Metrics details Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver By establishing a simple method of discriminating between apoptosis and necrosis in the liver we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient To further investigate what type of necrosis is involved in the initial necrotic cell death we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice which is lacking in a terminal executor of necroptosis necroptosis inhibition failed to block the onset of necrotic cell death while ferroptosis inhibition protected hepatocytes from necrotic death almost completely and suppressed the subsequent infiltration of immune cells and inflammatory reaction the amount of oxidized phosphatidylethanolamine was increased in the liver sample of the CDE diet-fed mice These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis the CDE model utilizes the supplementation of ethionine instead of methionine deficiency which is a methionine analog for methionine starvation We first determined the timeframe for the onset of steatohepatitis based on elevation of serum markers for liver damage We then aimed to identify the initial cell death that occurred within this timeframe We have also established a method for distinguishing apoptosis and necrosis easily in the liver and have evaluated the type of hepatocyte cell death by using cell death-related inhibitory agents and knockout mice in this model we discovered the role of ferroptosis as the initial cell death that triggers steatohepatitis the role of ferroptosis in liver disease remains poorly understood we focus on the type of cell death that initially occurs at the earliest stage of NASH Our data reveal that ferroptosis is the type of necrosis that precedes the other type of cell death thus giving cues to initiate inflammation in this model Our findings suggest the role of ferroptosis as a trigger at the onset of steatohepatitis and fibrosis in the liver were evaluated after choline-deficient Six mice were used for analyses at each time point a Representative images of lipid accumulation in the liver by Oil Red O staining after 0 b Measurement of serum liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after the CDE feeding (n = 6; **P < 0.01 c Fluorescence images of infiltration of immune cells by immunohistochemistry of liver sections for CD11b The number of CD11b-positive cells was evaluated in 20 non-overlapping fields of view for each biological sample The data are shown as the mean ± SEM (n = 6; **P < 0.01) d Gene expression analysis of inflammatory cytokines by quantitative reverse transcriptase PCR (RT-PCR) All data are normalized to Gapdh and shown as the means ± SEM (n = 6 e Representative images of liver fibrosis by Picro-Sirius Red staining f Expression analysis of fibrosis-related genes by quantitative RT-PCR All data are normalized to Gapdh and shown as the means ± SEM (n = 6; *P < 0.05 a Measurement of serum liver injury markers after 24 h of control (vehicle) and CCl4 treatment (n = 4; *P < 0.05) b HE staining of sections from control and CCl4-treated livers c Fluorescence images of PI- and CC3-stained cells in control and CCl4-treated livers d Fluorescence images of PI- and CC3-stained cells in the liver after 2 days of normal diet or the CDE diet feeding suggesting that hepatic necrosis initially begins around the portal vein after CDE feeding a Experimental design for identifying apoptosis and necrosis at the onset of steatohepatitis b Western blot analysis of CC3 protein in the liver extracts after the CDE diet feeding c Detection of apoptotic and necrotic cells in the liver after 14 to 18 h post CDE diet feeding by immunohistochemistry for CC3 and the in vivo necrosis assay d Measurement of serum liver injury markers after CDE diet feeding e Gene expression analysis of TNFα after the CDE diet feeding by quantitative RT-PCR All data are normalized to Gapdh and shown as the mean ± SEM (n = 5; *P < 0.05 a Experimental scheme for the administration of inhibitory agent b Serum liver injury marker levels of vehicle or Nec-1s (5 mg/kg) treated mice after 18 h of the CDE diet feeding (n = 6) c Detection of necrotic cells in the CDE-fed mouse treated with vehicle or Nec-1s The number of PI-positive cells was evaluated in 10 non-overlapping fields of view for each biological sample The data are shown as the means ± SEM (n = 6) d Gene expression analysis of inflammatory cytokines by quantitative RT-PCR All data are normalized to Gapdh and shown as the means ± SEM (n = 6) e Western blot analysis of phosphorylated RIPK3 (p-RIPK3) and β-actin in the liver extracts after the CDE diet feeding and ZVAD-FMK (L929 + TBZ) were used as control for necroptotic cell death a Serum liver injury marker levels of WT and Mlkl KO mice after 18 h of the CDE diet feeding (n = 5) b Detection of necrotic cells in the liver of WT and Mlkl KO mice at 18 h post CDE feeding by the in vivo necrosis assay The data are shown as the means ± SEM (n = 5) c Gene expression analysis of inflammatory cytokines by quantitative RT-PCR All data are normalized to Gapdh and shown as the means ± SEM (n = 5; *P < 0.05) a Serum liver injury marker levels in control (vehicle) or Trolox (100 mg/kg) treated mice (n = 9) b Detection of necrosis in the CDE-fed mice treated with vehicle or Trolox by the in vivo necrosis assay The data are shown as the means ± SEM (n = 9; *P < 0.05) c Immunohistochemical analysis of CDE-fed mouse with vehicle or Trolox for CD11b The number of CD11b-positive cells was evaluated in 10 non-overlapping fields of view for each mouse d Gene expression analysis of inflammatory cytokines by quantitative RT-PCR (n = 9) All data are normalized to Gapdh and shown as the means ± SEM (n = 9; *P < 0.05 e Measurement of oxidized PEs in the liver The level of each oxidized PE in the liver sample was compared among normal diet-fed mice and Trolox-treated CDE diet-fed mice (n = 4) a Serum liver injury marker levels in the CDE-fed mice treated with vehicle or DFO (100 mg/kg) (n = 9) The serum data are shown as dot plots and mean b Serum liver injury marker levels of the CDE-fed mice treated with vehicle or DFP (100 mg/kg) (n = 9; **P < 0.01 c Detection of necrotic cells in the CDE-fed mice treated with vehicle or DFP The data are shown as the means ± SEM (n = 9; **P < 0.01) d Immunohistochemical analysis of CDE-fed mouse treated with vehicle or DFP for CD11b The number of CD11b-positive cells was evaluated in 10 non-overlapping fields of view for each biological sample The data are shown as the mean ± SEM (n = 9; *P < 0.05) e Gene expression analysis of inflammatory cytokines by quantitative RT-PCR The gene expression data are normalized to Gapdh and shown as the means ± SEM (n = 9; **P < 0.01 Along with the advance in understanding of each programmed cell death much attention has been paid to the pathophysiological roles of multiple types of cell death in a wide range of diseases including hepatic failure Unlike the detection method of apoptosis such as TUNEL or CC3 staining the difficulty of identifying necrosis in vivo has hampered the acquisition of regional information about apoptosis and necrosis in a wide area of injured tissue we designed a new strategy by applying the PI staining for in vivo analysis and showed that initial cell death occurred predominantly in the periportal area at the onset of steatohepatitis This method may be applicable to the other tissues These studies strongly suggest that the pathological progression of NASH is governed by multiple types of cell death their involvement in the initial hepatic cell death at the onset of steatohepatitis has remained unclear because it is difficult to specify when hepatocytes begin to die in fatty liver in long-term experimental settings Considering that the liver is the major organ of xenobiotic metabolism continuous administration of chemical agents may not be suitable for follow-up examination in chronic liver injury models Given that multiple types of cell death are involved in the progression of disease state in NASH the effect of ferroptosis inhibition after the onset of steatohepatitis may become limited in conventional NASH models in which the hepatic cell death occurs within a short period of time in the context of steatosis we focused on the initial cell death at the onset of steatohepatitis These results suggested that ferroptosis is the initial necrosis to be the trigger for steatohepatitis necroptosis inhibition by using Nec-1s or Mlkl KO mice could not block the initial necrotic cell death Mlkl KO mice showed a tendency of decreasing inflammatory cytokine expression implying that necroptosis in the non-parenchymal cells might be involved in the exacerbation of inflammation in NASH Although it is unclear whether ferroptosis is followed by necroptosis and apoptosis in the progression stage of NASH there is evidence supporting the hypothesis that ferroptosis occurs in parallel with the other types of cell death in NASH patients as described above Total inhibition of multiple cell deaths relevant to NASH pathogenesis could prove to be effective in the treatment of NASH elucidation of the correlation among multiple types of cell death in the pathological process of NASH is crucial our data suggested that ferroptosis plays a potential role as the trigger for steatohepatitis Ferroptosis is a promising therapeutic target for the prevention of onset of steatohepatitis it is necessary to take ferroptosis into account for the therapeutic strategy of NASH Our findings will provide a new insight into the type of cell death relevant to the pathogenesis of NASH Male C57BL/6J mice were purchased from CLEA Japan, Inc. Mlkl−/− mice were provided by M. Pasparakis and described prebiously41 All animal experiments were conducted in accordance with institutional procedures and admitted by the Animal Care and Use committee of the Institute for Quantitative Biosciences the University of Tokyo and Research Institute the National Center for Global Health and Medicine We used 5- to 6-week-old mice for feeding CDE diet (MP Biomedicals and harvested liver and blood samples for further analyses ALT and AST were measured using Transaminase C-II Test Wako (Wako Japan) according to the manufacturer’s instruction USA) was dissolved in phosphate-buffered saline (PBS) at a final concentration of 25 µg/ml For the detection of necrotic cells in the liver the PI solution was injected into mice intravenously via the tail vein The liver samples were harvested and snap frozen in liquid nitrogen 10 min later The frozen block was cut into slices with 8 µm thickness by using Microtome Cryostat HM 525 (Thermo Fisher Scientific Industries the samples were counterstained with Hoechst Merged images were captured using BZ-X710:BZ-X Viewer (KEYENCE The number of PI-positive cells was counted using Hybrid Cell Count function in the Dual Signal Extraction mode of BZ-X Analyzer An average value of 10 random images per mouse was treated as a representative value for the mouse We injected CCl4 dissolved in olive oil (2 mg/kg; Wako) into 8-week-old mice intraperitoneally and harvested liver samples after 24 h we collected blood samples for measurement of serum AST and ALT We used 5- to 6-week-old mice for this experiment The inhibitor was injected intraperitoneally in four doses every 2 h from 10 to 16 h and then mice were sacrificed for evaluation at 18 h the in vivo necrosis assay was performed as described before The final dose of used inhibitor used is as follows: Necrostatin-1s (5 mg/kg; Focus Biomolecules Trolox was initially dissolved in a small amount of DMSO (Sigma) Nec-1s and Rosiglitazone were first dissolved in DMSO wild-type (WT) and Mlkl KO mice were analyzed as described above the sections were stained with Mayer’s hematoxylin solution (Wako) after rinsing with PBS the sections were washed with running water stained with Oil Red O staining solution (SIGMA) the sections were washed with running water and mounted with fluoromount (Cosmo Bio fixed samples with 4% paraformaldehyde were blocked with PBS containing 1% bovine serum albumin and 0.3% Triton X-100 The second antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 647 (Thermo Fisher Scientific All images of tissue sections were captured using BZ-X710:BZ-X Viewer (Keyence Total RNA was extracted from liver tissues with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The cDNA was synthesized from RNA with PrimeScriptTM RT Master Mix (Takara, Japan) for quantitative RT-PCR. The expression level normalized to Gapdh was analyzed by using the LightCycler 480 (Roche, Basel, Switzerland). The primer sequences are listed in Table 1 Proteins were extracted with RIPA lysis buffer from liver tissues and each concentration was measured by Bradford protein assay Protein samples were electrophoresed on polyacrylamide gels and transferred onto PVDF membranes Membranes were blocked with 5% skim milk in PBS and incubated with anti-MLKL Rat Ab (#MABC604; EMD Millipore anti-Phospho-MLKL (Ser345) Rabbit Ab (Mouse specific) (#62233; CST anti-Phospho-RIP3 (Thr231/Ser232) rabbit antibody (Mouse specific) (#57220; CST membranes were incubated with secondary antibody at room temperature for 60 min and washed with TBST The immunoblot was then imaged according to the manufacturer’s instructions from ClarityTM Western ECL Substrate (Bio Rad and semi-quantitatively measured by Multi Gauge (Fuji Film L929 cells were cultured in the presence of 20 μM ZVAD-FMK for 30 min followed by the addition of 20 ng/ml of mouse TNFα and 1 μM BV the cell lysate was recovered for western blotting the liver of mice (0.1 g) were homogenized with 1 ml methanol including an internal standard (17:0-lysoPC) using a glass homogenizer and extracted after stand for 1 h at on ice 5 min) to remove cellular and protein materials then the supernatant was diluted with 10 volumes of water and then adjusted to pH 3.0 with 0.1 N HCl The samples were applied to preconditioned (20 ml of methanol and 20 ml of water) C18 Sep-Pak cartridges (500 mg; Waters and washed with 20 ml of water to exclude nonvolatile ions followed by 10 ml of hexane to exclude cholesterols and neutral lipids The samples were eluted with 10 ml of methanol to obtain oxidized phospholipids The lipid extracts were dried under a gentle stream of nitrogen The LC-ESI-MS/MS analysis was carried out using a QTRAP 4500 quadropole linear ion trap hybrid mass spectrometer (AB Sciex Canada) with a Nexera XR high-performance liquid chromatography (Shimadzu Co. The sample was subjected to LC-ESI-MS/MS analysis using the XBridge BEH C18 column (Waters) and the phospholipid fractions were separated by a step gradient with mobile phase A (acetonitrile/methanol/water = 2:2:1 v/v/v containing 0.1% formic acid and 0.028% ammonia): mobile phase B (isopropanol containing 0.1% formic acid and 0.028% ammonia) ratios of 100:0 (0–5 min) and 100:0 (60–75 min) at a flow rate of 70 μL/min and a column temperature of 30 °C The multiple reaction monitoring was carried out to detect specific oxidized phospholipids MS/MS analysis was carried out in negative ion mode with the following settings deprotonated ions ([M-H]−) were selected as a precursor ion and the peroxidized fatty acyl chain were selected as product ions ([M-H-H2O]−) Statistical analyses were performed using GraphPad Prism software Statistical significance between two groups was evaluated using a two-tailed Mann–Whitney U-test P < 0.05 was considered statistically significant The exact number of biological samples was described in each figure legend There was no exclusion of outliers in all experiments Group allocation was performed without any bias Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity Clinical features and outcomes of cirrhosis due to non-alcoholic steatohepatitis compared with cirrhosis caused by chronic hepatitis C Molecular mechanisms and new treatment strategies for non-alcoholic steatohepatitis (NASH) The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis and liver progenitor cell dynamics in two common mouse models of chronic liver injury Role of adipose tissue in methionine-choline-deficient model of non-alcoholic steatohepatitis (NASH) Rodent nutritional model of non-alcoholic steatohepatitis: species Interventional potential of recombinant feline hepatocyte growth factor in a mouse model of non-alcoholic steatohepatitis Ferroptosis: an iron-dependent form of nonapoptotic cell death Regulation of ferroptotic cancer cell death by GPX4 Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition Ablation of ferroptosis regulator glutathione peroxidase 4 in forebrain neurons promotes cognitive impairment and neurodegeneration Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice Stem/progenitor cells in liver development An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat Mfsd2a+hepatocytes repopulate the liver during injury and regeneration Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis Relationship between the pattern of hepatic iron deposition and histological severity in nonalcoholic fatty liver disease Non-alcoholic fatty liver disease: is iron relevant Intracellular labile iron modulates adhesion of human monocytes to human endothelial cells Apoptosis: a review of programmed cell death Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor Salmonella induces macrophage death by caspase-1-dependent necrosis Relationship between changes in serum levels of keratin 18 and changes in liver histology in children and adults with nonalcoholic fatty liver disease Caspase 3 inactivation protects against hepatic cell death and ameliorates fibrogenesis in a diet-induced NASH model Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice Pathogenesis of nonalcoholic steatohepatitis Serum levels of oxidative stress markers in patients with type 2 diabetes mellitus and non-alcoholic steatohepatitis Non-alcoholic fatty liver disease in an area of southern Italy: main clinical or placebo for nonalcoholic steatohepatitis Venesection for non-alcoholic fatty liver disease unresponsive to lifestyle counselling-a propensity score-adjusted observational study Non-alcoholic steatohepatitis and iron: increased prevalence of mutations of the HFE gene in non-alcoholic steatohepatitis Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis Semaphorin 3E secreted by damaged hepatocytes regulates the sinusoidal regeneration and liver fibrosis during liver regeneration Download references We also thank the members of the Miyajima and Okochi laboratory for discussion and advices This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants JP26110007 (to M.T.) JP15H01386 and JP17H05513 (to H.I.); Japan Agency for Medical Research and Development (AMED) under Grant Number JP18gm1210002 (to M.T Present address: Centre for Heart Research The Westmead Institute for Medical Research National Center for Global Health and Medicine The authors declare that they have no conflict of interest 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/s41419-019-1678-y Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article but the work behind the scenes at the teams and their suppliers has continued at a rapid pace testing 36 engine oil and 40 race fuel variations over the winter who is at the heart of the ExxonMobil Formula 1 project Published on 25 March 2019 by Niels Hendrix Follow Max on social media and keep informed The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V., its licensors, and contributors. All rights are reserved, including those for text and data mining, AI training, and similar technologies. For all open access content, the relevant licensing terms apply. Your Ads Privacy ChoicesIMDb A new key visual has been released to promote the final episode of Life With an Ordinary Guy Who Reincarnated Into a Total Fantasy Knockout The 12th episode series finale will air on March 30th The anime adaptation is based on a manga series written by Yuu Tsurusaki and illustrated by Shin Ikezawa It was first published in November 2019 in Cycomics x Ura Shounen Sunday Comics magazine Eight tankoubon volumes have been published as of March 2022 The main cast includes M.A.O as Hinata Tachibana Rie Kugimiya as Ai to Bi no Megami (Goddess of Love and Beauty) Kaito Ishikawa as Schwarz von Liechtenstein Lohengramm Sayaka Yamai (ODDTAXI episode director) is directing the anime while Toshimitsu Takeuchi (Yu-Gi-Oh Aoi Yamato (ODDTAXI animation director) is the series’ character designer and Takeshi Watanabe (The Duke of Death and His Maid co-composer) is the music composer OLM Team Yoshioka is handling the animation production Crunchyroll is streaming the the anime series and describes it as: Childhood friends Tachibana Hinata and Jinguji Tsukasa were living the everyday life of office workers they were sent flying into another world by a mysterious being Jinguji sees his best friend has been turned into a beautiful blonde-haired The adorableness of Tachibana’s female form completely flummoxes Tachibana But these two are each others’ best friends To keep their relationship from being destroyed they must defeat the Demon Lord as quickly as possible and return to their original forms Source: Official Anime Twitter Metrics details Phosphorodiamidate morpholino oligonucleotides (PMOs) are a promising type of antisense oligonucleotides but their challenging synthesis makes them difficult to access This research presents an efficient synthetic approach for PMOs using the H-phosphonate approach The use of phosphonium-type condensing reagents significantly reduced coupling times compared with the current synthetic approach phosphonium-type condensing reagents facilitated the fragment condensation of PMO synthesizing up to 8-mer containing all four nucleobases with remarkable coupling efficacy This is the first report on the convergent synthesis of PMOs This approach would facilitate the large-scale synthesis of PMOs and accelerate their popularity and accessibility as a next-generation therapy Current approaches for the synthesis of PMO derivatives Although this approach is effective for synthesizing TMOs it is not applicable to the synthesis of PMOs This method gave the phsphoramidite derivative (C) as an intermediate the desired phosphorodiamidate linkage of PMO is not obtained this approach required the preactivation of H-phosphonate monomers (D) due to the low efficiency of the internucleotidic bond formation reaction of the existing approach the fragment condensation of PMOs has not yet been investigated we developed an efficient synthetic approach for PMOs using H-phosphonate monoester derivatives as monomer units The phosphonium-type condensing reagents provided the direct formation of an H-phosphonamidate linkage and this approach enabled the fragment condensation PyNTP showed a better result for the condensation reaction The lower NMR yield using MNTP was due to the formation of by-products Due to the higher activity as a condensing reagent the activation of 2c by MNTP might have led to the overactivation and caused inferior condensing efficacy and PyNTP were selected as condensing reagents for further investigation we achieved the direct condensation of H-phosphonate monoester and the amino group of the morpholino nucleoside the condensation reaction was completed within 20 min Although we attempted to isolate the H-phosphonamidate 2-mer 3cc 3cc was unstable and readily hydrolyzed during extraction and purification by silica gel column chromatography we attempted to convert the obtained H-phosphonamidate linkage to a stable phosphorodiamidate linkage as a one-pot reaction after the condensation reaction The desired product and a triaminophosphine oxide derivative were not separated by silica gel column chromatography despite our attempts to isolate phosphorodiamidate 2-mers 4 the crude mixtures of 4 were employed for synthesizing 2-mer fragments without further purification Reagents and conditions: (i) 3-cyanopyridine (10 equiv) a phosphorodiamidate 4-mer 9gtca containing four types of nucleobases (A and T) using 2-mer fragments (5gt and 7ca) and PyNTP as a condensing reagent 4-mers (9cccc and 9gtca) were employed for the next reaction Reagents and conditions: (i): 3-cyanopyridine (10 equiv) we synthesized a 6-mer 15 by two different synthetic routes (Route A and B) to investigate the effect of a steric hindrance caused by the length of fragments we condensed the 5ʹ-H-phosphonate 2-mer fragment 7cc with the amino group of 4-mer fragment 10cccc we condensed the 5ʹ-H-phosphonate 4-mer fragment 12cccc with the amino group of 2-mer fragment 5cc we used PyNTP as a condensing reagent for the condensation reaction to produce the desired 6-mer 15 with a 95% HPLC yield (entry 1) the condensation reaction using PyNTP as a condensing reagent did not proceed sufficiently we observed that longer 5ʹ-H-phosphonate fragments resulted in lower condensation efficacy This data showed that the steric hindrance of 5ʹ-H-phosphonate fragments was crucial for the condensation efficacy the effect of the steric hindrance of fragments bearing an amino group was less notable compared with 5ʹ-H-phosphonate fragments To overcome the steric hindrance of 5ʹ-H-phosphonate fragments we performed the condensation reaction of route B using MNTP which has a higher activity as a condensing reagent than PyNTP (entry 3) The use of MNTP caused side reactions and was ineffective in synthesizing 2-mer; however it gave the best result in the convergent synthesis due to its high activity we synthesized 8-mer 16 using PyNTP and MNTP as condensing reagents (entry 4 and 5 MNTP gave higher condensation efficacy (92% we synthesized another 8-mer 17 bearing all four nucleobases using MNTP as a condensing reagent (route D) which yielded an 8-mer 17 with a 92% HPLC yield These finding demonstrate that for the fragment condensation the selection of condensing reagents is crucial and MNTP is the optimal condensing reagent for the convergent synthesis of PMOs HPLC results showed a significant difference in lipophilicity between the 8-mer and 4-mer fragments (See SI) we investigated the impact of a steric hindrance caused by the length of fragments and demonstrated that the steric hindrance of 5ʹ-H-phosphonate fragments was crucial for the condensation efficacy we were able to overcome this issue and obtained 8-mer with outstanding HPLC yields Reagents and conditions: (i) 3-cyanopyridine (40 equiv) 2 h; (iii) concentrated aqueous NH3–EtOH (3:1 16 h; (iv) reverse phase column chromatography This study’s approach is an effective alternative for synthesizing PMOs The synthesis of various P-modified PMOs using the H-phosphonamidate derivative should be studied further The following procedure was used for the fragment condensation 2-mer or 4-mer fragment bearing the 3ʹ-NH group (5 μmol) and 2-mer or 4-mer fragment bearing an H-phosphonate monoester on 5ʹ-OH group (7.5 μmol) was dried by repeated coevaporation with dry pyridine and dissolved in a mixture of dry pyridine (50 μL) and acetonitrile (50 μL) A condensing reagent (30 μmol) was added to the solution at 0 °C and the mixture was stirred for 20 min at 0 °C 100 μmol) and a 9.5 M dimethylamine aqueous solution (20 μL 190 μmol) was added at 0 °C and the mixture was stirred for 1 min at 0 °C the mixture was diluted with CHCl3 (3 mL) and coevaporated with CHCl3 (3 × 3 mL) RP-HPLC was performed with a linear gradient of 0–60% MeCN for 60 min in a 0.1 M triethylammonium acetate buffer (pH 7.0) at 50 °C at a flow rate of 0.5 mL/min using a C18 column (100 Å The condensation yields were estimated by the area rations of the 4-mer 6-mer or 8-mer to unreacted 2-mer or 4-mer fragment bearing the 3ʹ-NH group The data that support the findings of this study are available in the Supporting Information of this article RNA therapeutics: Beyond RNA interference and antisense oligonucleotides In vivo and in vitro studies of antisense oligonucleotides—A review Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide Morpholino antisense oligomers: The case for an RNase H-independent structural type and S-DNA compared: Impact of structure and mechanism of action on off-target effects and sequence specificity Safety pharmacology and genotoxicity evaluation of AVI-4658 Dose-dependent restoration of dystrophin expression in cardiac muscle of dystrophic mice by systemically delivered morpholino Preclinical PK and PD studies on 2′-O-methyl-phosphorothioate RNA antisense oligonucleotides in the mdx mouse model Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras Development of an efficient method for phosphorodiamidate bond formation by using inorganic salts Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers Synthesis of phosphorodiamidate morpholino oligonucleotides using trityl and fmoc chemistry in an automated oligo synthesizer Synthesis of phosphorodiamidate morpholino oligonucleotides by H-phosphonate method Synthetic studies on the preparation of nucleoside N-alkyl-H-phosphonamidates Studies on reactions of nucleoside H-phosphonates with bifunctional reagents Synthesis of nucleoside N-alkylphosphonamidates Synthesis of nucleoside phosphoramidate and nucleoside phosphoramidothioate analogues via H-phosphonamidate intermediates Development of a new synthetic method for oligodeoxynucleotides using 3ʹ-H-phosphonamidate derivatives Development of kilogram-scale convergent liquid-phase synthesis of oligonucleotides Chemical synthesis of oligodeoxyribonucleotides using N-unprotected H-phosphonate monomers and carbonium and phosphonium condensing reagents: O-selective phosphonylation and condensation 1,3-Dimethyl-2-(3-nitro-1,2,4-triazol-1-yl)-2-pyrrolidin-1-yl-1,3,2-diazaphospholidinium hexafluorophosphate (MNTP): A powerful condensing reagent for phosphate and phosphonate esters How to get the most out of two phosphorus chemistries Atherton, F., Openshaw, H. & Todd, A. 174. Studies on phosphorylation. Part II. The reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. A new method for the phosphorylation of amines. J. Chem. Soc. 660–663. https://doi.org/10.1039/JR9450000660 (1945) Atherton, F. & Todd, A. 129. Studies on phosphorylation. Part III. Further observations on the reaction of phosphites with polyhalogen compounds in presence of bases and its application to the phosphorylation of alcohols. J. Chem. Soc. 674–678. https://doi.org/10.1039/JR9470000674 (1947) Download references We thank Ms. Fukiko Hasegawa, Dr. Yayoi Yoshimura (Tokyo University of Science), and Takayoshi Torii (Ajinomoto Co.) for the mass spectrum measurements. We would like to thank Enago (https://www.enago.jp) for the English language review the establishment of university fellowships towards the creation of science technology innovation Grant Number JPMJFS2144 and AMED under Grant Number JP21ae0121025 Research Institute for Bioscience Products and Fine Chemicals All authors have approved the final version of the manuscript The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41598-023-38698-2 Sign up for the Nature Briefing newsletter — what matters in science Metrics details USP9X variants have been reported in patients with X-linked intellectual disability we report two female patients with intellectual disability and pigment abnormalities along Blaschko lines Targeted resequencing identified two novel heterozygous variants Our findings provide further evidence that USP9X variants cause intellectual disability we identified novel USP9X variants in two female patients with XLID by using targeted resequencing The patient exhibited upswept and curly hair Pigment changes along Blaschko lines bilaterally on the upper arms (d) and neck She suffered from recurrent otitis media with effusion after 12 months of age She was able to lift her head at 4 months of age Her developmental quotient was 71 at 17 months Ophthalmological evaluations revealed astigmatism Ultrasonographic examination of the heart and abdomen showed normal findings Other normal laboratory tests included thyroid function (free T4 The candidate variant was confirmed by Sanger sequencing Novel heterozygous variants identified in two female patients. USP9X is predicted to contain a ubiquitin-specific protease domain, as determined by SMART (http://smart.embl-heidelberg.de/) Electropherogram for each patient and her parents All patients with USP9X variants exhibited ID Some patients with ID also showed pigment abnormalities along Blaschko lines and recurrent respiratory tract infections were observed in some of the previously reported patients but not in our patients Further analysis is required to determine the phenotype–genotype correlation we identified heterozygous USP9X variants in two female patients Our report provides further evidence that USP9X variants are associated with XLID The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.2621, https://doi.org/10.6084/m9.figshare.hgv.2624 Epigenetic etiology of intellectual disability Genetic studies in intellectual disability and related disorders X-linked intellectual disability update 2017 USP9X destabilizes pVHL and promotes cell proliferation exhibits frameshift mutations in colorectal cancers Loss of Usp9x disrupts cortical architecture hippocampal development and TGFbeta-mediated axonogenesis Novel COL4A1 mutation in a fetus with early prenatal onset of schizencephaly De novo loss-of-function mutations in USP9X cause a female-specific recognizable syndrome with developmental delay and congenital malformations Two females with mutations in USP9X highlight the variable expressivity of the intellectual disability syndrome Mutations in USP9X are associated with X-linked intellectual disability and disrupt neuronal cell migration and growth Download references 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/s41439-019-0081-7 Metrics details Coffin–Siris syndrome (CSS) is a congenital disorder characterized by growth deficiency characteristic facial features and hypoplastic nails of the fifth fingers and/or toes We previously identified mutations in five genes encoding subunits of the BAF complex Here we perform whole-exome sequencing in additional CSS patients identifying de novo SOX11 mutations in two patients with a mild CSS phenotype sox11a/b knockdown in zebrafish causes brain abnormalities potentially explaining the brain phenotype of CSS SOX11 is the downstream transcriptional factor of the PAX6–BAF complex highlighting the importance of the BAF complex and SOX11 transcriptional network in brain development further genetic investigation is required to fully address the genetic picture of CSS Here we apply whole-exome sequencing (WES) to 92 CSS patients and identify two de novo SOX11 mutations in two unrelated patients sox11 knockdown experiments in zebrafish result in a smaller head and significant mortality which were partially rescued by human wild-type SOX11 messenger RNA (mRNA) Two missense mutations in the HMG domain (blue box) occur at evolutionarily conserved amino acids (b) Free energy changes on the indicated mutations calculated by FoldX software Luciferase reporter assays measured transcriptional activity of the GDF5 promoter (−448/+319) (UCSC genome browser hg19: chr20: 34025709-34026457) in HeLa (left) and ATDC5 (right) cells HeLa or ATDC5 cells were co-transfected with WT or mutant (S60P and Y116C) SOX11 expression vector and reporter constructs containing either GDF5 promoter or empty vector (pGL3-basic) Relative luciferase activities compared with empty vector are presented as mean±s.d with each experiment performed in triplicate (upper) Immunoblot analysis of transfected HeLa and ATDC5 cell extracts showing wild-type (WT) or mutant (S60P and Y116C) SOX11 proteins (lower) both SOX11 mutants reduced GDF5 transcriptional activities in HeLa and ATDC5 cells (a) Embryos were injected with sox11-MO alone or with sox11- and tp53-MO or with sox11-MO and in vitro transcribed human SOX11 (hSOX11) mRNA (WT Injected embryos were categorized as normal The lethal and affected phenotype in sox11a/b-MO-injected embryos was partially rescued by WT hSOX11 mRNA overexpression All experiments were performed more than twice and evaluated statistically with a Student’s t-test (b) Head size ratios of embryos with control- or with sox11a/b- and tp53-MO or sox11a/b-MO and hSOX11 mRNA (WT or mutant) at 48 hpf (n≥10) (average of control-MO as 1) Dorsal views of midbrain width were measured (c) Brain cell death in MO-injected embryos at 30 hpf using acridine orange staining (lateral view) sox11 morphants show increased cell death in the CNS Quantification of acridine orange intensities in morphants are shown graphically (right It is interesting that mutations of SOX11 and other BAF subunit genes are mutually exclusive in CSS SOX11 mutations appear to be a rare cause of CSS as only 2 out of 92 patients (2.2%) showed SOX11 abnormality and to be limited to the mild end of CSS phenotype Abnormality of the upstream BAF complex tends to show a more severe phenotype compared with that of a downstream SOX11 mutation which may indicate rather specific effects of SOX11 mutations on the CSS phenotype mutations in both BAF complex genes and SOX11 result in the same phenotype (CSS) providing strong support for the BAF complex and SOX11 function in a common pathway and play an important role in human brain development Patients were seen by their attending clinical geneticists DNA samples were isolated from peripheral blood leukocytes using standard methods Informed consent was obtained from the parents of the patients for experimental protocols and displaying participants’ facial appearances in publications This study was approved by the institutional review board of Yokohama City University School of Medicine including 71 patients from a previous cohort and 21 new patients and resultant data presented as average values with s.d TaqMan quantitative real-time PCR was performed using cDNAs from adult (Human MTC Panel I Pre-designed TaqMan probes for human SOX11 (Hs00167060_m1 PCR was performed on a Rotor-Gene Q (QIAGEN CA) and expression levels normalized to ACTB Kidney expression was used as the standard (1 × ) Mouse neuroblastoma 2A (Neuro-2A) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)–high glucose GlutaMAX supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (Life Technologies Co.) Neuro-2A cells were plated into 24-well plates Each expression construct (200 ng) was transfected into Neuro-2A cells using X-tremeGENE 9 DNA Transfection Reagent (Roche Diagnostics cells were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min at room temperature and permeabilized in 0.1% Triton X-100/PBS for 5 min at room temperature C-terminal V5-6xHis-tagged SOX11 proteins were detected using a mouse anti-V5 primary antibody (1:200; Life Technologies Co.) and an Alexa Fluor 546 Goat Anti-Mouse IgG secondary antibody (1:1,000; Life Technologies Co.) Smears were mounted in Vectashield mounting medium with DAPI (Vector Lab. Confocal images were acquired using a Fluoview FV1000-D microscope (Olympus HeLa cells were cultured in DMEM-high glucose supplemented with penicillin (50 units ml−1) ATDC5 cells were cultured in DMEM/Ham’s F-12 (1:1) supplemented with the above antibiotics and 5% FBS and transfections performed using TransIT-LT1 (Takara Japan) with pGL3 reporter (500 ng per well) effector (250 ng per well) and pRL-SV40 internal control (6 ng per well) vectors cells were harvested and luciferase activities measured using the PicaGene Dual SeaPansy Luminescence Kit (TOYO B-Net Production of WT and mutant SOX11 proteins was assessed by immunoblot analysis with monoclonal anti-FLAG M2 HRP antibody (1:3,000; Sigma) OR) and injected (or co-injected) into one- to two-cell-stage embryos at a final concentration of 0.1 or 0.2 mM capped human SOX11 mRNAs transcribed in vitro from pEF6/V5-His B constructs were prepared using the mMessage mMachine T7 ULTRA Transcription Kit (Ambion For each MO knockdown and rescue experiment embryos from the same clutch were used as experimental subjects and controls Approximately 1 μg of capped RNA was injected per embryo The experiment was authorized by the institutional committee of fish experiments in the National Research Institute of Fisheries Science were fixed overnight in 4% PFA with PBS at 4 °C and stored in 100% methanol at −20 °C Samples were incubated in 100% acetone at −20 °C for 20 min the embryos were rinsed three times with PBS containing 0.1% Tween-20 Samples were then permeabilized by treatment with 0.5% Triton X-100 and 0.1% sodium citrate in PBS for 15 min Embryos were subjected to the TUNEL assay by using the ApopTag Red in situ Apoptosis Detection Kit (Merck KGaA Millipore Germany) according to the manufacture’s instruction All animals were photographed under the same conditions using a LSM510 confocal microscope (Carl Zeiss acridine orange-positive cells were quantitated using a selection tool in Adobe Photoshop for a colour range chosen by green colour selection of regions showing visually positive acridine orange staining defined head regions were selected in each embryo and chosen pixel numbers calculated using the image histogram calculation embryos at 48 hpf were fixed in 4% PFA overnight at 4 °C and dehydrated in methanol at −20 °C embryos at 48 hpf were fixed in Dent’s fixative (80% methanol and 20% dimethyl sulphoxide) overnight at 4 °C Embryos were permeabilized with proteinase K followed by postfixation with 4% PFA and washed with PBSTX (PBS containing 0.5% Triton X-100) After treating with 4% normal goat serum (NGS) in PBSTX for 2 h at room temperature embryos were incubated with mouse anti-HuC/D (1:500 Life Technologies Co.) or mouse anti-acetylated tubulin (1:1,000 Sigma) antibodies in 4% NGS/PBSTX overnight at 4 °C Embryos were washed five times with PBSTX for 10 min each and incubated with goat anti-mouse