Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.ArticleJOURNAL OF POULTRY SCIENCE. 51(1):87-95 (2014)journal articl

A Radiobrominated Tyrosine Kinase Inhibitor for EGFR with L858R/T790M Mutations in Lung Carcinoma.

Activating double mutations L858R/T790M in the epidermal growth factor receptor(EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as apositron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the corresponding substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated [77Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N-chlorosuccinimide. Although we aimed to provide a novel PET imaging probe, 77Br was used as an alternative radionuclide for 76Br. We fundamentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975 (IC50 0.18 0.06 M) comparable to that of CO-1686 (IC50 0.14 0.05 M). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686. These results indicate that the binding site of the radiotracers should be identical to that of CO-1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 0.17) was higher than that in H441 tumor (3.71 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification

A at Single Nucleotide Polymorphism-358 Is Required for G at -420 to Confer the Highest Plasma Resistin in the General Japanese Population

Insulin resistance is a feature of type 2 diabetes. Resistin, secreted from adipocytes, causes insulin resistance in mice. We previously reported that the G/G genotype of single nucleotide polymorphism (SNP) at −420 (rs1862513) in the human resistin gene (RETN) increased susceptibility to type 2 diabetes by enhancing its promoter activity. Plasma resistin was highest in Japanese subjects with G/G genotype, followed by C/G, and C/C. In this study, we cross-sectionally analyzed plasma resistin and SNPs in the RETN region in 2,019 community-dwelling Japanese subjects. Plasma resistin was associated with SNP-638 (rs34861192), SNP-537 (rs34124816), SNP-420, SNP-358 (rs3219175), SNP+299 (rs3745367), and SNP+1263 (rs3745369) (P<10−13 in all cases). SNP-638, SNP -420, SNP-358, and SNP+157 were in the same linkage disequilibrium (LD) block. SNP-358 and SNP-638 were nearly in complete LD (r2 = 0.98), and were tightly correlated with SNP-420 (r2 = 0.50, and 0.51, respectively). The correlation between either SNP-358 (or SNP-638) or SNP-420 and plasma resistin appeared to be strong (risk alleles for high plasma resistin; A at SNP-358, r2 = 0.5224, P = 4.94×10−324; G at SNP-420, r2 = 0.2616, P = 1.71×10−133). In haplotypes determined by SNP-420 and SNP-358, the estimated frequencies for C-G, G-A, and G-G were 0.6700, 0.2005, and 0.1284, respectively, and C-A was rare (0.0011), suggesting that subjects with A at −358, generally had G at −420. This G-A haplotype conferred the highest plasma resistin (8.24 ng/ml difference/allele compared to C-G, P<0.0001). In THP-1 cells, the RETN promoter with the G-A haplotype showed the highest activity. Nuclear proteins specifically recognized one base difference at SNP-358, but not at SNP-638. Therefore, A at -358 is required for G at −420 to confer the highest plasma resistin in the general Japanese population. In Caucasians, the association between SNP-420 and plasma resistin is not strong, and A at −358 may not exist, suggesting that SNP-358 could explain this ethnic difference

A radiobrominated tyrosine kinase inhibitor for egfr with l858r/t790m mutations in lung carcinoma

金沢大学疾患モデル総合研究センターActivating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third‐generation EGFR‐TKIs, such as osimertinib and rociletinib (CO‐1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine‐labeled CO‐ 1686 as a positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated‐CO1686 (BrCO1686) was synthesized by the condensation of N‐(3‐[{2‐chloro‐5‐(trifluoromethyl)pyrimidin‐4‐yl}amino]‐5‐bromophenyl) acrylamide with the corresponding substituted 1‐(4‐[4‐amino‐3‐methoxyphenyl]piperazine‐1‐yl)ethan‐1‐one. The radi-obrominated [77 Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N‐chlorosuccinimide. Although we aimed to provide a novel PET imaging probe,77Br was used as an alternative radionuclide for76Br. We fun-damentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non‐small‐cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild‐type EGFR). The BrCO1686 showed high cy-totoxicity toward H1975 (IC50 0.18 ± 0.06 μM) comparable to that of CO‐1686 (IC50 0.14 ± 0.05 μM). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO‐1686. These results indicate that the binding site of the radiotracers should be identical to that of CO‐1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 ± 0.17) was higher than that in H441 tumor (3.71 ± 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild‐type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.CC-BY 4.

