Herein we report a simple chemical route for the preparation of (Nd–Pr)2Fe14B magnetic powder using monazite concentrate as a precursor. Th, U, Sm, and La impurities were removed from monazite leachate by roasting, solvent extraction and leaching the concentrate. Purified leachate consisting of Nd and Pr Chlorides was added to the FeCl3 solution, and the solution produced was co-precipitated with NaOH. RE and Fe hydroxide precipitates were converted to the oxides by annealing at 700 °C. Boric acid and CaH2 were added in the RE and Fe oxides produced, and this mixture was reduced and diffused to (Nd–Pr)2Fe14B. Magnetic properties of the (Nd–Pr)2Fe14B produced were enhanced by introducing antiferromagnetic coupling, induced by Dy addition and efficient removal of CaO byproduct through ball milling in ethanol which increased the BHmax from 3.9 to 11.45 MGOe. Process reported is energy efficient, environment-friendly, time saving and low-cost.Syntheses of Nd Crystalline phosphate ore in monazite has high chemical and thermal stability, therefore it is quite difficult to extract RE from it. Generally, two chemical methods (acid digestion and leaching process) are commercially applied to obtain the RE from monazite. These processes require high temperature (230 °C) and sulfuric acid to roast the concentrate. Roasting is followed by digestion in which a large quantity of inorganic bases  hydroxide are used9. After roasting, digestion, multiple solvent extractions, leaching, and precipitation, RE-oxides are obtained10. RE-oxides are reduced to the RE metals by molten salt electrolysis. Reduced RE's are alloyed to the (Nd-RE)2Fe14B  after addition of Fe and B. Two major commercialized routes for the production of Nd2Fe14B magnetic powder are powder metallurgy and melt spinning process. Both of these processes are complicated and need high temperature for alloying steps . Conclusively it takes long time, huge amount of energy and resources to obtain pure RE metals from the monazite and then synthesis of Nd2Fe14B magnetic powder from them. Among permanent magnets, Nd2Fe14B hard magnets exhibit the highest magnetic properties such as magnetic remanence and energy density18. Hence Nd2Fe14B magnets have huge expanding a market, and it is important to introduce a low-cost, eco-friendly, energy-efficient and time saving method for their production  have a wide variety of applications in high-tech products and permanent magnets, hence their demand is increasing rapidly. Monazite is a rich source of various RE, since 70% of the monazite consists of the oxides and phosphates of various RE, mainly of Ce, La, Nd, Pr, and Smion Fig. .2Fe14B (RE = Nd and Pr) from monazite concentrate. Monazite concentrate was refined by roasting, solvent extraction, and leaching in HCl. Obtained leachate was mixed with FeCl3, then mixture was co-precipitated (to produce RE and Fe hydroxides) and annealed to produce RE and Fe oxides. Boric acid and CaH2 were added in the RE and Fe oxides and metal oxides were converted to the (Nd–Pr)2Fe14B by reduction-diffusion (R-D) process. Magnetic properties of the (Nd–Pr)2Fe14B produced are improved by Dy addition and  ball milling in ethanol.Herein we report a novel, simple, and convenient chemical method to synthesize (Nd–Pr)3·6H2O), sodium hydroxide (NaOH), boric acid (H3BO3), calcium hydride (CaH2), dysprosium (III) chloride hexahydrate (DyCl3·6H2O), ethyl alcohol (C2H5OH), sulfuric acid (H2SO4), hexane (C6H6) and acetone (CH3COCH3) were obtained from Sigma-Aldrich Co.Iron (III) chloride hexahydrate . The morphology, size, and elemental distribution of the intermediate and final product particles were observed with scanning electron microscopy  and transmission electron microscopy  operated at 200 kV embedded with energy-dispersive X-ray spectroscopy (EDS). Crystal structure analysis was done with X-ray diffractometer (Rigaku MiniFlex) with Cu-Kα source, with