30-CA014051

Response Properties of Nucleus Reticularis Lateralis Neurons After Electroacupuncture Stimulation in Rats

A descending inhibitory mechanism from the periaqueductal gray (PAG) to the spinal cord through the nucleus raphe magnus (NRM) is strongly involved in endogenous analgesic system produced by acupuncture stimulation. In addition to the PAG to NRM system which descends in the medial pathway of the brain stem, the nucleus reticularis lateralis (NRL) situated in the lateral part of the brain stem is reported to play an important role in modulating centrifugal antinociceptive action. In the present study, to clarify the role of NRL in acupuncture analgesia, we investigated the response properties of NRL neurons to acupuncture stimulation. The majority of NRM-projecting NRL neurons were inhibited by electroacupuncture stimulation. This effect was antagonized by ionophoretic application of naloxone, indicating that endogenous opioids act directly onto these NRL neurons. By contrast, about half of spinal projecting NRL neurons were excited by electroacupuncture stimulation, suggesting that part of the NRL neurons may modulate pain transmission directly at the spinal level

Strain-induced creation and switching of anion vacancy layers in perovskite oxynitrides

Using strain to control oxynitride properties. 京都大学プレスリリース. 2020-12-01.原子空孔の配列を制御する新手法の発見. 京都大学プレスリリース. 2020-12-02.Perovskite oxides can host various anion-vacancy orders, which greatly change their properties, but the order pattern is still difficult to manipulate. Separately, lattice strain between thin film oxides and a substrate induces improved functions and novel states of matter, while little attention has been paid to changes in chemical composition. Here we combine these two aspects to achieve strain-induced creation and switching of anion-vacancy patterns in perovskite films. Epitaxial SrVO3 films are topochemically converted to anion-deficient oxynitrides by ammonia treatment, where the direction or periodicity of defect planes is altered depending on the substrate employed, unlike the known change in crystal orientation. First-principles calculations verified its biaxial strain effect. Like oxide heterostructures, the oxynitride has a superlattice of insulating and metallic blocks. Given the abundance of perovskite families, this study provides new opportunities to design superlattices by chemically modifying simple perovskite oxides with tunable anion-vacancy patterns through epitaxial lattice strain

Design and formation of SiC (0001)/SiO2 interfaces via Si deposition followed by low-temperature oxidation and high-temperature nitridation

逆転の発想でSiCパワー半導体の高品質化に成功 --非酸化による酸化膜形成で高品質化10倍--. 京都大学プレスリリース. 2020-08-24.We report an effective approach to reduce defects at a SiC/SiO2 interface. Since oxidation of SiC may inevitably lead to defect creation, the idea is to form the interface without oxidizing SiC. Our method consists of four steps: (i) H2 etching of SiC, (ii) Si deposition, (iii) low-temperature (~750 °C) oxidation of Si to form SiO2, and (iv) high-temperature (~1600 °C) N2 annealing to introduce nitrogen atoms. The interface state density estimated by a high (1 MHz)–low method is in the order of 1010 cm−2 eV−1, two orders of magnitude lower than that of an interface formed by SiC oxidation

CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis

Abstract Background A micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991. Results Rats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control. These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts. Conclusions It was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects

Recombinant cells in the lung increase with age via de novo recombination events and clonal expansion

Homologous recombination (HR) is a critical DNA repair pathway, which is usually error-free, but can sometimes lead to cancer-promoting mutations. Despite the importance of HR as a driver of mutations, the spontaneous frequency of such mutations has proven difficult to study. To gain insight to location, cell type, and subsequent proliferation of mutated cells, we used the Rosa26 Direct Repeat (RaDR) mice for in situ detection and quantification of recombinant cells in the lung. We developed a method for automated enumeration of recombinant cells in lung tissue using the Metafer 4 slide-scanning platform. The mean spontaneous HR frequencies of the lung tissue in young and aged mice were 2 × 10−6and 30 × 10−6, respectively, which is consistent with our previous reports that mutated cells accumulate with age. In addition, by using the capability of Metafer 4 to mark the position of fluorescent cells, we found that recombinant cells from the aged mice formed clusters in the lung tissue, likely due to clonal expansion of a single mutant cell. The recombinant cells primarily consisted of alveolar epithelial type II or club (previously known as Clara) cells, both of which have the potential to give rise to cancer. This approach to tissue image analysis reveals the location and cell types that have undergone HR. Being able to quantify mutant cells in situ within lung tissue opens doors to studies of exposure-induced mutations and clonal expansion, giving rise to new opportunities for understanding how genetic and environmental factors cause tumorigenic mutations. Environ. Mol. Mutagen. 58:135–145, 2017. © 2017 Wiley Periodicals, Inc

Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society

Abstract The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail

Structural characterization of two solute-binding proteins for N,N′-diacetylchitobiose/N,N′,N′′-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the “Venus flytrap” mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria