The new trailer features the opening theme song “CALLING†” by Kaede Higuchi Kenkyo na Circle writes the I Was Reincarnated as the 7th Prince so I Can Take My Time Perfecting My Magical Ability light novel series, which features illustrations by Meru. Kenkyo na Circle began the novel on the Shosetsuka ni Naro in October 2019, while Kodansha acquired and began publishing it in print in July 2020. There is also a manga adaptation by Yosuke Kokuzawa. Kodansha USA is publishing the manga in English describing the story:Prince Lloyd wasn’t always a prince…in fact his previous life is one he remembers perfectly: he was a sorcerer But his new life has its own sets of challenges…including being a 10-year-old Source: I Was Reincarnated as the 7th Prince Official Website© Kenkyo na Circle KODANSHA / “I Was Reincarnated as the 7th Prince so I Can Take My Time Perfecting My Magical Ability” Production Committee Metrics details ArtA/ArtB (ArtAB) is the second reported toxin that catalyses ADP-ribosylation of pertussis toxin-sensitive G proteins; however the mechanistic basis of its toxicity is unknown we describe the purification of ArtAB toxins from these organisms and the characterization of their biological and physicochemical properties (A) Induction of ArtA expression in Salmonella strains by treatment with mitomycin C (MMC) Overnight cultures grown in syncase broth with ( + ) or without (−) 0.5 μg/ml MMC were centrifuged to separate the cells The supernatant was passed through a 0.22-μm filter and concentrated 15-fold using a Vivaspin 10 K (GE Healthcare) Total protein contents from the supernatants of S Typhimurium strain KST10 were resolved by SDS-PAGE and probed with an antibody against the 14-a.a peptide corresponding to the Arg10–His23 sequence of S (B) SDS-PAGE analysis of purified ArtABs from Salmonella strains The gel was stained using a silver staining kit (C,D) ADP-ribosylation of Ptx-sensitive G proteins by ArtABs; Ptx-sensitive G proteins from bovine brain (0.1 μg) were incubated with biotinylated NAD and purified ArtABs (100 ng) isolated from the indicated strain for 1 h at 37 °C and then ADP-ribosylated proteins were analysed by 12.5% SDS-PAGE (C) and ADP-ribosylation in the presence or absence of 20 mM DTT (D) (E) Western blot analysis of purified ArtABs and ArtAB-Sb were probed with rabbit anti-ArtAB The purified proteins catalysed ADP-ribosylation of the 41-kDa α subunit of the Ptx-sensitive heterotrimeric G protein from bovine brain (Fig. 1C), and this activity was dithiothreitol (DTT)-dependent (Fig. 1D) ArtA and pentameric ArtB subunits of ArtAB (A) Separation of ArtA and ArtB by Mono-Q anion exchange chromatography The dashed line shows the increasing salt gradient (0 to 0.5 M NaCl in 30 mM Tris-HCl (B) SDS-PAGE analysis of the protein content of the ArtA and ArtB chromatographic fractions obtained by Mono-Q anion exchange chromatography These along with purified ArtAB were separated by electrophoresis in the presence or absence of 100 mM DTT Molecular weight standards (kDa) are indicated on the left; positions of the toxin subunits are indicated on the right (C) Schematic illustration of ArtAB subunit structure Percentage of surviving mice following challenge with indicated amounts of ArtAB over time BALB/c mice (n = 5–8) were challenged with graded doses of ArtABs (ArtAB-DT104 or ArtAB-Sb) and observed daily over a 2-week period ArtABs catalyse ADP-ribosylation of G proteins in RAW 264.7 cells (A) ADP-ribosylation of G proteins by ArtA-DT104 western blot showing biotin-ADP ribose labelling of Ptx-sensitive G proteins from the bovine brain and RAW 264.7 cell membranes (3.6 μg) by ArtA-DT104 expressed in vitro (2 μl) or Ptx (100 ng) and ADP-ribosylated proteins were detected by western blotting using peroxidase-conjugated streptavidin as described in Materials and Methods western blot of the RAW 264.7 cell membrane fraction probed with anti-Gαi1 and -Gαi2 IgG antibodies membrane fraction of RAW 264.7 cells; lane 2 Ptx-sensitive G proteins from the bovine brain incubated with ArtA expressed in vitro; lane 4 RAW 264.7 cell membranes incubated with in vitro-expressed ArtA-DT104; lane 5 RAW 264.7 cell membranes incubated with Ptx (B–E) In vitro ADP-ribosylation of cell membrane proteins after preincubation of RAW 264.7 cells with ArtAB-DT104 or Ptx RAW 264.7 cells were incubated with the toxins indicated in upper row (Toxin preincubated with intact cells) for 16 h at 37 °C The membranes from the pretreated cells were then incubated with the toxins indicated in bottom row (Toxin incubated with membrane; in vitro-expressed ArtA or Ptx) in the presence of biotin-NAD ADP-ribosylated proteins were detected by western blotting as described above Results are shown for cells preincubated with different concentrations of ArtAB-DT104 (B); with no toxin pre-treatment (C); with 500 ng of ArtAB-DT104 (D); and with 500 ng of Ptx (E) toxin with membrane indicates which toxin was added along with biotinylated NAD to the membrane fractions during the in vitro membrane labelling ArtAB also belongs to the AB5 family; however CHO cells treated with ArtAB-DT104 and ArtAB-SW exhibited clustered growth suggesting that its B subunit may not be recognized by the receptor and is therefore not internalized by CHO cells ArtA is the only known Salmonella enzyme that catalyses ADP-ribosylation of mammalian G proteins Reactive oxygen species generated and released by host cells may induce artAB expression by intracellular bacteria Since we do not yet have evidence for ArtAB expression in vivo further studies on the regulation and expression of the toxin are required to clarify the importance of ArtAB in the virulence of Salmonella and 3-week-old female BALB/c mice were purchased from Hokudo Co Animals were maintained and used in accordance with the Guidelines for the Care and Use of Laboratory Animals of the National Institute of Animal Health CHO cells and the RAW 264.7 murine macrophage cell line were obtained from the American Type Culture Collection (Manassas CHO cells were cultured in Ham’s F12 medium and RAW 264.7 cells were cultured in Dulbecco’s Minimal Essential Medium supplemented with 10% foetal calf serum and 100 U/ml penicillin G at 37 °C in a humidified atmosphere of 5% CO2 Culture supernatant filtrate (400 ml) was acidified to pH 6.0 with concentrated HCl before adding 6 ml of Affi-Gel Blue equilibrated with 0.25 M phosphate buffer (pH 6.0) The resultant slurry was stirred at 4 °C for 18 h The resin was allowed to settle for 1 h and after siphoning off the supernatant solution it was packed into an empty 10-ml column with a Luer lock connection (MoBiTec Germany) and washed with 0.25 M sodium phosphate (pH 6.0) and then 0.05 M Tris-HCl (pH 7.4) before elution with 0.05 M Tris-HCl (pH 7.4) containing 0.75 M MgCl2 Fractions were collected and assayed by western blotting using a rabbit antibody against a 14-a.a and buffer exchange was performed with 10 mM phosphate buffer (pH 6.0) Proteins were loaded on a 5-ml CHT-I hydroxyapatite column (Bio-Rad) equilibrated with 10 mM phosphate buffer (pH 6.0) washed with 10 mM phosphate buffer (pH 6.0) followed by 0.1 M phosphate buffer (pH 7.0) and eluted with 0.1 M phosphate buffer (pH 7.0) containing 0.5 M NaCl The buffer in the eluted fraction containing ArtAB was exchanged with 1.5 M ammonium sulphate in 50 mM phosphate buffer (pH 7.0) the proteins were loaded on to a Resource PHE Hydrophobic Interaction Column (GE Healthcare) and eluted with a linear gradient of 1.5 to 0 M ammonium sulphate in 50 mM phosphate buffer solution (pH 7.0) using a fast protein liquid chromatography system (GE Healthcare) concentrated in phosphate-buffered saline (PBS) bongori did not bind to the hydroxyapatite gel under these conditions purification was performed using a Superpose 6 Column (10/300GL; GE Healthcare) with 50 mM phosphate buffer (pH 7.0) and 0.15 M NaCl For the preparative separation of ArtAB from DT104 the MonoQ Column (GE Healthcare) was loaded with purified ArtAB in a buffer containing 30 mM Tris-HCl (pH 8.8) Proteins were eluted with 20 ml of a linear gradient (0–500 mM) of NaCl in the same sample buffer Protein concentrations were determined using a Bio-Rad protein assay and ArtB were obtained from rabbits and mice injected subcutaneously with a 1:1 solution of purified antigen and Titer-Max-Gold (CytRx Animals were bled 2 weeks after the last injection IgG in sera from immunized animals was purified using a HiTrap Protein G HP Column (GE Healthcare) following the manufacturer’s instructions Anti-Gαi rabbit serum was purchased from Calbiochem (San Diego ArtAB in the culture filtrate was measured by sandwich (capture) ELISA The plate was coated with rabbit anti-ArtAB IgG (10 μg/ml in PBS containing 137 mM NaCl and 10 mM phosphate buffer at 100 μl/well) at 4 °C for 18 h The plate was blocked with 3% bovine serum albumin in PBS containing 0.02% NaN3 (blocking buffer; 400 μl/well) at room temperature for at least 2 h and washed twice with 400 μl of PBS Culture supernatant was passed through a 0.22-μm membrane filter and standards were prepared with the blocking buffer; these preparations were added at 50 μl/well Mouse anti-ArtAB IgG (diluted 1:200 in blocking buffer; 100 μl/well) was added and incubated at room temperature for 2 h then removed by washing three times (400 μl per wash) with PBS Goat anti-mouse IgG conjugated with horseradish peroxidase (diluted 