radiation wavelength of 0.15418 nm. For intermediate products, TEM, TEM-EDS, and HRTEM characterizations were performed on JEM-2100F by JEOL Ltd. For TEM analysis, RE and Fe oxide particles were dispersed in ethanol. Magnetic properties were measured by physical property measurement system  in the vibrating sample magnetometer mode. JEOL JEM-ARM200F Cs-corrected TEM was used to obtain “High-angle annular dark-field imaging scanning transmission electron microscopy” (HAADF-STEM) and “Low-angle annular dark-field imaging scanning transmission electron microscopy” (LAADF-STEM) images.Our study is mainly reporting an experimental process; hence the results and discussion section will also explain the experimental process in detail. To avoid the repetition of the information, experimental process has been moved to the supporting information. However, a flow chart of the experimental process is provided as Fig. 2SO4. Concentration of both the Th and U was recorded as ~ 0.42 g/L in the slurry. Efficiency of the process was determined by various factors e.g., leaching time, leaching temperature and acid to slurry ratio. Experimental conditions were changed in various experiments and optimum conditions were determined. In optimized process, acid to slurry weight ratio was kept as 1:3 and slurry was roasted at 220 °C for 90 min. Following equation explains the chemical reactions those took place during the roasting step. Monazite concentrate used in this work mainly consisted of phosphates and oxides of RE, Th, and U. At first, the concentrate was ground and sieved, as particle size approached 44–500 μm. Almost half of the sample passed through 150 mesh (105 μm) sieve. Sample was mixed with water in such a way that slurry with density of a 100 g/L was formed. The first step was the removal of Th and U from monazite by roasting in (6 N) HTo separate U and Th in the form of sulfate salts, Primene JM-T was used. Detail of the solvent extraction process is described in the supporting information.19. During the solvent extraction, sulfates of both the U and Th remained dissolved in the aqueous phase and were separated.Primene JM-T is a popular reagent used for the solvent extraction of RE. Molecules of the Primene JM-T make organometallic complex with the RE sulfate selectively3 produced were leached in HCl, hence leachate containing La (12.84 g/L), Pr (1.720 g/L), Nd (5.35 g/L), and Sm (less than 0.01 g/L) was obtained. Sm and La were removed by solvent extraction with Cyanex-572. Detail of the solvent extraction process is described in the supporting information.After the extraction of Th and U, in the next step, La and Sm were removed. Solution obtained from the first step was precipitated with (3 M) NaOH until pH value approached 10. RE(OH)20, which dimerizes and preferentially separates the Nd and Pr from Sm and La. By using Cyanex-572, almost 99.94% of the Sm and La were removed from the leachate.Cyanex-572 is mixture of phosphinic and phosphonic acidsIt was evaluated by ICP-AES analysis that the solution obtained after second solvent extraction, consisted of mixtures of chlorides of various RE Table . This so3 was added in the monazite leachate solution in such a way that RE:Fe (RE = Nd + Pr) ratio was kept as 15:77. For the synthesis of (Nd–Pr)2Fe14B by R-D, physical contact between constituent elements in the precursor is very important, because it leads to efficient solid-state diffusion. Hence in this work, co-precipitation was employed for the synthesis of oxide powder. Initial pH of the monazite leachate was 2.64. As NaOH solution was added, red precipitates of Fe (OH)3 were produced and dissolved again immediately. pH of the solution changed slowly until 4 and then after adding very little NaOH, it suddenly raised to the 7.FeClAll RE and Fe Chlorides were converted to hydroxides as presented in the equations below:Monazite leachate was co-precipitated at pH 