fluorescein isothiocyanate secondary antibody diluted in 2% NGS/PBSTX for 2 h at room temperature embryos were mounted in 2% methylcellulose and examined using a Fluoview FV1000-D confocal microscope (Olympus) Accession codes: Exome sequence data for CSS patients have been deposited in the Human Genetic Variation Browser under the accession code HGV0000001 ( http://www.genome.med.kyoto-u.ac.jp/SnpDB/repository/HGV0000001.html) Access to this data is controlled by the Yokohama City University Data Access Committee De novo SOX11 mutations cause Coffin–Siris syndrome From neural development to cognition: unexpected roles for chromatin Mutations affecting components of the SWI/SNF complex cause Coffin-Siris syndrome Coffin-Siris syndrome is a SWI/SNF complex disorder Mutations in SWI/SNF chromatin remodeling complex gene ARID1B cause Coffin-Siris syndrome Coffin-Siris syndrome and the BAF complex: genotype-phenotype study in 63 patients A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome Clinical correlations of mutations affecting six components of the SWI/SNF complex: detailed description of 21 patients and a review of the literature The crystal structure of the Sox4 HMG domain-DNA complex suggests a mechanism for positional interdependence in DNA recognition Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations The FoldX web server: an online force field Performance of protein stability predictors SOX11 contributes to the regulation of GDF5 in joint maintenance Gene targeting reveals a widespread role for the high-mobility-group transcription factor Sox11 in tissue remodeling Expression of sox11 gene duplicates in zebrafish suggests the reciprocal loss of ancestral gene expression patterns in development The transcription factor protein sox11 enhances early osteoblast differentiation by facilitating proliferation and the survival of mesenchymal and osteoblast progenitors tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors The sox family of transcription factors: versatile regulators of stem and progenitor cell fate SOX10 mutations in patients with Waardenburg-Hirschsprung disease Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9 The BAF complex interacts with Pax6 in adult neural progenitors to establish a neurogenic cross-regulatory transcriptional network The SWISS-MODEL Repository and associated resources Embryonic senescence and laminopathies in a progeroid zebrafish model Download references We thank the individuals and their families for participation in this study We also thank Nobuko Watanabe for her technical assistance This work was supported by the Ministry of Health Labour and Welfare of Japan; the Japan Society for the Promotion of Science (a Grant-in-Aid for Scientific Research (B) and a Grant-in-Aid for Scientific Research (A)); the Takeda Science Foundation; the fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems; the Strategic Research Program for Brain Sciences; and a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) from the Ministry of Education New Delhi is also appreciated for funding support for the DNA banking facility Yoshinori Tsurusaki and Eriko Koshimizu: These authors contributed equally to this work Yokohama City University Graduate School of Medicine Sanjay Gandhi Postgraduate Institute of Medical Sciences Osaka Medical Center and Research Institute for Maternal and Child Health National Research Institute of Fisheries Science Nagasaki University Graduate School of Biomedical Sciences Satoshi Watanabe & Koh-ichiro Yoshiura collected samples and provided subjects’ clinical information The authors declare no competing financial interests Supplementary Figures 1-7 and Supplementary Tables 1-2 (PDF 1036 kb) Reprints and permissions Download citation Metrics details Glycine encephalopathy (GCE) is a rare autosomal recessive disorder caused by defects in the glycine cleavage complex Here we report a patient with GCE and elevated level of glycine in both the serum and the cerebrospinal fluid Trio-based whole-exome sequencing identified novel compound heterozygous mutations (c.738-2A>G and c.929T>C (p.Met310Thr)) in LIAS three homozygous mutations have been reported in LIAS All previously reported GCE patients also show elevated level of serum glycine Our data further supports LIAS mutations as a genetic cause for GCE we report a GCE individual with novel compound heterozygous mutations in LIAS A 21-year-old female patient was born to healthy nonconsanguineous parents length 48.5 cm (mean) and occipitofrontal circumference 33.8 cm (75th percentile) Her initial development was normal until 18 months Her elder sister died of herpes simplex virus encephalitis at the age of 18 months T2-weighted brain magnetic resonance imaging (MRI) of the individual with LIAS mutations (a) Examined at disease onset (19 months old) High-intensity areas are present in the caudate nucleus and the putamen The high-intensity areas have expanded into the white matter High-intensity areas are noted not only in the basal nucleus but also in the subcortical white matter White matter atrophy is predominant in the frontal region The institutional review boards of Yokohama City University School of Medicine and Tokyo Women’s Medical University approved the study Informed consent was obtained from the family All variants within exons or ±2 bp from exon–intron boundaries and registered in dbSNP137 the National Heart Lung and Blood Institute Exome Sequencing Project Exome Variant Server (NHLBI-ESP 6500) or our in-house (exome data from 575 Japanese individuals) databases Variants were confirmed by Sanger sequencing with an ABI PRISM 3500xl or ABI3130xl autosequencer (Life Technologies Lymphoblastoid cell lines derived from the patient and a healthy control were established After incubation with 1.5 μl of dimethyl sulfoxide(as a negative control) or 1.5 μl of the protein synthesis inhibitor cycloheximide (100 mg ml−1 in dimethyl sulfoxide) (Sigma-Aldrich Total RNA was isolated from treated lymphoblastoid cells using the RNeasy Plus Mini Kit (Qiagen Germany) and was used for reverse transcription with the Super Script III First-Strand Synthesis System (Life Technologies) One microliter of synthesized cDNA was amplified by PCR PCR products were electrophoresed on a 5%–20% gradient polyacrylamide gel stained with ethidium bromide and quantitatively measured using a FluorChem 8900 (Alpha Innotech Three independent experiments were performed Statistical analyses were performed using analysis of variance Each PCR band was cloned into the pCR4-TOPO vector (Life Technologies) and sequenced using ABI 3130xl autosequencer Novel compound heterozygous mutations (c.738-2A>G and c.929T>C (p.M310T)) identified in the patient The upper and middle panels show the LIAS gene structure that comprises 11 exons The lower panel shows evolutionary conservation of the mutated amino acid through six different species The altered nucleotides are highlighted in gray boxes Three previously reported mutations (p.R249H p.E159K and p.D215E) are highlighted in gray (b) Mutation mapping on the crystal structure of lipoyl synthase 2 from T The mutation site is shown as sticks with translucent spheres in red in the whole (left) and the close-up (right) views of the enzyme structure [4Fe-4S] clusters and dithiothreitol (DTT) are depicted in color-coded sticks: green for C Some hydrophobic side chains forming a core with Leu241 are shown as translucent spheres The amino acid number in the parentheses is that for human LIAS Black dashed lines indicate hydrogen bonds A full color version of this figure is available at the Journal of Human Genetics journal online the p.Leu241Thr (p.Met310Thr) mutation is likely to impair the enzymatic activity due to destabilized S-adenosylmethionine binding (a) Reverse transcription-PCR (RT-PCR) analysis showing two PCR products (422-bp (isoforms 1 and 2) and 293-bp (isoform 3)) were observed in a healthy control individual a 344-bp product (corresponding to isoforms 1 and 2) and a 215-bp product (corresponding to isoform 3) were detected in cycloheximide (CHX)-treated cells from the patient at a higher level compared with untreated and dimethyl sulfoxide (DMSO)-treated cells (b) Densitometric data of the RT-PCR products represented as mean±s.d *P<0.05 by analysis of variance (ANOVA) (c) Sequencing of the 422-bp wild type (WT) product only found missense mutation allele based on the electropherogram (d) Schematic presentation of RT-PCR products Sequencing of the 344-bp product showed a 68-bp insertion from intron 7 and exon 8 skipping producing a premature stop codon at amino acid position 250 Sequencing of the 215-bp product showed a 68-bp insertion from intron 7 and exons 7 and 8 skipping producing a premature stop codon at amino acid position 207 one patient (p.Asp215Glu) with hypotonia and seizures showed constant development and residual lipoylated proteins suggesting that some lipoic acid synthetase activity is retained residual lipoic acid synthetase activity may be correlated with phenotypic severity a glycine level is elevated in all patients serum glycine levels may not be correlated with LIAS genotypes Regardless of some biochemical and clinical data it is difficult to extract clinical difference between our case of heterozygous mutations and reported cases of homozygous missense mutations due to the limited number of patients (n=4) Further accumulation of patients with LIAS mutations is needed LIAS mutant phenotype is relatively milder compared with the common GCE The acute onset of encephalopathy in our patient is interesting We assumed that the stressful condition by the upper respiratory infection would have been the trigger of disease progression as sometimes seen in other cases of vanishing white matter a patient with compound heterozygous missense and splice site mutations in LIAS Further analysis of LIAS-related patients is needed to delineate the phenotypic spectrum and phenotype–genotype correlation Late-onset nonketotic hyperglycinemia with a heterozygous novel point mutation of the GLDC gene Identification of the mutations in the T-protein gene causing typical and atypical nonketotic hyperglycinemia Structural and expression analyses of normal and mutant mRNA encoding glycine decarboxylase: three-base deletion in mRNA causes nonketotic hyperglycinemia The glycine cleavage system: structure of a cDNA encoding human H-protein and partial characterization of its gene in patients with hyperglycinemias Characteristic MRI findings in neonatal nonketotic hyperglycinemia due to sequence changes in GLDC gene encoding the enzyme glycine decarboxylase Variant non ketotic hyperglycinemia is caused by mutations in LIAS Lipoic acid synthetase deficiency causes neonatal-onset epilepsy De novo SOX11 mutations cause Coffin-Siris syndrome Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions Protein structure prediction on the Web: a case study using the Phyre server Endogenous production of lipoic acid is essential for mouse development Prediction of long-term outcome in glycine encephalopathy: a clinical survey Download references We thank the patients and their families for their participation in this study We also thank Nobuko Watanabe for her excellent technical assistance Labour and Welfare of Japan; the Japan Society for the Promotion of Science (Grants-in-Aid for Scientific Research (A) (B) (C)); the Takeda Science Foundation; the Japan Science and Technology Agency (the fund for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation Systems); the Ministry of Education Science and Technology of Japan (the Strategic Research Program for Brain Sciences); and a Grant-in-Aid for Scientific Research on Innovative Areas (Transcription Cycle) The authors declare no conflicts of interest Download citation Metrics details A Corrigendum to this article was published on 26 October 2015 Joubert syndrome (JS) and related disorders (JSRD) are autosomal recessive and X-linked disorders characterized by hypoplasia of the cerebellar vermis with a characteristic ‘molar tooth sign’ on brain imaging and accompanying neurological symptoms including episodic hyperpnoea JSRD are clinically and genetically heterogeneous We applied whole-exome sequencing (WES) to five JSRD families and found mutations in all: either CEP290 Compared with conventional Sanger sequencing WES appears to be advantageous with regard to speed and cost supporting its potential utility in molecular diagnosis it can be very difficult to identify the causative mutations in individual cases Familial pedigree and brain MRI of the patients The molar tooth sign is visible in all patients (arrowheads) indicating that the V4 library offered superior coverage to the SureSelectXT library around the regions of the JSRD genes On the basis of our in-house 135 exome data the allele frequency of the mutation was 1/270 allele (0.74%) indicating that it may be a rare variant in Japanese The other mutations were not found in our in-house 135 exome data WES would also be suitable for the diagnosis of JSRD Though the read-coverage of the old version of SureSelect did not sufficiently collect genomic DNAs for four genes (INPP5E the performance of the V4 (51 Mb) library was satisfactory for all genes as exome capture technology is based on hybridization it can be refractory to homologous regions so other methods such as multiplex PCR amplification and multiple microdroplet PCR technology could be useful in addition we were able to identify causative mutations in five non-consanguineous families with JSRD using WES implying that WES or other next-generation sequencing technologies will be a main factor of molecular diagnosis encoding inositol polyphosphate-5-phosphatase E link phosphatidyl inositol signaling to the ciliopathies Mutations in TMEM216 perturb ciliogenesis and cause Joubert Abnormal cerebellar development and axonal decussation due to mutations in AHI1 in Joubert syndrome The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome cause pleiotropic forms of Joubert syndrome Mutations in the gene encoding the basal body protein RPGRIP1L Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290 OFD1 is mutated in X-linked Joubert syndrome and interacts with LCA5-encoded lebercilin TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum Mutations in KIF7 link Joubert syndrome with Sonic Hedgehog signaling and microtubule dynamics A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium Evolutionarily assembled cis-regulatory module at a human ciliopathy locus Mutations in C5ORF42 Cause Joubert Syndrome in the French Canadian Population Application of next generation sequencing to molecular diagnosis of inherited diseases (e-pub ahead of print 11 May 2012; doi:10.1007/128_2012_325) Download references This work was supported by research grants from the Ministry of Health the Japan Science and Technology Agency (N Matsumoto) the Strategic Research Program for Brain Sciences (N Matsumoto) and a Grant-in-Aid for Scientific Research on Innovative Areas-(Transcription cycle)-from the Ministry of Education Science and Technology of Japan (N Matsumoto) a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (N Matsumoto) a Grant-in-Aid for Young Scientist from Japan Society for the Promotion of Science (HS N Miyake) and a grant from the Takeda Science Foundation (N Matsumoto Gunma University Graduate School of Medicine National Center for Child Health and Development The URLs for data presented herein are as follows Novoalign, http://www.novocraft.com/main/index.php Burrows-Wheeler Aligner, http://bio-bwa.sourceforge.net/ SIFT, http://sift.jcvi.org/ PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/ Mutation Taster, http://neurocore.charite.de/MutationTaster/ Supplementary Information accompanies the paper on Journal of Human Genetics website Download citation Nerdy office worker Hinata has once again blundered at a singles mixer While he's grumbling about it on the way home a naked goddess appears out of nowhere and sends the two friends to another world--while also turning Hinata into a hot young woman Hinata and Jinguji set off on a journey to defeat the demon king even if Hinata's new feminine form gives them...unexpected feelings Can they save this fantasy world before their lifelong bromance becomes a romance Although at first blush this can look like a sneaky way to avoid writing a same-sex relationship the truth is a bit different: it's not that Jinguji can't fathom himself in a relationship with his best friend Tachibana; it's that he can't quite picture that relationship if Tachibana suddenly turned into a woman thus making it more “acceptable.” But even that's way too serious a description for this because it's a ridiculous story about two guys in their thirties who get isekai'd away by a peculiar goddess and have to figure out both the whole isekai thing and whether or not their sudden attraction to each other is the product of the goddess' curse I'm having a hard time writing about this because every time I set down what this is about on paper a lot of the humor comes from Jinguji desperately trying to deny how incredibly attracted he is to Tachibana while conveniently forgetting that he wondered back in Japan if he wants to be with him romantically – and that he doesn't want him to find a girlfriend Both the writing and the art play this aspect up really well and one scene where Tachibana and Jinguji are trying desperately not to show how much they want to hold hands is excellently drawn The book is at its best when it's leaning into the absurdity of the story and world from the utterly terrifying bunny-bear they meet in the woods to the truth about said bunny-bear and their ill-fated meeting with a noble elf Even the way Tachibana gets a crash course in being a woman when a group of bandits become enamored with them is pretty great and it's much more rote than the rest of the story The pacing here helps – the book moves quickly and the jokes don't wear out their welcome but I don't love that Jinguji twice uses the word “slut” to refer to women he doesn't like It's presumably to show that he's what used to be called a “woman hater,” which is at least partially coded language for queer but also has connotations of someone who prefers to be single the word is so loaded that I feel like a better one could have been found this is both fun in its own right and a good accompaniment to its anime adaptation Kadokawa will also publish the Heaven Burns Red Comic Anthology on September 27. The comic anthology will feature a cover illustration by P Goto, and stories from eight manga creators including Juri Misaki and Sakaki Yoshioka A special program will stream on G's Channel website on August 31 at 8:00 p.m JST to celebrate the comic anthology's release The game is Maeda's first completely new game in 13 years mysterious life forms called "Cancer" are attacking Earth and the planet is on the verge of a true crisis Humans tried to fight back with their weapons and countries disappeared in the horrors of the war the Cancer control the majority of land on the planet Only those who wield the Seraph can defeat the Cancer and the women in the squads carry the weight of humanity's hope on their shoulders She puts everything she has into fighting against the Cancer Sources: Kadokawa, Comic Natalie Seven Seas Entertainment has announced the license acquisition of five new manga series debuting in English for the very first time They include Life with an Ordinary Guy Who Reincarnated into a Total Fantasy Knockout The Knight Captain is the New Princess-to-Be Here are the full details for each new manga license:  The first new manga license from Seven Seas is the isekai comedy series Life with an Ordinary Guy Who Reincarnated into a Total Fantasy Knockout, written by Yuu Tsurusaki and illustrated by Shin Ikezawa. The series has also inspired a 12-episode anime adaptation that aired from January 12 The manga was first launched by Cygames and Shogakukan in Cycomi magazine on November 18 It is published under the Cycomi x Ura Sunday imprint and has released seven tankoubon volumes in total as of June 2022 An eighth volume is scheduled to be released on November 17 The first volume will be released for the first time in North America in September 2023 and will be available on print and digital platforms as single volume editions Seven Seas describes the synopsis of Life with an Ordinary Guy Who Reincarnated into a Total Fantasy Knockout as:  Tachibana Hinata and Jinguji Tsukasa are two young men who have been friends since childhood they’re sent flying into another world by a mysterious being–and now Tachibana is a beautiful blonde girl When Jinguji angrily demands that they be returned home and go back to normal and the only way out is for them to go on a quest and defeat the demon king But…now the two friends can’t stop thinking about how hot the other is Will they save the world before their lifelong bromance becomes a romance Seven Seas has also licensed Yasuko Yamaru’s The Knight Captain is the New Princess-to-Be shoujo manga series The romantic comedy tale follows a dashing royal bodyguard who pretends to be engaged to her childhood friend The Knight Captain is the New Princess-to-Be was first published in Hakusensha’s LaLa shoujo manga magazine on February 22 Three tankoubon volumes have been released as of June 2022 Comico also publishes the manga in English as Grand Master Knight Has Become the Princess through its Pocket Comics website and app The first volume of the series will be released in August 2023 as single volume editions An eBook version will also be made available on various digital platforms “Lady Chris,” was born into a noble family and treated more or less like a boy growing up she’s not only captain of the imperial guards–she personally protects Prince Leonardo who has been a dear friend since childhood demands that Leo find a suitable girl to marry he says that he’s already found one: Chris but figures that Leo doesn’t really love her like that; it’s probably just some ploy to keep his father happy she starts to wonder if maybe her princely pal does see her as more than a friend Another new manga acquisition is Cat on the Hero’s Lap written by Kousuke Iijima and illustrated by Shiori It is described as a hilarious fantasy adventure tale about a hero and his favorite cat on his lap The series was first launched in Shogakukan’s Manga ONE app and Ura Sunday website on June 26 Three tankoubon volumes have been released as of October 2022 Volume one will be released sometime in November 2023 and be available on print and digital platforms as single large-trim editions Seven Seas describes the story of Cat on the Hero’s Lap as:  The hero Ledo and his companions have a mission: defeat the Demon Lord once and for all taking down foes with only her fists and feet; Grace the wizard harnesses powerful magic to blast away enemies in their path Ledo faces an unexpected challenge in their quest: a big following the party wherever they go…and jumping into Ledo’s lap whenever it’s free Everyone knows you can’t just dump a cat off your lap in good conscience If Ledo pushes this sweet kitty away while it’s happily making biscuits will he be any better than the monsters he’s trying to fight A swords and sorcery story for fans of cats and comedy Hidenori Yamaji’s Soara and the House of Monsters will also be released for the first time in English This delightful and intricately illustrated fantasy tale follows a girl who was raised to fight monsters but starts building comfortable homes for them instead The series was first launched in Shogakukan’s monthly Shounen Sunday S and Sunday Webry website on November 25 A single tankoubon volume has been released as of May 12 with a second volume due for release on November 10 The first volume will be released in October 2023 on both print and digital formats as single large-trim editions The synopsis of Soara and the House of Monsters is described by Seven Seas as:  Soara is a young orphan girl who was raised by knights and trained to battle the monsters that constantly attacked their kingdom But by the time Soara is ready to join the fight herself peace has been declared and her blade is no longer needed Searching for a new home and a new purpose Soara finds herself working with Krik to build comfortable homes for them will she discover a new home and family for herself Hideo Yamamoto’s Homunculus will also be released by Seven Seas for the first time in English The supernatural horror manga inspired a 2021 live-action Netflix film adaptation directed by Takashi Shimizu Yamamoto has also created the Ichi the Killer and Voyeur manga series Homunculus ran in Shogakukan’s Weekly Big Comic Spirits seinen magazine from March 17 15 tankoubon volumes have been released in total The first two volumes will be released as an oversized omnibus edition in June 2023 The entire series will be available as five large-trim omnibus paperbacks Seven Seas describes the synopsis of Homunculus as:  Between spending his days with the homeless and his nights in his vehicle he has little to his name–but not so little that he’ll agree to be the subject of a scientific experiment An unnerving medical student is stalking him and offering to pay Nakoshi a significant sum to test trepanation: the ability to draw out a sixth sense by drilling into the skull he finally agrees to cash and the operating table until Nakoshi realizes his vision has warped in his left eye…showing him the twisted homunculus inside every human NYK has been gradually offloading older tanker tonnage of late the 2002-built ship is actually its youngest VLCC in its fleet one of Thailand’s largest independent tanker owners Nathalin’s fleet today now comprises 23 ships Don't have an account? Fantasy Bishoujo Juniku Ojisan to (Fabiniku) anime revealed a new trailer 2022.The new trailer introduces the additional cast Previously, it was revealed that M・A・O is voicing Hyuga’s female form (Hinata), while Satoshi Hino is Tsukasa Rie Kugimiya is the Goddess of Love and Beauty Kento Ito is voicing Hyuga’s male form.A new key visual which features all of the characters is now available Hyuga Tachibana and Tsukasa Jinguji are 32-year old salarymen they are suddenly teleported to another world a goddess summoned them and somehow Hyuga became a beautiful blonde woman Tsukasa soon realizes that he has feelings for Hyuga’s new form they can’t pursue a romantic relationship because they are best friends!The duo must defeat the Demon Lord and return to their real forms before they fall in love with each other This is a story about a dull old man and a beautiful ex-man OLM’s Team Yoshioka is animating the series Sayaka Yamai is directing the Fabiniku anime with Toshimitsu Takeuchi on series composition while Takeshi Watanabe is composing the music Fantasy Bishoujo Juniku Ojisan to (With a Fogie Reincarnated as a Pretty Fantasy Girl) is a Japanese manga series by Yuu Tsurusaki (author) and Chibimaru (illustrator). The author/illustrator duo is also married. The manga started serialization in 2019 on Cygames’ Cycomi manga app.Source: Official Website©Yuu Tsurusaki Chibimaru (Shin Ikezawa)/Cygames/Fabiniku Production Committee and the January 11 premiere date for the anime Hikaru Tohno voices the character Satina (left in images above), while Amane Shindō is voicing Ultina (right) Yoshiki Fukuyama performs the anime's opening theme song "Akatsuki no Salaryman" (Salaryman at Dawn) while idol group Luce Twinkle Wink performs the anime's ending song "'FA'NTASY to!" The anime will premiere on TV Tokyo on January 11, and will premiere on BS TV Tokyo on January 14 The manga's story begins when a 32-year-old unpopular salaryman is transported alongside his handsome friend to a fantasy world While his friend has been transported without change the salaryman now has the body of a beautiful girl he must go on an adventure with his friend to defeat the world's demon lord Tsurusaki and Ikezawa launched the manga in Cycomi in November 2019, and Shogakukan published the manga's fifth compiled book volume on October 18 Sources: Fantasy Bishōjo Juniku Ojisan to anime's website, Comic Natalie Please activate JavaScript function on your browser the official logo for the G7 Japan 2016 Ise-Shima Summit was officially selected Creator (Supreme Award for Excellence)Shiho Utsunomiya (third grade Oita Prefectural Tsurusaki Technical High School)  Message from the creator:“The design features a red disc at the center which represents the circle of the sun and features in Japan’s national flag This central motif is circled by cherry blossom petals - an iconic symbol of Japan The petals signify the seven participating G7 countries of Japan The blue crescent in the background represents the ocean surrounding Ise-Shima - the venue of the G7 Japan 2016 Summit The logo evokes the image of a globally interconnected ocean in the hope that the nations of the world will unite for peace.” Chairman of the Logo Selection Committee for the G7 Ise-Shima Summit:“The composition strikes a perfect balance between the cherry blossom petals and the blue sea in expressing the beauty of nature in Japan I believe that the work is appropriate for conveying the splendor of Japan.” Others who received Awards for Excellence are:Rui Nishikawa (first grade Mie Prefectural Suginoko School for Special Needs Education; Junior High School Department) Matsuyama Municipal Takahama Junior High School) Incorporated Educational Institution Umemura Gakuen Mie Junior High School) Apphia Yu is directing the English dub with assistant Michelle Rojas. Ray Wilkins is the ADR engineer, and Peter Hawkinson is the assistant The anime premiered on January 11. Crunchyroll streamed the anime as it aired in Japan Yoshiki Fukuyama performed the anime's opening theme song "Akatsuki no Salaryman" (Salaryman at Dawn) while idol group Luce Twinkle Wink performed the anime's ending song "'FA'NTASY to!" Tsurusaki and Ikezawa launched the manga in Cycomi in November 2019, and Shogakukan published the manga's seventh compiled book volume on June 17 Source: Crunchyroll (Liam Dempsey) Tsurusaki and Ikezawa launched the manga in Cycomi in November 2019, and Shogakukan published the manga's third compiled book volume in December Shogakukan will publish the fourth volume on Wednesday Sources: Cycomi's Twitter account, Dengeki Online The TV anime adaptation of Yū Tsurusaki and Shin Ikezawa’s Fantasy Bishōjo Juniku Ojisan to (With a Fogie Reincarnated as a Pretty Fantasy Girl) manga revealed a key visual The anime series will debut in January 2022 The series centers around a 32-year-old unpopular salaryman who is summoned to another world by a naked goddess together with his handsome best friend he has to go on a journey to defeat the demon king Tsurusaki and Ikezawa launched the manga in Cygames’ Cycomi online manga site in November 2019 The manga’s 5th volume was published on October 18 Official Site Official Twitter 2025 years 3 month 24 Date 12 when 19 minutes Please note that this article contains advertisements It has been announced that original photos and handwritten profiles of Hinatazaka46's fifth generation members will be released every day at noon starting today with Ohno Manami and Tsurusaki Nika being the first to be released Nika Tsurusaki March 