1:500 in blocking buffer; 100 μl/well) was added and incubated at room temperature for 2 h followed by three washes (400 μl per wash) with PBS Tetramethylbenzidine-H2O2 (TMB Peroxidase EIA Substrate Kit; Bio-Rad) was added and the reaction was terminated by adding 100 μl of 1 N H2SO4 The optical density at 450 nm was recorded Proteins were separated by 12.5% or 15% SDS-PAGE on a 0.1% SDS-Tris-glycine running buffer system and stained with Coomassie blue or silver using a silver staining kit (Daiichi Pure Chemical SDS-PAGE gels were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) in a buffer containing 20% methanol Membranes were incubated for 30 min in blocking buffer consisting of 1% Western Blocking Reagent (Roche Molecular Diagnostics USA) in maleic acid buffer (100 mM maleic acid and 150 mM NaCl The membranes were incubated with rabbit antiserum (1:1000 in maleic acid buffer) and then alkaline phosphatase-conjugated anti-rabbit IgG (Bio-Rad) diluted 1:10,000 in maleic acid buffer Bands were visualized using a chemiluminescent substrate kit (Bio-Rad) cells grown in 75-cm2 flasks were washed twice with PBS Pellets were resuspended in a solution of 0.25 M sucrose and homogenized using a sample-grinding kit (GE Healthcare) Homogenates were centrifuged at 600 × g for 10 min to remove nuclei and unbroken cells and the supernatant was centrifuged at 40,000 × g for 20 min; the sediment that separated from the supernatant fraction was resuspended in a solution of 20 mM Tris-HCl (pH 7.5) and 5 mM MgCl2 and stored at −80 °C ArtAB neutralization in BALB/c mice was assayed by i.p injection of 0.2-ml mixtures of 2 μg of ArtAB and 100 μg of rabbit anti-ArtAB IgG preincubated at 37 °C for 18 h mice were injected with the same amount of ArtAB incubated with IgG from non-immunized rabbit serum CHO cells grown to confluence in flasks were trypsinized and diluted in F-12 medium with 5% foetal calf serum to a concentration of approximately 2 × 104 cells/ml A 150-μl volume aliquot of the suspension was added to 8 wells of a flat-bottomed microtiter plate After allowing 4 h for attachment and stabilization The HA test was performed as described elsewhere15 50 μl of 0.7% chicken erythrocytes in PBS (v/v) was added to 50 μl of the test substance or Ptx serially diluted with PBS in a microplate The preparation was incubated at room temperature for 60 min The minimum amount of sample causing complete agglutination of the red blood cells was recorded 5-week-old female BALB/c mice (n = 4–10) were administered the test substance by i.p tail vein blood samples were drawn and leukocytes were counted serum insulin in day 3 blood samples was measured 15 min after injection of 50% glucose (0.5 ml) using an ELISA kit (Morinaga Institute of Biological Science Japan) according to the manufacturer’s instructions RAW 264.7 cells were harvested and subcultured in a 24-well plate at a density of 1.0 × 105 cells/well the medium was replaced with 0.5 ml of fresh medium containing toxins Cultures were incubated for an additional 24 h and washed twice with 1 ml of buffered saline solution containing 137 mM NaCl 0.4 ml of buffered saline supplemented with 1 mM MgCl2 and 1 mM IBMX was added to the cultures which were incubated at 37 °C for 30 min; this incubation was followed by a 15-min incubation at 37 °C with 10 μM isoproterenol or 10 μM forskolin and/or 50 μM LPA The medium was next removed by aspiration and cells were washed with PBS We next added 0.4 ml of lysis reagent from the cAMP Enzyme Immunoassay System Kit (GE Healthcare) The cAMP assay was performed according to the manufacturer’s instructions Differences were evaluated using one-way ANOVA with Tukey’s test or Dunnett’s test All P values were calculated using GraphPad Prism version 6.0 and were interpreted as significant at values less than 0.05 All animal procedures were carried out in strict accordance with local guidelines and with ethical approval from the National Institute of Animal Health An abundance of bacterial ADP-ribosyltransferases-implications for the origin of exotoxins and their human homologues A simple method of estimating fifty per cent endpoints Download references This work was supported by a grant from the Japan Society for the Promotion of Science KAKENHI (no We thank Youtaro Soga and Masashi Nagato for their technical assistance with experiments We thank Hiroshi Tsunemitsu and Tomohito Hayashi for constructive discussions Bacterial and Parasitic Diseases Research Division purified the ArtAB proteins and conducted the animal and cell experiments All authors read and approved the final manuscript The authors declare that they have no competing interests Download citation DOI: https://doi.org/10.1038/s41598-017-02517-2 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research Researchers from Tokyo Medical and Dental University (TMDU) have developed novel compounds with potential as drug treatments for COVID-19 by modifying a previous “hit” compound that was active against the SARS-CoV virus Japan – The ongoing COVID-19 pandemic While the vaccination program is advancing drug treatments for COVID-19 are still highly important for those who become infected a team at Tokyo Medical and Dental University (TMDU) National Center for Global Health and Medicine (NCGM) and Kumamoto University has designed and synthesized compounds that have the potential to be novel drugs targeting SARS-CoV-2 The SARS-CoV-2 virus contains an enzyme called the “main protease” that cleaves other proteins encoded in the SARS-CoV-2 genome as part of viral activity and replication Mpro is an important and appealing target for drugs treating COVID-19 because it is both essential for viral replication and very different from any human molecules so drugs targeting Mpro are likely to have few side effects and be very effective When testing a panel of compounds known to have inhibitory activity against SARS-CoV the virus responsible for the 2002 SARS outbreak the team identified a compound named 5h/YH-53 that showed some activity inhibiting SARS-CoV-2 Mpro they used 5h as a starting point to develop other compounds with increased efficiency and stability “Our strategy involved introducing fluorine atoms into the part of the molecule responsible for inhibiting Mpro to increase its binding affinity as well as replacing a bond within 5h that is easily broken down by the liver with a different structure to increase biostability,” explains lead author Kohei Tsuji compound 3 showed high potency and was able to block SARS-CoV-2 infection in vitro without any viral breakthrough,” explains senior author Hirokazu Tamamura a derivative of compound 3 in which an easily broken-down amide bond had been replaced with a stable thioamide bond also showed remarkable anti-SARS-CoV-2 activity.” Although compound 4 had lower Mpro inhibitory activity than compound 3 the increased stability meant that the overall activity of compound 4 was comparable to that of compound 3 When they tested these novel compounds on a variety of strains of SARS-CoV-2 compound 3 was as effective on mutant strains of the virus as on the ancestral Wuhan strain neither compound 3 or 4 showed any toxicity to cultured cells These data suggest that these compounds show high potential as drug treatments for COVID-19 A repertory of drug choice is important for treating disease and so the development of efficient drugs to target the novel SARS-CoV-2 virus is highly important This work identifies two compounds as potential drugs and further development of these compounds continues It also proves the principle that easily broken-down amide bonds can be replaced with thioamide bonds in drug development to increase the stability of the resulting compounds this is an important advance in both the wider drug development field as well as for drugs to treat COVID-19 10.1016/j.isci.2022.105365 Potent and biostable inhibitors of the main protease of SARS-CoV-2 are not responsible for the accuracy of news releases posted to EurekAlert by contributing institutions or for the use of any information through the EurekAlert system Copyright © 2025 by the American Association for the Advancement of Science (AAAS) 2019Ink Teaser 2019 We were not aware that Kazu and company were making a movie this year.. but now that the teaser dropped it is all we can think about Yuri Okubo and Yutaro Miyazawa for what should be quite the flick Keep an eye out for more as we go into fall More from SNOWBOARDER Magazine here! TOKYO — Avant-garde pianist and composer Toshi Ichiyanagi, who studied with John Cage and went on to lead Japan’s advances in experimental modern music, has died. He was 89. Mr. Ichiyanagi, who was married to Yoko Ono before she married John Lennon, died Friday, according to the Kanagawa Arts Foundation, where Mr. Ichiyanagi had served as general artistic director. The cause of death was not given. “We would like to express our sincerest gratitude to all those who loved him during his lifetime,” the foundation’s chairman, Kazumi Tamamura, said in a statement Saturday. Mr. Ichiyanagi studied at The Juilliard School in New York and emerged a pioneer, using free-spirited compositional techniques that left much to chance, incorporating not only traditional Japanese elements and instruments but also electronic music. He was known for collaborations that defied the boundaries of genres, working with Jasper Johns and Merce Cunningham, as well as innovative Japanese artists like architect Kisho Kurokawa and poet-playwright Shuji Terayama, as well as with Ono, with whom he was married for several years starting in the mid-1950s. “In my creation, I have been trying to let various elements, which have often been considered separately as contrast and opposite in music, coexist and penetrate each other,” Mr. Ichiyanagi once said in an artist statement. Japanese traditional music inspired and emboldened him, he said, because it was not preoccupied with the usual definitions of music as “temporal art,” or what he called “divisions,” such as relative and absolute, or new and old. Modern music was more about “substantial space, in order to restore the spiritual richness that music provides,” he said. Among his well-known works for orchestra is his turbulently provocative “Berlin Renshi.” Renshi is a kind of Japanese collaborative poetry that is more open-ended free verse than older forms like “renku.” In 1989, Mr. Ichiyanagi formed the Tokyo International Music Ensemble — The New Tradition (TIME), an orchestral group focused on traditional instruments and “shomyo,” a style of Buddhist chanting. His music traveled freely across influences and cultures, transitioning seamlessly from minimalist avant-garde to Western opera. Mr. Ichiyanagi toured around the world, premiering his compositions at Carnegie Hall in New York and the Théâtre des Champs-Élysées, Paris. The National Theater of Japan also commissioned him for several works. He remained prolific over the years, producing Concerto for marimba and orchestra in 2013, and Piano Concerto No. 6 in 2016, which Mr. Ichiyanagi performed solo at a Tokyo festival. Mr. Ichiyanagi received numerous awards, including the Alexander Gretchaninov Prize from Juilliard, L’ordre des Arts et des Lettres of the French Republic and the Order of the Rising Sun, Gold Rays with Rosette and the Medal of Purple Ribbon from the Japanese government. Born in Kobe to a musical family, Mr. Ichiyanagi showed promise as a composer at a young age. He won a major competition in Japan before moving to the U.S. as a teen, when such moves were still relatively rare in postwar Japan. A private funeral is being held with family. A public ceremony in his honor is in the works, being arranged by his son, Japanese media reports said. Yuri Kageyama is on Twitter https://twitter.com/yurikageyama Home Delivery Gift Subscriptions Log In Manage My Account Customer Service Delivery Issues Feedback News Tips Help & FAQs Staff List Advertise Newsletters View the ePaper Order Back Issues News in Education Search the Archives Privacy Policy Terms of Service Terms of Purchase Work at Boston Globe Media Internship Program Co-op Program Do Not Sell My Personal Information Metrics details The development of genome editing systems based on the Cas9 endonuclease has greatly facilitated gene knockouts and targeted genetic alterations Precise editing of target genes without off-target effects is crucial to prevent adverse effects in clinical applications Although several methods have been reported to result in less off-target effects associated with the CRISPR technology these often exhibit lower editing efficiency and innocuous CRISPR technology is still required Anti-CRISPR proteins are natural inhibitors of CRISPR-Cas systems derived from bacteriophages was fused with the N terminal region of human Cdt1 that is degraded specifically in S and G2 the phases of the cell cycle when homology-directed repair (HDR) is dominant Co-expression of SpyCas9 and AcrIIA4-Cdt1 not only increases the frequency of HDR but also suppress off-targets effects the combination of SpyCas9 and AcrIIA4-Cdt1 is a cell cycle-dependent Cas9 activation system for accurate and efficient genome editing The wide application of CRISPR technology is expected in the fields of agriculture biotechnology and others in the coming years Although CRISPR technology is the most useful method for genome editing off-target effects that cause unexpected mutations at pseudo-target DNA sequences could occur similarly to those seen using as ZFN and TALEN in addition to endeavor to increase the efficiency of precise editing at on-targets off-target effects should be carefully addressed when genome editing tools are used The method could provide a way for avoiding cytotoxicity HDR activity was only marginally increased which was probably due to the amount of Cas9-Geminin (1–110) fusion was not fully recovered in the S phase after degradation in the G1 phase when anti-CRISPR expression can be controlled by cell cycle the activity of Cas9 endonuclease could also be controlled in the cells we fused the anti-CRISPR AcrIIA4 with the N-terminal region of human chromatin licensing and DNA replication factor 1 (hCdt1) for activation in the S/G2 phases and inactivation in the G1 phase hCdt1 is degraded by ubiquitin-mediated proteolysis through the SCFSkp2 complex in the S/G2 phases The cell cycle dependent Cas9 activation system was validated using SpyCas9 endonuclease and AcrIIA4 in the cells the system displayed autonomous Cas9 activity switch dependent on the cell cycle a Description of anti-CRISPR mediated cell cycle specific Cas9 activation system. In G1 phase, AcrIIA4 which is a known inhibitor of SpyCas9 inhibits Cas9-sgRNA by binding the complex. In S/G2/M phases, AcrIIA4 is degraded because of S phase degradation domain from Cdt1, and the SpyCas9-sgRNA complex is activated. b Constructed episomal vector and hypothesized expression change of AcrIIA4-Cdt1 and SpyCas9. The molar ratio of plasmid (SpyCas9:AcrIIA4) changed from 1:1 to 1:5 The cleavage activity of SpyCas9 was calculated using the T7E1 assay Mutation was detected by T7E1 assay and microchip electrophoresis The editing efficiency was calculated by the formula; 100 × (1–sqrt(1 – (b + c)/(a + b + c))) where “a” is the integrated intensity of the undigested PCR product and “b” and “c” are the integrated intensities of each cleavage product a Method to confirm the HDR efficiency using XhoI restriction enzyme b Results of target HDR (left) and off-target mutation (right) Each PCR product amplified from extracted genomic DNA reacted with XhoI for HDR or T7E1 for off-target mutation The editing efficiency was calculated by the formula; 100 × ((b + c)/(a + b + c)) for target HDR 100 × (1–sqrt(1 – (b + c)/(a + b + c))) for off-target mutation Significance in difference was tested by Student’s t-test AcrIIA4-Cdt1 was constructed for precise genome editing using SpyCas9 derived from Streptococcus pyogenes. The target mutation rate by NHEJ was decreased when SpyCas9-sgRNA and AcrIIA4-Cdt1 were co-expressed due to SpyCas9 inhibition by AcrIIA4. A dose-dependent increase of SpyCas9 inactivation by AcrIIA4-Cdt1 was also confirmed (Fig. 3) it was difficult to efficiently control the activity of SpyCas9 by using different vectors having SpyCas9 or anti-CRISPR possibly due to variable amounts of plasmids in each cell an episomal vector coding SpyCas9 and AcrIIA4-Cdt1 genes via self-cleaving peptide 2A was newly constructed It was confirmed that the amount of ArIIA4-Cdt1 was dependent on cell cycle increased in G1 and decreased in the S/G2/M phases indicating that Cdt1 could be captured in the proteasome degradation in the cells Change of SpyCas9 expression level was not evident even when simultaneously expressed with AcrIIA4-Cdt1 AcrIIA4-Cdt1 showed efficient reduction of mutagenesis by NHEJ and off-target effects the efficiency of HDR was increased by the use of AcrIIA4-Cdt1 with SpyCas9 These results suggest that the degradation of anti-CRISPR at the S/G2 phase activates SpyCas9 and promotes DNA repair through HDR The use of ssODN as a template enhanced HDR efficiency a further step of SpyCas9 cleavage to make a short double stranded DNA could become a bottle neck of efficiency it is considered that ssODN can be used more efficiently as a template in the S/G2 phase in the AcrIIA4-Cdt1 and SpyCas9 co-expressing cells the use of ssODN increase target HDR/NHEJ ratio compared with the use of SpyCas9 alone which means target NHEJ showed higher efficiency than HDR and HDR efficiency could be increased by optimizing the homology arm of ssODN The first is the rapid recovery of active SpyCas9 from its suppressed status by anti-CRISPR This enables SpyCas9 to cleave the target sequences promptly after cells enter the S phase The second is the effect of SpyCas9 activity by fusing Geminin to its N-terminus SpyCas9 is in the native form except for the addition of a proline residue at the N-terminus after T2A peptide cleavage 293A cells (Thermo Fisher ScientificA) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2 After introduction of the episomal vector encoding Cas9 or AcrIIA4-Cdt1–2A-Cas9 by Lipofectamine 3000 the cells were selected using 350 μg/mL Hygromycin B solution (FUJIFILM Wako) for 3–7 days Lipofectamine 3000 (Thermo Fisher Scientific) was used for western blots and assessment of plasmid amount Neon® Transfection System 10 μL kit (Thermo Fisher Scientific) was used to assess endogenous HDR activity In western blot analysis and time lapse observation 500 ng of plasmid DNA were transfected by lipofection into 293A cells grown to 80–90% confluency repair template plasmid and sgRNA plasmid (each 250 ng) were transfected into 293A cells grown to 80–90% confluency by lipofection In the HDR assessment using ssODN as the template 50 pmol of ssODN and 250 ng of