7, 8, 9, 10, 11, 12, and 13 in separate experiments. After co-precipitation at pH 7, 8, 9 and 10, some of the precipitates were suspended in the solution and it took more than 2 h for them to settle down Fig. . But aft3+ forms highly acidic complexes in monazite leachate. After the addition of NaOH, Fe forms aqua complexes [Fe(H2O)3(OH)3]. [Fe(H2O)3(OH)3] is not stable at pH below 7, however, when pH exceeds 7, neutral complexes of Fe precipitate out forming hydroxides. This is the reason that percentage yield increases with increasing the pH. Similarly, RECl3 is converted to [RE(H2O)3(OH)3]. By increasing pH more Nd and Pr were precipitated. Colors of Fe3+, Nd3+, and Pr3+ are brick red, pink, and light green, respectively. Figure 2O)3(OH)3] as the color of the hydroxide mixture slightly turns brown. Hydroxides of both Fe and RE produced by co-precipitation were amorphous and could not be detected in XRD. [RE(H2O)3(OH)3] and [Fe(H2O)3(OH)3] were converted to the REFeO3 (mainly (NdFeO3)) and Fe2O3 by annealing at 700 °C in the presence of air. Oxides produced at various pH were characterized by TEM 3].exes [FeHO3(OH)3].exes [FeHO3(OH)3].exes [FeHO3(OH)3].exes [FeHO3(OH)3].2 and then pressed into pellet form. Pellet was annealed at 1000 °C in the inert environment for the complete R-D of the oxides.Co-precipitation at pH 13 gave the highest percentage yield (~ 99%) of RE and Fe oxides Fig. , hence o3.6H2O was added in monazite solution before co-precipitation. The rest of the experiment was performed the same as the previous experiment. Dy content consequently enhanced the magnetic properties of the final product. Product obtained in this experiment RE2Fe14B will is labeled as (Nd–Pr)1.5Dy0.5Fe14B.In a separate experiment, DyCl2 were produced as by-product. Produced CaO is helpful to control the particle size by Ostwald ripening during the diffusion and stops the formation of oversized (Nd–Pr)2Fe14B particles21. However, being non-magnetic CaO byproduct reduces the magnetic properties of the magnetic particles. (Nd–Pr)2Fe14B and, (Nd–Pr)1.5Dy0.5Fe14B were washed with water to remove the CaO. But water reacted with the CaO and formed Ca(OH)2 which is sparingly soluble in water. Consequently, this non-magnetic Ca(OH)2 was left in the (Nd–Pr)2Fe14B after washing.During the R-D, CaO and H1.5Dy0.5Fe14B was ball milled in ethanol. CaO removal by ethanol ball milling in this work is different from the study that we reported earlier22. In our previous study, milling was performed in a planetary ball milling machine at 300 rpm for 3 h which was quite useful but still had few disadvantages. Milling in the close vessel at high rpm for 3 h increased the temperature of the vessel, which may lead to the partial oxidation of the product. Hence a separate cooling system was attached with the milling apparatus. Secondly, planetary ball milling cannot be used at industrial scale as efficiently as table ball milling. Furthermore, milling at intense conditions as above contaminates the balls and bowl of the milling apparatus and it is not convenient to wash the apparatus after every washing. Hence, in this study common table ball milling apparatus with glass vial (as milling jar) was used, which is much cheaper as compared to the Pulverisette-6 planetary ball mill machine, that was used in the previous study. Most importantly it was found that that table ball mill removed CaO as effectively as the planetary ball mill.For the more efficient removal of the CaO, in another experiment, (Nd–Pr)1.5Dy0.5Fe14B+ CaO) was ground and dispersed in the ethanol (20 ml) in a glass vial. Zirconia balls (70 g) were added to the above mixture and the vial was sealed with the Teflon tap after closing the lid. Ball milling was performed for 3 h at 180 rpm in the open air and then the product obtained was washed with the water thrice to separate the non-magnetic by-products. After the water washing, product