3th (Tuesday): Niina Sakai Yu Sato March 3th (Wednesday): Izuki Shimoda Saki Katayama March 3th (Thursday): Ota Mizuki Takai Rika March 3th (Friday): Sakura Matsuo The photos show the members with different expressions than in their profile videos Their handwritten profiles include personal details such as their birthdays their favorite Hinatazaka46 songs and music videos The fifth generation of Hinatazaka46 consists of 2024 members who will join Hinatazaka8 after passing the new member auditions held in August 46 they attracted a lot of attention on social media It was also announced that the 4 fifth-generation members will be introduced at Hinatazaka46's "10th Hina Birthday Festival" live concert to be held at Yokohama Stadium on Saturday We can't take our eyes off their future success ■ Hinatazaka46 XNUMXth Generation (in order of announcement) Manami Ohno Profile video:https://youtu.be/JA9ML18irK0 Born in Tokyo Niko Tsurusaki Profile video:https://youtu.be/GkFHwHvPPto Born in Kanagawa Prefecture Niina Sakai Profile video:https://youtu.be/22AtHPetHFM Born in Kanagawa Prefecture Yu Sato Profile video:https://youtu.be/QXQUKkvSrCQ Born in Fukuoka Prefecture Izuki Shimoda Profile video:https://youtu.be/u4KlgbAoyF0 From Chiba Prefecture Saki Katayama Profile video:https://youtu.be/WQuKs-yYWaI From Saitama Prefecture Mizuki Ota Profile video:https://youtu.be/iIEroRlhjq0 Born in Osaka Prefecture Rika Takai Profile video:https://youtu.be/-BFjsHEY8tk From Hyogo Prefecture Sakura Matsuo Profile video:https://youtu.be/PuBfEY7WAyw Born in Kanagawa Prefecture Kuramori Hinano Profile video:https://youtu.be/HWEDcjZa9tU Born in Osaka Prefecture ■ Hinatazaka46 XNUMXth Generation Teaser Movie View on youtube ■ Hinatazaka46 XNUMXth Generation Unveiling Site https://www.hinatazaka46.com/5th_generation/ <Related information> ■Latest Live "The 6th Hina Birthday Celebration" April 2025th and 4th, 5 Yokohama Stadium, Kanagawa Prefecture https://www.hinatazaka46.com/6th-anniversary/ ■Hinatazaka46 official website https://www.hinatazaka46.com/ ■Hinatazaka46 OFFICIAL YouTube CHANNEL https://www.youtube.com/@46officialyoutubechannel99 ■YouTube channel "Hinatazaka Channel" https://www.youtube.com/@hinatazakachannel ■ Hinatazaka46 Official X @hinatazaka46 ■ Hinatazaka46 Official TikTok @hinatazakanews TOMOO's new song "LUCKY" has been chosen as the ending theme for KyoAni's new work "CITY THE ANIMATION" Maki Goto performs an additional performance for her one-day 1th anniversary live tour Fans go wild with her stunning dancing and singing from her latest songs to Morning Musume's classics 2025 years 5 month 3 Date 19 when 22 minutes {excerpt}<\/p><\/li>"},"theme":{"name":""}}What's New Sakurazaka46 XNUMXth generation members' newly taken photos & handwritten profiles released! The XNUMXth batch includes Yamada Momomi and Katsumata Haru! 2025 years 5 month 4 Date 12 when 16 minutes 2025 years 5 month 3 Date 21 when 21 minutes 2025 years 5 month 3 Date 19 when 43 minutes 2025 years 5 month 3 Date 19 when 27 minutes 2025 years 5 month 3 Date 19 when 11 minutes 2025 years 5 month 3 Date 18 when 56 minutes 2025 years 5 month 3 Date 18 when 17 minutes 2025 years 5 month 3 Date 18 when 11 minutes 2025 years 5 month 3 Date 18 when 07 minutes Based on a Japanese light novel series written by Yukiya Murasaki and Exemplified by Takahiro Tsurusaki,’ How To Summon a Demon Lord‘ or’Isekai Maou into Shoukan Shoujo no Dorei Majutsu’ is a Dream isekai anime with ecchi and harem Topics The narrative revolves around a Japanese hikikomori gamer named Takuma Sakamoto who’s summoned into the world of his favorite MMORPG,’ Cross Reverie,’ with a panthers girl named Rem and an elf girl named Shera Although they meant to make him their servant due to a magical ring that the protagonist owns the charm ricochets off him and returns to the girls who unexpectedly discover they have collars around their necks Takuma begins using the personality of his in-game character If season 1 is all about Diablo settling from the new universe and finally making friends season 2 sees him dealing with complex issues and dangerous enemies ‘How Not to Summon a Demon Lord’ season 1 was initially conducted from July 5 If you’re curious to understand when how To Summon a Demon Lord’ season 3 will come out ‘How To Summon a Demon Lord’ season two (also written as’How Not to Summon a Demon Lord Ω’) premiered on April 9 and aired 10 episodes before concluding on June 11 which was produced by Ajia-do Cartoon Works (‘Nintama Rantarou’) season 2 has been developed by Okuruto Noboru (‘The Girl in Twilight’) in collaboration with Tezuka Productions (‘Astro Boy’) Satoshi Kuwahara helmed the directorial team in season 2 with Kazuyuki Fudeyasu directing the writing team In terms of a prospective how Not to Summon a Demon Lord’ season 3 Neither the producers nor any of the studios involved with the project have made any official statement on the topic yet a massive fanbase has developed around the isekai anime genre propelling most jobs that deal with the notion to mainstream popularity ‘How Not to Summon a Demon Lord’ is no exception this popularity ensured that the show would find another season If the anime was able to replicate the success of its debut year in season 2 there’s no explanation as to why it will not get another year The producers can also opt to produce a cinematic sequel to the TV anime a trend that’s become quite prevalent in recent decades with projects like the black Clover’ movie and’Kimetsu no Yaiba Movie: Mugen Ressha-hen.’ The first 6 volumes of the source material are adapted for the anime Murasaki and Tsurusaki have published 13 volumes to date there is loads of content where an upcoming season could be developed Considering the intermediate season between the first two seasons was approximately 3 years we can suppose that’s Not to Summon a Demon Lord’ season 3 will probably come sometime in 2024 FUNimation’s How NOT to Summon a Demon Lord Season 2 English dub was released in the spring 2021 arcade season The main Japanese voice cast can be returning for Diablo (Masaaki Mizunaka) Joining the new cast for the second season was Miku Ito as Lumachina Weselia Here’s the How NOT to Summon a Demon Lord dub cast: FUNimation’s How NOT to Summon a Demon Lord Season 3 Fragrant hasn’t been announced yet How to observe How NOT to Summon a Demon Lord Omega uncensored The anime TV series never reaches the level of this Redo of Healer anime (see our post about Redo of Healer Season two ) but lots of the scenes continue to be censored Anime fans wanting to understand how to watch How NOT to Summon a Demon Lord uncensored will need to turn to the Blu-Ray/DVD release to see the complete uncut version because there’s not any AT-X version How NOT to Summon a Demon Lord Season 1 Blu-ray/DVD box set released on February 24 will be published in three Blu-ray/DVD volumes The gaps in the very first season between the censored and uncensored versions were frequently quite subtle because not one of the episodes had actual full nudity suggestive scenes were blocked out entirely it has relied more on conveniently positioned environmental props instead of invasive black bars and decals which divert from the encounter Besides eliminating light glares and black bars it is unsure whether Lumachina’s tentacles will probably be changed What’s known is that anime fans are going to have the ability to see How NOT to Summon a Demon Lord Season 2 uncensored online with streaming sites There will be a”Dual Summon Version” (or W Summon Version) with prolonged scenes and re-edits The uncensored version will be split into a Petit Demon King variation and a Serious Demon King variant with the latter being a director’s cut of this next season By Epic Dope Staff No Comments on Isekai Fabiniku Gets Anime: Juniku and His Friend’s Chaotic Comedy on Screen! We all often say things we don’t mean out of frustration Isekai Bishoujo Juniku Ojisan is about a 32-year-old man who wishes to become a woman due to not being able to get a girlfriend Now the fun starts when this childish wish of his is actually granted The Fantasy Bishoujo Juniku Ojisan to manga by Yu Tsurusaki and Shin Ikezawa is getting an anime adaptation as announced by Cycomi’s Twitter account Crazy transsexual comedy from Cycomics“With an uncle who is a beautiful girl in another world”Ma-sa-ka-no https://cycomi.page.link/fabiniku A visual was released to announce the news It shows the protagonist after he is converted into a woman and his handsome best friend Tsukasa Jinguji reading the news and getting shocked Monday morning is another world beautiful girl uncle Babiniku uncle update date!What a mysterious thing happenedTVADMeToDecisionSamadhi! 🥳🥳🥳🥳🥳🥳 Hyooooooo Yu Tsurusaki also made a visual to celebrate the announcement The visual has the protagonist drawn in Tsurusaki’s style The manga takes on the concept of transsexuality and the theme of gender-bending to give us a comic plot in the world of isekai manga It has incredible comic timing and artwork Shin Ikezawa really did put a good amount of work into character design and illustration it really does take on an important theme that people should talk about more Yu Tsurusaki kept that in mind and carefully fabricated a plot that people can enjoy without being offensive The manga was launched in November 2019 and began serialization in Cycomi The fourth compiled book volume of the comedy series will be published on May 19th I can’t wait for the anime to come out because I really enjoyed the manga and to see my favorite characters come to life is always exciting Fantasy Bishoujo Juniku Ojisan to is a Japanese manga created by Tsurusaki and Chibimaru They first serialized the manga on the Cycomics website in November 2019 and Comic Pixiv began featuring individual chapters in March 2020 The story follows a dull old man and his handsome best friend who are summoned to another world by a goddess he has to go on a journey with his best friend to defeat the demon king! Source: Cycomi Twitter Our talented team of Freelance writers - Always on the lookout - pour their energies into a wide range of topics bringing to our audience what they crave - fun up-to-date news All content cited is derived from their respective sources. If you believe we have used your copyrighted content without permission, send us an email at [email protected] and we will remove it immediately You must be logged in to post a comment