sgRNA plasmid were transfected into 5 × 104 cells using a pulse voltage of 1245 V The products were stored 4 °C in 3% dimethylsulfoxide until used PCR conditions of other genes followed manufacturers’ manuals PCR fragment DNA was purified using the QIAquick PCR Purification Kit (Qiagen) Fragment DNA (200 ng) was annealed in 19 μL of a solution containing ed 2 μL of 10 × NEBuffer 2 (NEB) using 95 °C for 10 min The annealed DNA received 1 μL of T7 endonuclease 1 and was incubated at 37 °C for 1 h Reacted samples were purified by the QIAquick PCR Purification Kit (Qiagen) DNA fragments were analyzed using MultiNA (SHIMADZU) where “a” and “b” represent the areas of cleaved fragments and “c” is the area of an uncleaved fragment 200 ng of amplified DNA reacted with 0.5 μL of XhoI or HindIII (NEB) in Cutsmart buffer (NEB) and 1 × bovine serum albumin (NEB) at 37 °C for 1 h (XhoI) or 3 h (HindIII) Reacted samples were purified by ethanol precipitation The indel efficiency of indel was calculated as: where “a” and “b” represent the areas of cleaved fragments and “c” represents the area of an uncleaved fragment 293A cells were seeded on 24-well plates at 4 × 104 cells/well and cultured in high-glucose DMEM (FUJIFILM Wako) containing 10% FBS and penicillin/streptomycin 500 ng of pFucci-G1 Orange Expression vector was transfected into 293 A cells using Lipofectamine 3000 following the manufacturer’s protocol transfected cells were seeded on 35 mm glass bottom dish (Greiner Bio-One) 24 h after transfection using phenol red free high-glucose DMEM (Thermo Fisher Scientific) containing 10% FBS and penicillin/streptomycin Expression of mKO2 was observed every 1 h up to 24 h by using FLUOVIEW FV10i (Olympus) 293A cells were seeded into 24-well plates at 4 × 104 cells/well and cultured in high-glucose DMEM containing 10% FBS and penicillin/streptomycin 500 ng of plasmids were transfected into cells using Lipofectamine 3000 following the manufacturer’s protocol and cells were cultured for a further 24 h Transfected cells were seeded on 35 mm glass bottom dish 24 h before observation Cells were fixed by 4% formaldehyde solution which was diluted from 16% formaldehyde solution (Thermo Fisher Scientific) into PBS for 10 min at room temperature (rt) Cells were permeabilized by 0.1% TritonX-100 (Merck Millipore) for 10 min at r.t and blocked by Blocking One (Nacalai tesque cells were incubated with anti-FLAG tag antibody (SIGMA) for 1 h at rt and anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody which is labeled by Alexa Fluor 594 (Thermo Fisher Scientific) for 30 min at rt Nucleus of cells were stained by Hoechst 33258 (DOJINDO) for 15 min at rt Observation of stained cells was performed using FLUOVIEW FV10i In vitro biochemical experiments were performed three independent times All other data (if any) are available upon reasonable request Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA and provide additional tools for evolutionary studies Multiplex genome engineering using CRISPR/Cas systems RNA-programmed genome editing in human cells RNA-guided human genome engineering via Cas9 Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system Efficient genome editing in zebrafish using a CRISPR-Cas system Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells Manipulating the mammalian genome by homologous recombination Regulation of DNA double-strand break repair pathway choice Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery A Cas9 variant for efficient generation of indel-free knockin or gene-corrected human pluripotent stem cells Post-translational regulation of Cas9 during G1 enhances homology-directed repair Inhibition mechanism of an anti-CRISPR suppressor AcrIIA4 targeting SpyCas9 Disabling Cas9 by an anti-CRISPR DNA mimic Inhibition of CRISPR-Cas9 with bacteriophage proteins Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein Naturally occurring off-switches for CRISPR-Cas9 Solution structure and dynamics of anti-CRISPR AcrIIA4 A role for the nuclear-envelope in controlling DNA-replication within the cell-cycle Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase Cdt1 degradation to prevent DNA re-replication: conserved and non-conserved pathways Inhibition of eukaryotic DNA replication by geminin binding to Cdt1 Structural basis for inhibition of the replication licensing factor Cdt1 by geminin Visualizing spatiotemporal dynamics of multicellular cell-cycle progression Ubiquitin ligases: cell-cycle control and cancer Interwoven ubiquitination oscillators and control of cell cycle transitions Crystal structure of Cas9 in complex with guide RNA and target DNA A genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) screen Identifies NEK7 as an essential component of NLRP3 inflammasome activation A retroviral CRISPR-Cas9 system for cellular autism-associated phenotype discovery in developing neurons Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage Easy quantitative assessment of genome editing by sequence trace decomposition Easy quantification of template-directed CRISPR/Cas9 editing Donor DNA utilization during gene targeting with zinc-finger nucleases CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA ssODN-mediated knock-in with CRISPR-Cas for large genomic regions in zygotes high-resolution mapping of protein localization in mammalian brain by in vivo genome editing A scalable strategy for high-throughput GFP tagging of endogenous human proteins Illegitimate DNA integration in mammalian cells Nuclear-gene targeting by using single-stranded DNA avoids illegitimate DNA integration in Chlamydomonas reinhardtii PCR artifact in testing for homologous recombination in genomic editing in zebrafish Improving CRISPR-Cas nuclease specificity using truncated guide RNAs High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects Rationally engineered Cas9 nucleases with improved specificity Enhanced proofreading governs CRISPR-Cas9 targeting accuracy Systematic discovery of natural CRISPR-Cas12a inhibitors Discovery of widespread type I and type V CRISPR-Cas inhibitors Download references The authors would like to thank Ms. Mayumi Fujisawa and Ms. Maiko Hoshino for their technical assistance, and Editage (www.editage.com) for English language editing This work was supported in part by the New Energy and Industrial Technique Development Organization (NEDO) of Japan the Japan Society for the Promotion of Science (JSPS) KAKENHI (JP25410171 and JP16K01931 to W.N.) JSPS Fellows (17J08531 to D.M.) and Grants-in-Aid for Scientific Research on Innovative Areas (JP24119506 Institute of Biomaterials and Bioengineering Graduate School of Biomedical and Health Sciences provided the scientific direction and the overall experimental design for the studies Tokyo Medical and Dental University and Hiroshima University have filed a patent application broadly relevant to this work are the investigators of record listed on the patent application Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s42003-020-01340-2 Sign up for the Nature Briefing newsletter — what matters in science The previously announced I Was Reincarnated as the 7th Prince so I Can Take My Time Perfecting My Magical Ability anime has released a new trailer and visual ahead of its premiere this April Tsumugi Akita Animation Lab is animating the adaptation under the direction of Jin Tamamura Additional cast members include Lynn as Sylpha Previously announced cast include Makoto Koichi as Lloyd and Fairouz Ai as Grim Naoki Tozuka is doing both the series composition and scriptwriting while Naru Nishikori is credited as the chief animation director and character designer Yuichi Abe will serve as the art director and R・O・N as the music composer The theme songs are “Kyunrious” by Kaede Higuchi as the opening and “The Secret of Happiness” by Akane Kumada as the ending It is based on a light novel series written by Kenkyo na Circle and illustrated by Meru It started out as a web novel and was later picked up by Kodansha and published under the Ranobe Bunko imprint A manga adaptation by Yosuke Kokuzawa is also currently ongoing on the Magazine Pocket platform Source: Official Website©Kenkyo naCircle Kodansha/”The Seventh Prince” Production Committee Metrics details Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however the mechanism of anaerobic microbial decomposition of HSs is not completely understood no microorganisms capable of anaerobic decomposition of HSs have been isolated we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp HSAI-1 isolated from the deep terrestrial subsurface The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions as determined using high-performance size-exclusion chromatography Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances as well as the generation of aliphatic components HSAI-1 anaerobically decomposes and transforms HSs This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface Given that the bacterium and HAs were collected from subsurface environments anaerobic HS decomposition may occur widely in situ Most of the decomposition products were found in lower molecular mass fractions as expected We isolated a strictly anaerobic HS-decomposing bacterium following enrichment cultivation in media containing commercial HSs (Aldrich HAs). The 16S rRNA gene sequence of the isolate was most closely related to that of Clostridium puniceum DSM 2619T (98.9% similarity) (Supplementary Fig. 1); therefore The growth of strain HSAI-1 was monitored based on the increase in cell numbers in media containing Aldrich HAs or naturally occurring HAs from the Koetoi diatomite layer with or without 0.5% glucose (Supplementary Table 1) The initial cell density in each experiment was set at 1.2–1.5 × 107 cells mL−1 A 10-fold increase occurred in 8 days in cultures supplemented with glucose whereas only a small increase was observed when HAs were the sole carbon source cultures were supplemented with 0.5% glucose 14CO2 evolution in an anaerobic culture of Clostridium sp The 14CO2 in the headspace gas was measured via liquid scintillation counting of inoculated cultures (filled circles) Uninoculated control cultures (open circles) yielded little or no 14CO2 The data points are the mean values of triplicate samples ± standard deviations *P < 0.05 was considered significant in Student’s t-test HSs are decomposed anaerobically by Clostridium sp chromatograms of Aldrich HAs at 0 and 28 days chromatograms of Koetoi HAs at 0 and 28 days Each experimental group consisted of 5 culture setups (n = 5) One representative chromatogram from each experimental group is shown chromatograms from uninoculated control cultures are depicted in blue and those from inoculated cultures (using strain HSAI-1) are shown in magenta The tops of the main peaks with retention times ranging from 7.8 to 8.3 min are indicated with vertical lines: a dotted line for the uninoculated controls and a solid line for the inoculated cultures The peak indicated by the downward-pointing arrowhead corresponds to the high-molecular mass HAs in cultures inoculated with strain HSAI-1 Representative FT-IR spectra are shown for Aldrich HAs or Koetoi HAs (n = 3) (b,d) Cultures inoculated with Clostridium sp This previous study assumed the involvement of undefined sulfate-reducing bacteria whereas our study focused on pure cultures of a Clostridium species this bacterial strain may be able to degrade biopolymers under anaerobic conditions in the context of the previously mentioned studies If anaerobic bacteria are stimulated by assimilable organic carbon existing in the subsurface environment then the decomposition of HSs might occur in the deep subsurface environment the anaerobic degradation of HA by strain HSAI-1 might produce monoaromatic compounds as degradation products of the HAs Determining the compounds that are produced after the decomposition of HAs will be the focus of a future study these results indicated that carboxyl groups in both the Aldrich and Koetoi HAs and alcohol or polysaccharide-related substances in the Koetoi HAs are decomposed by strain HSAI-1 and CO2 is released thus supporting the possibility of HA decomposition Similar substances may have also been produced in our Clostridium cultures the biodegradation of persistent organic matter in the deep subsurface can be better defined strain HSAI-1 was deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH Germany) and NBRC (NITE Biological Resource Center National Institute of Technology and Evaluation Japan) under the accession numbers of DSM 100957 and NBRC 111506 The samples were stored in sterilised 1-L polypropylene bottles and transferred immediately to our laboratory The mixture of groundwater collected from the three sites was used for the enrichment and cultivation of methanogens an aliquot of the enriched culture was used as the inoculum for the enrichment and cultivation of HA-degrading bacteria Siliceous mudstone samples were collected from the Koetoi Formation which is composed of diatomite from the Miocene to Pliocene epochs and used for preparing HAs as described below HA-containing solutions were filter-sterilised using membrane filtration units (pore size 0.22 μm) before addition to culture media was calculated by dividing the field number (FN) by the magnification of the objective (100×); “dispersed area” was the effective size of the region over which the sample was dispersed (i.e. the area of the stained region on the Isopore membrane filter which was 64π mm2 in our study); “sample volume” was the volume of bacterial sample applied to the Isopore membrane filter for staining; and “dilution factor” represented the degree to which the formalin-fixed bacterial sample was diluted with 1 × PBS The resulting 14C-HA pellet was re-suspended in 1 ml of 0.1N NaOH with a radioactivity of 0.12 × 106 dpm which contained approximately 0.11% of the original 14C-labelled catechol *Radioactivity of 14CO2 in the whole part of headspace (13.6 mL) = Radioactivity of 14CO2 in 500 μl × 27.2 Culture media were centrifuged at 20,000 × g for 30 min to separate the culture supernatant and cell debris and then the culture supernatant was transferred to new test tubes 10N NaOH was added to the culture supernatant to increase the alkalinity of the solution (>pH 12.0) to dissolve the HA completely and the samples were shaken vigorously on a reciprocal shaker (Type MK201D; Yamato Scientific Co The samples were centrifuged at 20,000 × g for 20 min to separate the insoluble fraction and supernatant The resulting supernatant was mixed with 6N HCl to increase the acidity of the solution (<pH 2.0) to precipitate the acid-insoluble HAs After centrifugation at 20,000 × g for 20 min the HA precipitate was rinsed twice with 0.1N HCl and then once with water the rinsed HA precipitate was lyophilized under vacuum overnight The dried HAs were stored at −20 °C until use HAs were extracted from culture media as described above HPSEC analysis was conducted using a Prominence HPLC (SPD-M20A Japan) equipped with a photodiode array detector and a GL-W530 column (Hitachi Hitec by 300 mm) preceded by a guard column (Hitachi Hitec; 4.0 mm I.D the approximate upper exclusion limit was 50,000 Da (calibrated with pullulan) Isocratic elution with a mobile phase mixture of 10 mM Tris-HCl (pH 8.0) and 10 mM NaCl was performed at a flow rate of 1.0 mL min−1 at a constant temperature of 40 °C The column was calibrated using sodium polystyrene sulfonates (1.3–168 kDa) Elution profiles were recorded and the resulting data were reanalysed with LC Solution software (Shimadzu FT-IR analysis was carried out by the Osaka Kankyo Gijutsu Center Co. FT-IR spectra were measured with a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific Inc. USA.) scanning the 4000–400 cm−1 range with an average of 128 scans and a spectral resolution of 4 cm−1 Statistical comparisons between the control and test groups were performed using Student’s t-test Differences were considered significant at P < 0.05 Anaerobic decomposition of humic substances by Clostridium from the deep subsurface The Geologic History of the Carbon Cycle in Treatise on Geochemistry 2nd edn Geochemistry of Humic Substances in Lake Sediments Structural alteration of humic acids by Pseudomonas spp from deep terrestrial subsurface diatomite formations in northernmost Japan Biodegradation of soil humic acids by Streptomyces viridosporus Chemical and biological studies on environmental humic acids II Spectroscopic studies of humic acids from subsurface sediment samples collected across the Aegean Sea Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus Isolation of soil Streptomyces strains capable of degrading humic acids and analysis of their peroxidase activity Some aspects of the biochemistry of humic acid decomposition by fungi Comparison of decolorization by microorganisms of humic acids with different 13C NMR properties Natural Organic Matter and Humic Colloids in Soil and Environmental Chemistry (ed Nature and Distribution of Humic Matter In Humic Matter in Soil and the Environment: Principles and Controversies Structural alterations of humic acid fractions in a steel slag-compost fertilizer during fertilization Analysis by pyrolysis/methylation-gas chromatography/mass spectrometry Biodegradation and biological treatments of cellulose Microbiology of Environmental Engineering Systems in Environmental Biotechnology The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications Stability of organic carbon in deep soil layers controlled by fresh carbon supply Molecular structure in soil humic substances: The new view Persistence of soil organic matter as an ecosystem property Soil Humic Substances in Biopolymers (eds Steinbüchel A Aromatic and volatile acid intermediates observed during anaerobic metabolism of lignin-derived oligomers Quinone moieties act as electron acceptors in the reduction of humic substances by humics-reducing microorganisms Novel electrochemical approach to assess the redox properties of humic substances Humic substances as electron acceptors for microbial respiration Extracellular electron transfer through microbial reduction of solid-phase humic substances Humic acid reduction by Propionibacterium freudenreichii and other fermenting bacteria Anaerobic mineralization of toluene by enriched sediments with quinones and humus as terminal electron acceptors Biodegradation of natural and synthetic humic acids by the white rot fungus Phanerochaete chrysosporium Microbial degradation and transformation of humic acids from permanent meadow and forest soils Analytical determination of the microbial utilization and transformation of humic acids extracted from municipal refuse Lignin degradation by Streptomyces viridosporus: isolation and characterization of a new polymeric lignin degradation intermediate Anaerobic biodegradation