was rinsed with acetone twice then stored in the glove box in the inert environment. Final product obtained in this experiment was labeled as (Nd–Pr)1.5Dy0.5Fe14B (BM).In the current study, 1.5 g of R-D product ((Nd–Pr)2Fe14B, (Nd–Pr)1.5Dy0.5Fe14B, and (Nd–Pr)1.5Dy0.5Fe14B (BM) was performed. XRD patterns of all three products described above are very similar. This is because of their very similar crystal structures. However, the ethanol ball milled product did not show any Nd peak 1.5Dy0.5Fe14B and (Nd–Pr)1.5Dy0.5Fe14B (BM) was estimated as 2, 1.5 and 1.3 μm eak Fig. k. Most pcts Fig. ,12,13. Tcts Fig. ,12,13.Fi2Fe14B shows two mixed crystalline lattice fringes, one is related to (002) plane of (Nd–Pr)2Fe14B with the d-spacing value of 0.299 nm and another one is related to (121) plane of Nd phase with d- spacing value 0.256 nm, as shown in Fig. To study the micro-structure, LAADF-STEM and HAADF-STEM analysis was performed Fig. h–j. HAAD2Fe14B crystal lattice. Two main evidence for the Dy substitution in the Nd2Fe14B come from the XRD and HRTEM. In case of the (~ 25%) Dy substitution in Nd2Fe14B, XRD peaks are shifted towards right side, because of smaller size of Dy (as compared to the Nd)21. In our study, no peak shift was observed and there was no evidence of complete Dy substitution in the HRTEM analysis. However, there is a strong possibility that Dy is partially substituted in the Nd2Fe14B crystal lattice which is also evident in SEM–EDS. Usually, when Nd2Fe14B is prepared by the R-D method, added Dy prefers to substitute inside the crystal lattice. However, in our precursors there are 16 RE in the precursors, hence, it is very difficult to know that which RE will substitute inside the lattice and which will remain outside the lattice.There is no evidence that all (~ 25%) the Dy is substituted in the NdμB) are calculated (per formula unit of (Nd–Pr)2Fe14B from Ms values. Experimentally determined values of magnetic moment (μB) in this work are lower than the values predicted by theoretical calculation22 because experimental conditions conditions are not fixed as theoretical. Theoretical values were calculated assuming the temperature as 5 K , and it was assumed that all particles are single domain and un-oxidized (no washing effect).Magnetic properties of all the products together with hysteresis loop are given in Fig. 2Fe14B is not a straightforward case. Reduction in the Ms and enhancement in the coercivity has been reported after the Pr addition to the Nd2Fe14B24. But meanwhile contradictory results have also been observed. Zhang et al25 reported the reduction in squareness ratio  after Pr addition to the Nd2Fe14B. Imran et al26 quantitatively measured the enhancement of the magnetic moment (μB/formula unit), when 25% Pr was substituted in the Nd2Fe14B crystal lattice. They reported that overall magnetic moment was enhanced after the Pr substitution (because of the Nd–Pr ferromagnetic coupling).Studying the effect of Pr on the of magnetic properties of (Nd–Pr)2Fe14B based magnetic material. If the Pr is outside the Nd2Fe14B crystal and found on the grain boundary, it has negligible effect on the magnetic moment. However, in this case it decouples the Nd2Fe14B grain boundaries effectively. This decoupling enhances the coercivity . If Ms value is reduced this is mainly because of enhancement in the anisotropy energy and coercivity. However if the Pr is substituted in the (Nd–Pr)2Fe14B crystal lattice overall magnetic moment of the lattice will increase. Enhanced magnetic moment  will reduce anisotropy energy and coercivity. During the synthesis of (Nd–Pr)2Fe14B by R–D method  Pr is usually substituted inside the Nd2Fe14B crystal. Furthermore, HAADF-STEM image age Fig. h also ag1.5Dy0.5Fe14B in two different ways. If Dy substitutes in the Nd2Fe14B crystal, it couples antiferro-magnetically with the surrounding elements. However, if Dy is found