Purification and characterization of a 39kDa apurinic/apyrimidinic endonuclease from mouse ascites sarcoma cells.

&#60;P&#62;We purified an apurinic/apyrimidinic (AP) endonuclease from mouse ascites sarcoma (SR-C3H/He) cells. The enzyme showed nicking activity on acid-depurinated DNA but not on untreated, intact DNA. It also showed priming activity for DNA polymerase on both acid-depurinated and bleomycin-damaged DNA. The priming activity on bleomycin-damaged DNA was two times higher than that on an acid-depurinated DNA. The enzymatic properties indicate that the enzyme is a class II AP endonuclease having DNA 3' repair diesterase activity. The purified enzyme has a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal pH for AP endonuclease activity was 8.0 in 50 mM Tris-HCl buffer. The AP endonuclease activity depended on divalent cation such as Mg2+ and Co2+ ions, and was inhibited by 2 mM EDTA with no addition of the divalent cation. An appropriate concentration of sodium or potassium salt stimulated the activity. Partial digestion of the AP endonuclease with Staphylococcus aureus V8 protease produced 4 major peptide fragments which may be used for protein sequencing.&#60;/P&#62;</p

cDNA cloning of rat major AP endonuclease (APEX nuclease) and analyses of its mRNA expression in rat tissues.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic (AP) endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. It is also a redox factor (Ref-1), stimulating DNA binding activity of AP-1 binding proteins such as Fos and Jun. In the present paper, a cDNA for the enzyme was isolated from a rat brain cDNA library using mouse Apex cDNA as a probe and sequenced. The rat Apex cDNA was 1221 nucleotides (nt) long, with a 951-nt coding region. The amino acid sequence of rat APEX nuclease has 98.4% identity with mouse APEX nuclease. Using the rat Apex cDNA as a probe for Northern blot analysis, the size of rat Apex mRNA was shown to be approximately 1.5 kb. Its expression was compared in 9 rat organs on postnatal days 7 and 28. Although Apex mRNA was expressed ubiquitously, the levels varied significantly, suggesting organ- or tissue-specific expression of the Apex gene. The highest level was observed in the testis, relatively high levels in the thymus, spleen, kidney and brain, and the lowest level in the liver. The level of expression at postnatal day 28, with the exception of the testis, was almost the same as or lower in respective organs than that at postnatal day 7. Postnatal developmental changes of Apex mRNA expression in the testis and thymus were further studied. The expression in testis was markedly increased on postnatal days 21 and 28. The expression in thymus increased once at postnatal day 14, and then decreased. The developmental changes of Apex mRNA expression in testis and thymus suggest that APEX nuclease is involved in processes such as recombinational events.</p

Optimization of iterative reconstruction parameters with attenuation correction, scatter correction and resolution recovery in myocardial perfusion SPECT/CT

Objective: The aim of this study was to characterize the optimal reconstruction parameters for ordered-subset expectation maximization (OSEM) with attenuation correction, scatter correction, and depth-dependent resolution recovery (OSEMACSCRR). We assessed the optimal parameters for OSEMACSCRR in an anthropomorphic torso phantom study, and evaluated the validity of the reconstruction parameters in the groups of normal volunteers and patients with abnormal perfusion. Methods: Images of the anthropomorphic torso phantom, 9 normal volunteers and 7 patients undergoing myocardial perfusion SPECT were acquired with a SPECT/CT scanner. SPECT data comprised a 64 × 64 matrix with an acquisition pixel size of 6.6 mm. A normalized mean square error (NMSE) of the phantom image was calculated to determine both optimal OSEM update and a full width at half maximum (FWHM) of Gaussian filter. We validated the myocardial count, contrast and noise characteristic for clinical subjects derived from OSEMACSCRR processing. OSEM with depth-dependent resolution recovery (OSEMRR) and filtered back projection (FBP) were simultaneously performed to compare OSEMACSCRR. Results: The combination of OSEMACSCRR with 90-120 OSEM updates and Gaussian filter with 13.2-14.85 mm FWHM yielded low NMSE value in the phantom study. When we used OSEMACSCRR with 120 updates and Gaussian filter with 13.2 mm FWHM in the normal volunteers, myocardial contrast showed significantly higher value than that derived from 120 updates and 14.85 mm FWHM. OSEMACSCRR with the combination of 90-120 OSEM updates and 14.85 mm FWHM produced lowest % root mean square (RMS) noise. Regarding the defect contrast of patients with abnormal perfusion, OSEMACSCRR with the combination of 90-120 OSEM updates and 13.2 mm FWHM produced significantly higher value than that derived from 90-120 OSEM updates and 14.85 mm FWHM. OSEMACSCRR was superior to FBP for the % RMS noise (8.52 ± 1.08 vs. 9.55 ± 1.71, p = 0.02) and defect contrast (0.368 ± 0.061 vs. 0.327 ± 0.052, p = 0.01), respectively. Conclusions: Clinically optimized the number of OSEM updates and FWHM of Gaussian filter were (1) 120 updates and 13.2 mm, and (2) 90-120 updates and 14.85 mm on the OSEMACSCRR processing, respectively. Further assessment may be required to determine the optimal iterative reconstruction parameters in a larger patient population. © 2013 The Japanese Society of Nuclear Medicine

DECIGO pathfinder

DECIGO pathfinder (DPF) is a milestone satellite mission for DECIGO (DECi-hertz Interferometer Gravitational wave Observatory) which is a future space gravitational wave antenna. DECIGO is expected to provide us fruitful insights into the universe, in particular about dark energy, a formation mechanism of supermassive black holes, and the inflation of the universe. Since DECIGO will be an extremely large mission which will formed by three drag-free spacecraft with 1000m separation, it is significant to gain the technical feasibility of DECIGO before its planned launch in 2024. Thus, we are planning to launch two milestone missions: DPF and pre-DECIGO. The conceptual design and current status of the first milestone mission, DPF, are reviewed in this article

The status of DECIGO

DECIGO (DECi-hertz Interferometer Gravitational wave Observatory) is the planned Japanese space gravitational wave antenna, aiming to detect gravitational waves from astrophysically and cosmologically significant sources mainly between 0.1 Hz and 10 Hz and thus to open a new window for gravitational wave astronomy and for the universe. DECIGO will consists of three drag-free spacecraft arranged in an equilateral triangle with 1000 km arm lengths whose relative displacements are measured by a differential Fabry-Perot interferometer, and four units of triangular Fabry-Perot interferometers are arranged on heliocentric orbit around the sun. DECIGO is vary ambitious mission, we plan to launch DECIGO in era of 2030s after precursor satellite mission, B-DECIGO. B-DECIGO is essentially smaller version of DECIGO: B-DECIGO consists of three spacecraft arranged in an triangle with 100 km arm lengths orbiting 2000 km above the surface of the earth. It is hoped that the launch date will be late 2020s for the present

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target