of the lignin and polysaccharide components of lignocellulose and synthetic lignin by sediment microflora Anaerobic degradation of soluble fractions of [14C-lignin]lignocellulose Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan Microbial communities associated with acetate-rich gas-petroleum reservoir surface facilities Organic matter characterization in Methods of soil analysis Part 3 a novel methanogenic archaeon isolated from paddy field soil in Japan and DNA-DNA hybridization among Methanoculleus species The anaerobic mesophilic cellulolytic bacteria Differential staining of bacteria in clinical specimens using acridine orange buffered at low pH Enumerating bacterial cells on bioadhesive coated slides Nucleic Acid Techniques in Bacterial Systematics Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species The neighbor-joining method: a new method for reconstructing phylogenetic trees MEGA5: molecular evolutionary genetics analysis using maximum likelihood evolutionary distance and maximum parsimony methods The catalytic role of uranyl in formation of polycatechol complexes Download references This work was supported by the Ministry of the Economy We would like to acknowledge the Japan Atomic Energy Agency (JAEA) for the collection of subsurface groundwater samples at the Horonobe Underground Research Laboratory We are grateful to the Japan Petroleum Exploration Co. for their cooperation in sample collection We gratefully thank the Central Institute of Isotope Science for accommodating us with the facilities for the isotope experiments Masami Fukushima from the Graduate School of Engineering of Hokkaido University for technical advice regarding HA analysis We appreciate the technical assistance provided by Mr Horonobe Research Institute for the Subsurface Environment Northern Advancement Centre for Science and Technology Graduate School of Environmental Earth Science performed the experiments and wrote the manuscript with input from the other authors prepared an enrichment culture to isolate Clostridium sp strain HSAI-1 and discussed the content of the present study participated in discussions about the content of the study The authors declare no competing financial interests Download citation I Was Reincarnated as the 7th Prince so I Can Take My Time Perfecting My Magical Ability series is getting an anime adaptation by studio Tsumugi Akita Animation Lab Teaser visual and trailer have been revealed The main cast will include Makoto Koichi as Lloyd and Fairouz Ai as Grim I Was Reincarnated as the 7th Prince so I Can Take My Time Perfecting My Magical Ability is Japanese light novel series written by Kenkyo na Circle and illustrated by Meru It started out as a web novel and was later picked up by Kodansha and published under their Ranobe Bunko imprint Kodansha is publishing the manga in English and describes the plot as: Also read:My Daughter Left the Nest and Returned an S-Rank Adventurer Gets Anime the upcoming remake of the anime series Shaman King released a new promotional video the promotional video revealed the opening and closing theme songs The anime is slated to premiere on April 1 and will be produced by Bridge under the direction of Joji Furata The opening theme song is titled “Soul salvation” while the closing is titled “Boku no Yubisaki.” Fun fact: Megumi Hayashibara previously performed two opening and two closing theme songs for the 2001 anime.SynopsisShamans possess mysterious powers that allow them to commune with gods and even the dead…and Manta Oyamada’s about to learn all about them because his class just welcomed a new transfer student: Yoh Asakura a boy from way off in Izumo…and a shaman in training Synopsis via Kodansha ComicsAlso you can watch the new Shaman King promotional video here.In summary The new promotional video also revealed the opening and closing theme songs to be performed by Megumi Hayashibara Moreover, you can access all of our stories here!Source: Shaman King Official Website and Official Twitter©武井宏之・講談社／SHAMAN KING Project.・テレビ東京Copyright © 2021 Kodansha Advanced Media The second season of I Was Reincarnated as the 7th Prince So I Can Take My Time Perfecting My Magical Ability will premiere in July The anime's first season cast and staff are returning for the second season The anime's first season premiered on TV Tokyo and BS NTV in April 2024. Muse Asia streamed the anime as it aired in Japan The first anime season premiered by streaming exclusively in Japan on Amazon Prime Video and Hikari TV in January 2023. Muse Asia streamed the anime as it aired in Japan Source: Muse Asia's Facebook page (link 2) Kimamani Majutsu o Kiwamemasu) light novel series will premiere in July and also revealed a teaser visual and two new cast members The anime will have a special stage at the AnimeJapan 2025 event on March 22 Yōsuke Kokuzawa and character designer Meru launched a manga based on Kenkyo na Circle's novels in Kodansha's Magazine Pocket app in 2020 Kodansha will published the manga's 18th compiled book volume on February 7 Kenkyo na Circle premiered the series on the Shōsetsuka ni Narō (Let's Become Novelists) website in October 2019 Kodansha began publishing the light novels with illustrations by Meru in July 2020 The light novels' eighth volume shipped in Japan in July 2024 Popular GA Bunko novel Rakudai Kishi no Cavalry is getting an anime adaptation Through the anime was previously announced to be progressing as the third part in GA Bunko’s 10th anniversary project the animation production staff and cast have finally been announced It has also been revealed that the anime will be a TV anime but with the release of information on the staff and cast it finally feels like the anime is coming together who has created many hits including Baka and Test and Ef: A Tale of Memories and Sei Komatsubara is the character designer Silver Link and Nexus are producing the animation It will be interesting to see what kind of series this group of staff will create The cast as well will likely spark attention a student who transfers into a school for training Mage Knights Because of his ease at handling large roles one after the next there will be anticipation over how he will perform in this series Additional cast includes Shizuka Ishigami as Stella Vermillion a princess of the Vermillion Kingdom whom Ikki meets and who changes his fate Rakudai Kishi no Cavalry is a “school life sword action” novel written by Riku Misora and illustrated by Won Six volumes of the novel have been published and two volumes have been published so far by Square Enix the series has over 5 million copies in print Anime Rakudai Kishi no Cavalry Official Site [Main Staff]Director: Shin OnumaSeries Director: Jin TamamuraSeries Composer: Shogo YasukawaCharacter Designer: Sei KomatsubaraSound Director: Jin AketagawaAnimation Production: Silver Link [Main Cast]Ikki Kurogane: Ryota OsakaStella Vermillion: Shizuka Ishigami Shizuku Kurogane: Nao ToyamaNagi Arisuin: Shintaro AsanumaOthers Anime Rakudai Kishi no Cavalry© Riku Misora SB Creative / Rakudai Kishi no Cavalry Production Committee Source: animeanime There’s been little info about the new anime series *Rampo Kitan: Game of Laplace* since it was unveiled at Noitamina’s "Announcement Conference 2015" in November 2014 *A Dog of Flanders* donuts introduced to *World Masterpiece Theater* donut lineup on May 7 The official website for Shaman King Flowers, the sequel to the recent television anime of Hiroyuki Takei's Shaman King manga started streaming the anime's second promotional video on Tuesday and it revealed new and returning cast members The anime will premiere on January 9 on the TV Tokyo channel at 24:00 JST (effectively, January 10 at midnight JST or January 9 at 10:00 a.m. EST). It will then run on BS TV Tokyo is performing the anime's opening theme song "Turn the World." Sumire Uesaka is performing the ending theme song "Dear Panta Rhei." The new Shaman King anime premiered in April 2021. Netflix began streaming the anime worldwide in August 2022. The anime ended with 52 episodes The anime adapted all 35 volumes of the original manga's new complete edition, which Kodansha started publishing in print volumes in Japan in June 2020 The first anime adaptation of the manga premiered in 2001 Sources: Shaman King Flowers anime's website, Mainichi Shimbun's Mantan Web Your browser does not support JavaScript, or it is disabled．