outside the crystal lattice, it is found as Dy rich phase and there is no antiferro-magnetic coupling involved. Furthermore, paramagnetic behaviour of the Dy can enhance the magnetic moment of the final product. Dy might be oxidized during washing process and produced oxide, which acted as soft magnet. Hence, Dy or Dy2O3 phases enhanced the overall magnetic moment of the magnetic particles.Dy exhibits almost three times higher magnetic moment as compared to the Nd or Pr and it can affect the magnetic moment of (Nd–Pr)27:s is the saturation magnetization, µ is magnetic moment of Dy or Dy2O3 rich phase, µ0 is vacuum permeability constant, H is applied field, kB is Boltzmann’s constant, T is temperature, N is density of magnetically-active atoms in the Dy phase, and L(x) = coth(x) − 1/x27. Equation above states that magnetization of the Dy or Dy2O3 rich phase is directly proportional to the quantity as well as the magnetic moment of the Dy.The response of an ideal, homogeneous paramagnetic particles to applied external magnetic field is given by a Langevin function1.5Dy0.5Fe14B will decrease. On contrary, if more Dy is found outside a crystal lattice overall magnetic moment will increase. Enhanced magnetic moment in (Nd–Pr)1.5Dy0.5Fe14B suggests that there is more Dy outside the crystal lattice as compared to the inside. BSE-SEM and BSE-SEM–EDS images of (Nd–Pr)1.5Dy0.5Fe14B further support this statement 1.5Dy0.5Fe14B phase which is prominent with dark gray color. EDS measurement for site 1 and 2 are taken from that region. Site 3 represents the area which has high concentration of Pb and Sn. These two elements come from the solder. Site 4 indicates the presence of Dy phase, which the Dy phase outside the (Nd–Pr)1.5Dy0.5Fe14B crystal lattice. Conclusively, this Dy increases the magnetic moment of the final product.Hence, it is proposed that when more Dy is substituted inside the crystal lattice magnetic moment of (Nd–Pr)2Fe14B, Dy enhances anisotropy energy, Mr and BHmax33. In this work, Dy was also added to increase the anisotropy field and BHmax. BHmax of the (Nd–Pr)1.5Dy0.5Fe14B approached to 8.56 MGOe 1.5Dy0.5Fe14B was improved up to 11.45 MGOe  as compared to the (Nd–Pr)2Fe14B (5.24 kOe). The main reason behind the enhancement in coercivity is the stronger anisotropic field created by Dy. (Nd–Pr)1.5Dy0.5Fe14B had smaller particle size as compared to the (Nd–Pr)2Fe14B 2 which contaminated the final product when it was washed with water. Ca(OH)2 effectively decoupled the (Nd–Pr)2Fe14B grains and enhanced coercivity of both (Nd–Pr)2Fe14B and(Nd–Pr)1.5Dy0.5Fe14B. Ethanol ball milling increased the magnetic moment and BHmax of (Nd–Pr)1.5Dy0.5Fe14B (BM) but decreased the coercivity value down to 5.31 kOe μB), Mr (56.3 emu/g), and Ms (86.5 emu/g) were recorded for (Nd–Pr)2Fe14B. In contrast, Dy addition enhanced the magnetic moment (20.38 μB), Mr (70.6 emu/g), and Ms (104.2 emu/g) values of (Nd–Pr)1.5Dy0.5Fe14B.Among all three products, minimum values of magnetic moment 1.5Dy0.5Fe14B (BM) exhibited the maximum values of magnetic moment (21.67 μB), Mr (87.7 emu/g) and Ms (110.8 emu/g). Although Dy addition and Ca(OH)2 removal increased both Mr and Ms values but these factors enhanced the Mr value more as compared to the Ms value. This lead to the enhancement in squareness ratio, which was recorded as 0.65, 0.68 and 0.79 for (Nd–Pr)2Fe14B, (Nd–Pr)1.5Dy0.5Fe14B and (Nd–Pr)1.5Dy0.5Fe14B (BM) respectively.Removal of non-magnetic Ca(OH)2Fe14B particle were synthesized from monazite concentrate. At first Th, Sm, U, and La were separated from monazite by roasting, solvent extraction, and leaching in HCl. Produced monazite leachate mainly consisted of chlorides of Nd and Pr. After addition of FeCl3, Nd and Pr and Fe chlorides were converted to Nd, Pr and Fe oxide by co-precipitation and annealing. Finally, these oxides were reduced and diffused to produce (Nd–Pr)2Fe14B. Pr in the (Nd–Pr)2Fe14B reduced its energy product because of ferromagnetic coupling with Nd and Fe. Dy addition enhanced anisotropy energy, coercivity and Mr value. Meanwhile, Mr value of the water washed product was reduced because of the presence of the Ca(OH)2, the non-magnetic by-product of the R-D. Ethanol ball milling efficiently removed the CaO, hence BHmax was almost tripled (from 3.9 to 11.45 MGOe). Process reported is energy-efficient, environment-friendly, low cost and time-saving.In a convenient chemical method (Nd–Pr)Supplementary Information.


Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer’s disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.The The online version contains supplementary material available at 10.1186/s40478-021-01285-5. APP, PSEN1 and PSEN2. Sporadic AD is a multifactorial disease exhibiting a strong genetic component with an estimated attributable risk of 60–80%  (#42702), P{TRiP.HMS00147}attP2 Rab5 (#34832), UAS-Rab7.GFP (#42705), UASp-YFP.Rab7.Q67L (#24103), UASp-YFP.Rab7.T22N (#9778), UAS-Rab11-GFP (#8506), UASp-YFP.Rab11.Q70L (#9791), UASp-YFP.Rab11.S25N (#9792), P{TRiP.HMS01056}attP2 Vha68-2 (#64582), P{TRiP.HMS01442}attP2 VhaAC39-1 (#35029), UAS-mCherry:NLS (#38424), attP2 empty line (#8622), UASp-YFP.Rab9 (#9784), Rab1EYFP (#62539), Rab6EYFP (#62544), UAS-GFP.KDEL (#9898), UAS-ManII-EGFP (#65248), UAS-GFP-LAMP (#42714), Appl[d] (#43632), UAS-Appl (#38403), UAS-APP695 (#33796), UAS-YFP:Rab4 (#23269), UAS-YFP:Rab4[S22N] (#9768), UAS-YFP:Rab4[Q67L] (#9770), UAS-Rab4:mRFP (#8505) and UAS-mCD8:mRFP (#27400).Flies were raised at 25 °C under a light/dark cycle of 12 h/12 h (3000 lx) on standard fly medium , unless otherwise stated. UAS-BIN1iso1, UAS-BIN1iso1 ΔEx7, UAS-BIN1iso1 ΔCLAP, UAS-BIN1iso9, UAS-BIN1iso8 and UAS-dAmphA lines were generated in this work  in MOPS 1X buffer . After migration, samples were transferred on to nitrocellulose membranes using the Biorad Trans-blot transfert system kit (Biorad) according to the supplier technical recommendation . Next, membranes were incubated in milk  to block non-specific binding sites during 1 h at RT, followed by several washes. Immunoblotting was carried out with primary antibodies anti-BIN1 , anti-α-tubuline , anti-dAmph [Drosophila heads (n = 10) and thorax (n = 5) were dissected and crushed in ice-cold LDS lysis buffer  supplemented with reducing agent . Samples were centrifuged at 8500, 1/5000) and anti, 1/5000).CO2-anesthetized flies were placed in a 35 mm cell culture dish half-filled with 1% agarose and covered with water at 4 °C as described . Flies wFly heads were dissected and fixed in 4% paraformaldehyde phosphate buffer saline (PBS) for 20 min at room temperature. After washing, retinas were finely dissected, permeabilized and depigmented in 0.3% (v/v) Triton X-100 in PBS (0.3% PBT) overnight at 4 °C under gentle agitation. After blocking with 5% normal goat/donkey serum in 0.3% PBT, samples were incubated overnight at 4 °C with the primary antibodies diluted in 0.3% PBT. The following antibodies were used: anti-NA/K ATPase alpha subunit , anti-rhodopsin  and anti-GFP . After washing, they were incubated overnight at 4 °C with Alexa 555 Phalloidin anti-F-actin  and the secondary antibodies diluted in 0.3% PBT: Alexa 488 Donkey anti-guinea pig , Alexa 633 Goat anti-mouse . After washing, samples were incubated in 90% glycerol PBS for 30 min in the dark before being mounted in the same solution. Retinas were imaged with a LSM710 confocal microscope  equipped with a 40X oil objective.Drosophila eyes were dissected and fixed in 1% glutaraldehyde, 4% paraformaldehyde, 0.1 M sodium cacodylate buffer (pH 6.8) 30 min at room temperature and then overnight at 4 °C. After washing, eyes were post-fixed at room temperature in 1% OsO4 and 1.5% potassium ferricyanide for 1 h, then with 1% uranyl acetate for 45 min, both in distilled water at room temperature in the