増強されたECHアンテナシステムを用いたLHDにおけるECCD適用性の向上

The power injection system for electron cyclotron heating (ECH) and electron cyclotron current drive (ECCD) was modified and upgraded. An outside horizontal port 2-O on the Large Helical Device (LHD) was furnished with two antenna systems for the EC-waves of the frequencies of 77 and 154 GHz, respectively. In addition to them, two new antenna systems for 77 and 154 GHz waves were installed in the 2-O port. Each antenna in the 2-O port has wide range of EC-wave beam direction control so that these are suitable for ECCD which requires toroidally oblique EC-wave beam injection. In the LHD 18th experimental campaign in 2014-2015, an ECCD experiment with second harmonic resonance condition, on-axis magnetic field of 1.375 T for 77 GHz waves, was performed in which some combination patterns of two 77 GHz ECCDs were applied. The discharges of dual co- and dual counter-ECCDs showed remarkable plasma currents of ∼±26 kA in both of the co- and counter-directions, by 6 s pulse duration and injection powers of 366 and 365 kW. The new antenna has nearly the same capability for ECCD with that of the existing antenna. The improvement in the flexibility of the ways of applying plural ECCDs will offer a highly useful tool for investigations on the phenomena concerning with the plasma current such as magnetohydro-dynamics

Stable sustainment of plasmas with electron internal transport barrier by ECH in the LHD

The long pulse experiments in the Large Helical Device has made progress in sustainment of improved confinement states. It was found that steady-state sustainment of the plasmas with improved confinement at the core region, that is, electron internal transport barrier (e-ITB), was achieved with no significant difficulty. Sustainment of a plasma having e-ITB with the line average electron density ne_ave of 1.1 × 1019 m−3 and the central electron temperature Te0 of ∼3.5 keV for longer than 5 min only with 340 kW ECH power was successfully demonstrated

Progress of long pulse discharges by ECH in LHD

Using ion cyclotron heating and electron cyclotron heating (ECH), or solo ECH, trials of steady state plasma sustainment have been conducted in the superconducting helical/stellarator, large helical device (LHD) (Ida K et al 2015 Nucl. Fusion 55 104018). In recent years, the ECH system has been upgraded by applying newly developed 77 and 154 GHz gyrotrons. A new gas fueling system applied to the steady state operations in the LHD realized precise feedback control of the line average electron density even when the wall condition varied during long pulse discharges. Owing to these improvements in the ECH and the gas fueling systems, a stable 39 min discharge with a line average electron density ne_ave of 1.1  ×  1019 m−3, a central electron temperature Te0 of over 2.5 keV, and a central ion temperature Ti0 of 1.0 keV was successfully performed with ~350 kW EC-waves. The parameters are much improved from the previous 65 min discharge with ne_ave of 0.15  ×  1019 m−3 and Te0 of 1.7 keV, and the 30 min discharge with ne_ave of 0.7  ×  1019 m−3 and Te0 of 1.7 keV

Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA genes. In addition, among 72,027 uniquely mapped SNPs and insertions/deletions localized within human genes, 13,215 nonsynonymous SNPs, 315 nonsense SNPs, and 452 indels occurred in coding regions. Together with 25 polymorphic microsatellite repeats present in coding regions, they may alter protein structure, causing phenotypic effects or resulting in disease. The H-InvDB platform represents a substantial contribution to resources needed for the exploration of human biology and pathology

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