Please check the site policy for more information MAEBASHI--Gunma Prefecture is trying to cash in on its reputation as a relatively low-risk area for earthquakes floods and other natural disasters to entice companies to set up shop in this inland region of central Japan Telecom giant Nippon Telegraph and Telephone Corp is set to shortly transfer some functions of its main office currently handled in Tokyo to Takasaki famous for its Osaka Ohsho brand of frozen gyoza recently built a factory in the prefecture “Gunma Prefecture is known for its relatively lessened temblor risk,” noted a representative of Eat&Foods’ parent firm “The historical record also shows that typhoons and inundation from torrential downpours caused damage there on fewer occasions.” Eat&Foods has two factories in Itakura and started construction on a third plant there last December “Minimizing damage from natural disasters while ensuring stability of food production is essential for sustainable business activities,” the company official added “Reducing the danger of a disaster striking is one of the most important factors.” Gunma prefectural authorities emphasize its “lower disaster risk.” is whether Gunma Prefecture is really resistant to calamities the local government developed a map to show how many times temblors with an intensity of 4 or stronger on the Japanese seismic scale of 7 occurred between January 1919 and March 2022 in Gunma Prefecture and surrounding regions The data revealed that 73 such quakes hit Gunma 187 and 159 were reported in neighboring Tochigi Gunma Prefecture experiences fewer earthquakes The central government’s 2020 National Seismic Hazard Maps for Japan released in March last year by the Headquarters for Earthquake Research Promotion displays details of the projected risk of each region being hit by a quake measuring lower 6 or stronger within 30 years Among prefectural capitals in the Kanto region Maebashi logged the lowest risk of 6.4 percent Chiba at 62.3 percent and Mito at 80.6 percent Gunma Prefecture came up trumps when it came to the likelihood of being inundated by torrential rains According to land ministry statistics on flooding Gunma suffered 55.9 billion yen ($390 million) in flood damage over the 10 years from 2011 through 2020 about half or less the figures for Tokyo and five other prefectures in the Kanto region A nationwide geological ranking released in 2016 by ground survey firm Jibannet Co in Tokyo explains Gunma Prefecture’s ability to withstand natural disasters Jibannet calculated the overall geological security ranking for 10.9 million building plots The average level for each prefecture was evaluated The findings showed Gunma Prefecture scored 78.99 ranking second nationwide only after Okinawa Prefecture at 82.75 Other prefectures in northern Kanto earned high rankings with Tochigi placing fourth at 78.63 and Ibaraki seventh at 75.19 “Ash from ancient eruptions of volcanoes in the Kanto region helped form flat plateaus in many areas,” said a Jibannet representative in explaining the rankings Now that reduced disaster risks are backed up by a range of data Gunma Prefecture is going all-out to attract capital investment “Awareness of disaster risks has heightened since the Great East Japan Earthquake and tsunami in 2011 and recent large-scale floods,” said an official from the prefecture’s future investment and digital industry division “Companies are increasingly apt to pick out sites with fewer disasters on record in the hope of conducting stable The prefectural representative said a high level of safety offers an advantage in luring companies “We are stressing that our prefecture is characterized by fewer earthquakes and floods,” the official added EDITORIAL: Anti-disaster efforts need a check on Disaster Prevention Day EDITORIAL: How to respond to disasters key issue for LDP candidates Preparing for next emergency still the norm following 3/11 More than 1,000 residents still in temp housing from 2018 floods Information on the latest cherry blossom conditions Please right click to use your browser’s translation function.) A series based on diplomatic documents declassified by Japan’s Foreign Ministry Here is a collection of first-hand accounts by “hibakusha” atomic bomb survivors chefs and others involved in the field of food introduce their special recipes intertwined with their paths in life A series about Japanese-Americans and their memories of World War II In-house News and Messages No reproduction or republication without written permission Please view the main text area of the page by skipping the main menu. The page may not be displayed properly if the JavaScript is deactivated on your browser Japanese version Silva and Kalim are members of the Patch Tribe and Silva is one of the Ten Priests that officiate the Shaman Fight The anime will premiere on TV Tokyo and its affiliates in April, and will adapt all 35 volumes of the manga's new complete edition, which Kodansha started publishing in print volumes in Japan on June 17 Megumi Hayashibara is once again performing the anime's opening song "Soul salvation" and ending theme song "#Boku no Yubisaki" (My Fingertip) (She performed two opening songs and two ending songs for the 2001 anime.) Shueisha originally held the rights to the manga in Japan Sources: Shaman King anime's website, Comic Natalie The official website released a new commercial for the anime “ Tensei Shitara Dainana Ouji Datta no de Kimamani Majutsu o Kiwamemasu” ( I Was Reincarnated as the 7th Prince So I Can Take My Time Perfecting My Magical Ability ) Jin Tamamura (The Girl in Twilight) is directing the anime at Akita Anime Lab with a script by Naoki Tozuka the life of a magical freak came to an end after a terrible encounter with nobles who ended his life with a powerful spell His last wish had to be granted by reincarnating as Lloyd of Saloom Now he will be able to perfect his magic as he pleases… Kokuzawa and character designer Meru then launched a manga adaptation based on the Kenkyo na Circle novel on Kodansha's Magazine Pocket app in 2020 Kenkyo na Circle debuted 'Tensei Shitara Dainana' on the Shousetsuka ni Narou in October 2019 Kodansha began publishing light novels featuring Meru's illustrations in July 2020 Source: Official Website All images on this site belong to their respective owners The official Twitter account for the television anime of Kenkyo na Circle's I Was Reincarnated as the 7th Prince So I Can Take My Time Perfecting My Magical Ability (Tensei Shitara Dainana Ōji Datta no de Kimamani Majutsu o Kiwamemasu) light novel series announced four more cast members and the April 1 premiere for the anime on Tuesday The anime will premiere on TV Tokyo and BS NTV on April 1 at 24:00 (effectively, April 2 at midnight or April 1 at 11:00 a.m. EDT), and on AT-X on April 3 Kaede Higuchi performs the opening song "Kyunrious." Akane Kumada will perform the ending theme song "Happy no Himitsu" (The Secret of Happiness) Kodansha published the manga's 14th compiled book volume on February 9 The light novels' seventh volume shipped in Japan in November 29 Source: I Was Reincarnated as the 7th Prince anime's Twitter account Sentai Filmworks revealed in February that it was making an English dub for the anime. A teaser video showed Sentai Filmworks ADR Director Christopher Ayres working on the title The series premiered in Japan in October 2015 and Sentai Filmworks streamed the series on Hulu as it aired. The company announced in 2015 that it had licensed the anime The "school sword action" story revolves around Magic Knights modern magic-users who fight with weapons converted from their souls Ikki Kurogane goes to a school for these Magic Knights but he is the "Failed Knight" or "Worst One" who is failing because he has no magical skills a foreign princess and the "Number One" student Japan is well known for its beer, and many a skier has celebrated a successful day on the ski slopes with an ice cold Asahi, Kirin, Sapporo or Yebisu. But with Japan’s craft beer scene now hitting full stride it makes sense that we’re now seeing the emergence of smaller craft breweries in ski resort towns around the country Or the (somewhat) recent arrival of a foreign après-ski culture and a thirst for a greater variety of beers there are several breweries making their mark on ski resorts in Japan Dan Cockburn is living many people’s dream falling in love with the town and its now world-renowned winters With some brewing experience under his belt and feeling a desire for a greater variety of beer that he was used to back home in the UK “It quickly became apparent what I was meant to be doing in Hakuba and so we started the long road to building a brewery and getting it licensed.” This season will be the brewery’s fifth year in operation and with renovations carrying on over the summer which Cockburn says is currently his favourite rich and complex malt base is matched with the unique hop of Chinook” The Hakuba Amber is Dan’s current favourite (for now) modernised with American hops and then given a Japanese touch with the wonderfully clean mountain water we use We work up all our recipes to ignore the conventional style rules but to be accessible to the local Japanese as well as foreign visitors whilst being unique and interesting enough for the connoisseur It’s a fun challenge that involves regular taste-testing!” Shiga Kogen is mostly known for its skiing – it was a venue for the 1998 Winter Olympics – and its proximity to the famous Jigokudani snow monkeys though visitors to the area will likely have come across Shiga Kogen Beer A selection of Shiga Kogen beers. Image: snowmonkeytrip.com Since 2004, Head Brewer Sato Eigo has been making Shiga Kogen Beer at Tamamura Honten, a company with a 200-year history in sake production. As a Shiga Kogen local, he takes pride and inspiration from his natural surroundings; the brewery produces its own hops, sake rice, barley, wheat, buckwheat (soba) and blueberries. In a 2013 interview with The Japan Beer Times Sato proclaimed that “as this [Shiga Kogen] is a ski resort worthy of the world I want to make a beer here of equal stature” “We produce a lot of different beer and they’re all our babies” onsen and the “overwhelming kindness of the locals” In 2014 he opened Libushi – home to a taproom and brewery – though he has since expanded the brewery to a larger site also in Nozawa Tom naturally recommends their “Nozawa