dark. After washing, they were dehydrated with successive ethanol solutions. Eyes were infiltrated with epoxy resin (EMbed 812 from EMS) and were mounted in resin into silicone embedding molds. Polymerization was performed at 60 °C for 2 days. Ultrathin sections of 70–80 nm thickness were observed on formvar-coated grid with a Hitachi H7500 TEM , and images were acquired with a 1 Mpixel digital camera from AMT .hiPSCs  modified for BIN1 in exon 3 Fig.  were gen2+ and Mg2+. BIN1 WT and KO hiNPCs, thus generated, were maintained for up to 10 passages.In order to obtain hiNPCs, the embryoid body method detailed by Stemcell Technologies was used for the induction of BIN1 WT and KO hiPSCs. Following the generation of hiNPCs, these derived cells were maintained in treated cell culture dishes pre-coated with poly-L-ornithine (PLO) and laminin (5 µg/mL). PLO solution was made in water (0.001%) while laminin was diluted in PBS with Ca2D cultures comprising hiNs and hiAs were produced from hiNPCs. 60,000 hiNPCs/well were plated in 24-well cell imaging plates from Eppendorf (Cat # 0030741005) pre-coated with PLO (0.001%) and laminin (10 µg/mL). Cells were kept in 0.5 mL of NPC medium per well for 24 h. Following this, equal volume of complete BrainPhys medium  was added to each well to begin the process of differentiation. Subsequently, media was changed in the plates bi-weekly. The media change consisted of removing half of the existing medium in each well and replacing it with an equal volume of fresh complete BrainPhys medium. Mixed cultures of hiNs and hiAs were obtained at the end of 6 weeks from the start of the differentiation process.Cerebral Organoids were generated from hiPSCs at 80% confluency. hiPSCs were detached from the Vitronectin XF substrate using Gentle Cell Dissociation Reagent (Stemcell Technologies), centrifuged, pelleted and resuspended in Embryoid Body (EB) seeding medium (Stemcell Technologies) to form EBs. 9000 cells were plated per well in a 96-well round-bottom ultra-low attachment plate. After two days, 1 or 2 EBs were transferred to a well of a 24-well ultra-low attachment plate containing Induction Medium (Stemcell Technologies). The EBs were kept in the induction Medium for 2 days and next they were transferred into Matrigel (Corning) using an embedding surface. When the Matrigel polymerized, the EB were transferred to a 6-well ultra-low adherent plate with Expansion Medium (Stemcell Technologies). After 3 days, the medium was replaced by Maturation Medium (Stemcell Technologies) and the plate was placed in an orbital shaker (65 rpm speed). Complete media changes were done on a bi-weekly basis.Lentiviral constructs were produced by the Vect’UB platform within the TBM Core unit at University of Bordeaux, Bordeaux, France . All three lentiviral constructs harbored reporter tags for the expression of tdTomato protein. The lentiviral vectors used were the empty vector—436 (ID # 1770), 436-Bin1Iso1 (ID # 1771), and 436-Bin1Iso9 (ID # 1772). Lentiviral infections were done in 3-week old differentiation cultures obtained from hiNPCs. Viral transductions were performed at a multiplicity of infection (MOI) of 1. In brief, appropriate volumes of each construct were mixed in complete BrainPhys medium and 50 ul of the viral medium mix was then added to each well. Each of the 3 constructs were added in triplicate to respective wells for each of the BIN1 WT and KO cells. Infected cells were maintained for a further 3-week period with bi-weekly changes of half volume of medium in each well.Cells were fixed in PFA (4% w/v) for 10 min. Fixed cells were then washed with PBS 0.1 M. Cells were then blocked with blocking solution  at room temperature for 1 h under shaking conditions. Primary antibodies diluted in the blocking solution were then added and incubation was done overnight at 4ºC under shaking conditions. The following day, cells were washed with PBS 0.1 M 3 times for 10 min each before the addition of fluorophore-conjugated secondary antibodies in blocking solution for 2 h at room temperature under shaking conditions ensuring protection from light. 