Series” though he is particularly passionate about Libushi’s Barrel and Foeder aged beers His favourite of these is the King Kong Knee Drop a 9.5% stout aged in Ichiro’s malt whiskey barrels giving the ski resort that is known almost as much for its partying as its powder its first ever micro brewery located in Niseko Town where it overlooks the train station a Belgian ale and an aptly named Miyuki (Deep Powder) IPA Niseko Brewing Restaurant © Niseko Promotion Board Green season visitors are in for the real treat though with a variety of seasonal beers that are as colourful as the Hokkaido summer This season’s variety include a rosée biere which uses juice from nearby Subetsu cho – a fruit-growing region in Hokkaido that is known for its mineral-rich Myoko Kogen Beer’s Baked Tomato Spicy Ale Myoko’s Tatra-kan beer hall is one of the Japan’s oldest ski resort breweries launching in 1993 under the guidance of U Fleků – a 500-year-old brewery/restaurant in Prague the European influence on Tatra-kan’s menu and decor is overwhelming with a beer list that features a Czech pilsner a German hefeweizen (brewed using a Bavarian recipe) The brewery has earned several major accolades at the World Beer Awards most recently picking up gold medals in the 2019 Japan category for its pilsner The more adventurous drinkers amongst you should also look out for the Myoko Kogen Beer seasonal brews which this year featured a Baked Tomato Spicy Ale (pictured) Voting is now open – your chance to support the resorts accommodation and service providers that have delivered the best experiences to their guests You’ll also have the chance to win a $150 eGift voucher from backcountry.com VOTE NOW Δdocument.getElementById( "ak_js_1" ).setAttribute( "value" Ski Asia’s bi-monthly newsletter with the latest news Crunchyroll will stream the anime as it airs in Japan Kodansha published the manga's 11th compiled book volume on May 9 The light novels' sixth volume shipped in Japan in December 2022 Sources: I Was Reincarnated as the 7th Prince So I Can Take My Time Perfecting My Magical Ability anime's website, Comic Natalie Nagi searches for her sister in the parallel world She has led a difficult life in a cruel world and cannot relate to the normal lives of Asuka and the others She begins to open up to Asuka and her friends as she travels with them The game will launch in October for iOS and Android devices, as well as for PCs (through browsers via DMM Games) The game shares the anime's original scenario writer Shogo Yasukawa (Food Wars! Shokugeki no Soma, Mitsuboshi Colors, Hyperdimension Neptunia) is writing and supervising the anime's scripts. Writer Hiroshi Yamamoto is credited for "SF setting." Ryu☆ (pronounced "Ryūsei," aka Bemani game series composer Ryūtarō Nakahara) is the music producer, and Kenji Itō (Romancing SaGa, Puzzle & Dragons games) is writing the main theme song. Michi is singing the opening theme song "Soranetarium," and Ami Wajima is singing the ending theme song "Kowarekake no Radio" (Broken Radio) Sources: The Girl in Twilight game's official website, MoCa News The official website for Animax's original television anime The Girl in Twilight (Akanesasu Shōjo) revealed a third key visual on Thursday for the anime. Manga creator Masakazu Katsura drew the illustration Sources: The Girl in Twilight anime's website, Comic Natalie Shaman King Funbari Chronicle is currently the hottest anime turn-based RPG that saw its release in Japan by Studio Z Inc We understand players are excited and wanted to know what are the most powerful Shaman’s to use Don’t worry we got you covered with this tier list for the best Shamans in Shaman King Funbari Chronicle Pleas note that players will be given a 10x free gacha draws after the tutorial and this gacha can be rerolled until you get your favourite characters so the reroll process is just super quick!!! But be aware that not all the characters in this tier list are available in the initial reroll summons there are characters that are exclusive in the normal Rare Gacha The normal gacha only features a 2% drop rate for 3-star characters is a strong DPS unit that pairs well with Yoh Asakura Technique type She provides an Attack boost to all allies and recover the HP of all allies every turn She also increases 50% defense of all allies with her 3rd ability Yata No Kagami is a strong single target DPS unit that is able to boost the attack of allies This Attack boost further increases if there is Anna Kyoyama Technique in the team He is one of the hardest-hitting Shamans in the game currently with his Mystery Skill multiplier being 1000% damage to a single enemy He also increases his Defense by 50% for 3 turns with his 2nd skill Amidamaru Shield *Noted that ★3[Lose While Protecting] Yoh Asakura is not available in the initial reroll summons is a great debuffer that can apply Poisons and Bleed to all enemies using his Mystery Skill His 3rd ability Bone Bind is an AOE disabling skill that make the targets sleep and applies Bleeding he’s one of the best debuffers in the game He can apply Poisons even with his basic attacks is a 3-Star Shaman which players can obtain by completing the Funbari Dash Missions She is a great debuffer and good supporter She can inflict 1 stack of Sleep paralysis on a single target while dealing 750% damage using her Mystery skill She also heals an ally using her 2nd ability Tamao’s allowance Her 3rd skill cleanses all debuffs on an ally but this skill can be enhanced to cleanse debuff from all allies Her easy-to-get nature also increase her evaluation is  a great control type unit has he can AOE freeze all enemies using his Mystery Skill He also inflicts Critical Hit Rate down and Defense down debuffs using his 3rd ability This Is The Power of Nature *Noted that ★3 [The Power to Challenge] Horohoro is not available in the initial reroll summons is a good DPS unit that deals 700% damage while inflicting the Incapacitation effect on the target He pairs with Doren and if Doren is in the team He also provides a 25% Attack boost for all allies for 3 turns with his 3rd ability Cheesecake His 2nd skill grants him Avoidance for 3 turns *Noted that ★3 [Nothing Good Happens Even If  You Fall For Me] Horohoro is not available in the initial reroll summons is a single targeted damaging ability that increases his own self-healing ability for 3 turns He can tank for your team as he Provokes all enemies and grants Guts buff to herself using his 3rd ability Inspiration and Conch Shell Guts buff gives her a 50% chance to not die when dealt with a fatal hit She also can reduce the attack power of a single target using her 2nd skill The Spirit of the Trainee *Noted that★3 [Unemployed] Mikihisa Asakura is not available in the initial reroll summons ★3 [Won’t Let the Food Escape] Marion Fauna  can deal 700% damage to a single target while inflicting 20% attack down using her Mystery skill Her 3rd ability Don’t Let the Food Escape reduces the defense of a single target Her 2nd skill Delta Magnum is an AOE targeted attack that deals 75% damage to all enemies *Noted that★3 [Won’t Let the Food Escape] Marion Fauna is not available in the initial reroll summons deals AOE targeted damage to all enemies using her Mystery Skill but his multipliers are low at 500% Lyserg increases his Mystery Gauge and deals damage to all enemies with his 3rd ability Pendulum Wave His 1st skill is a single targeted damaging skill that can inflict Sleep on the target *Noted that★3 [Dowsing Revolution] Lyserg Diethel  is not available in the initial reroll summons Shaman King Funbari Chronicle – 5 Tips to Help You Get Started Official Site Official Twitter The sequel series to the original 2001 Shaman King anime Fans of the original anime and the source materials can expect to see the new series when it arrives on Netflix on April 21 Shaman King: Flowers was first released in January this year in Japan It is an adaptation of the same-name manga series by Hiroyuki Takei and it continues the narrative of the original series the new series follows the adventures of Hana Asakura (Anna and Yoh’s son) and his adventures in the Shaman world Hana has grown up to become a rather lazy and brutal kid who neglects his studies, skips classes, and gets into gang fights—that is until he learns about the upcoming battle among Shaman Kings From what we know so far, Shaman King: Flowers adapted all 29 chapters of the manga into 13 episodes of the anime which isn’t all that different from the 2021 reboot of the original considering that the same anime studio that worked on the reboot also handled the production of the new series with Takeshi Furuta as the director and Shoji Yonemura in charge of the anime’s script much of the core voice cast from the original is reprising their roles in Shaman King Flowers The reboot introduced Satohiko Sano’s replacement as character designer which resulted in some big changes to the character design like Tamamura looking older and sporting longer hair The massively popular franchise still has plenty of material that hasn’t been adapted into the anime form and it’s highly unlikely that Shaman King: Flowers will be the last anime release given the number of spin-offs the original manga has spawned ' + scriptOptions._localizedStrings.webview_notification_text + ' " + scriptOptions._localizedStrings.redirect_overlay_title + " " + scriptOptions._localizedStrings.redirect_overlay_text + "