3 washes with PBS were done for 10 min each at room temperature under shaking conditions with protection from light. Hoechst 33258 nucleic acid stain was added to PBS 0.1 M in the second wash. Cells were mounted with fluoromount and imaged directly in the cell imaging plates. All images were acquired using an LSM 880 Confocal Scanning Microscope housed at the Imaging Platform of the Institut Pasteur de Lille using the ZEISS ZEN Imaging Software. Image acquisition was done at 40X for the various cellular markers in Fig. Cerebral organoids were fixed in PFA (4% w/v) for 30 min at 4 °C followed by three washes with PBS 0.1 M. Cerebral organoids were then placed in sucrose solution (30% w/v) overnight before being embedded in O.C.T (Tissue-Tek). Embedded tissue was sectioned at 20 μm using a cryostat and mounted slides were stored at − 80 °C until immunostaining was performed. Mounted tissue was removed from storage and warmed by placing at room temperature for 30 min. Tissue were rehydrated and washed with room temperature PBS 0.1 M 3 times for 5 min. Slides were then washed once with PBS with 0.2% Triton X-100 for 15 min. Tissue was blocked using 10% of donkey serum in PBS 0.1 M for 1 h at room temperature. After blocking, primary antibodies were added to 0.2% Triton X-100 and 10% of donkey serum in PBS 0.1 M at appropriate dilutions and incubated overnight at 4 °C. The next day, slides were washed with PBS 0.1 M 3 times for 5 min each with gentle shaking. Subsequently, slides were incubated with fluorophore-conjugated secondary antibodies in 0.2% Triton X-100 and 10% of donkey serum in PBS 0.1 M for 2 h at room temperature in the dark. After secondary antibody incubation, slides were washed 3 times with PBS for 5 min with gentle shanking. Nuclei were visualized by incubating the tissue for 5 min with Hoechst stain in PBS 0.1 M. Sections were mounted using aqueous mounting medium (Polysciences). Images were acquired using an LSM 880 Confocal Scanning Microscope in concert with the ZEISS ZEN imaging software housed at the Imaging Platform of the Pasteur Institute, Lille. For EEA1 quantifications, we selected 5 random regions in the surface (50-250 µm) of each cerebral organoid, where a high density of MAP2-positive cells could be distinguished. Images were acquired using a 63X objective in 2–3 different sections obtained from at least 3 cerebral organoids of each genotype.hiPSCs were detected with antibodies for SOX2 (RRID:AB_2651000) and SSEA4 (RRID:AB_2651001) using the Molecular Probes™ Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit . Antibodies used for immunocytochemistry/immunohistochemistry were EEA1 , MAP2 , RFP , SOX2 , NESTIN , and GFAP . All fluorophore-tagged secondary antibodies were sourced from Jacskon ImmunoResearch Europe Ltd.Samples from the 2D cultures or brain organoids were collected in RIPA buffer containing protease inhibitors  and sonicated two times at 60–70% during 10 s before use for the western blotting analyses.Protein quantification was performed using the BCA protein assay (Thermo Scientific). In total, 10 μg of protein from extracts were separated in SDS–polyacrylamide gels 4–12%  and transferred to nitrocellulose membranes (Bio-Rad). Next, membranes were incubated in milk  to block non-specific binding sites during 1 h at RT, followed by several washes with TTBS. Immunoblottings were carried out with primary antibodies anti-BIN1 , anti-APP   and anti-β-ACTIN  overnight at 4 °C on 20 RPM agitation. The membranes were washed three times in TTBS, followed by incubation with HRP-conjugated secondary antibodies anti-mouse  and anti-rabbit  overnight at 4 °C on 20 rpm. The membranes were washed three times in TTBS, and the immunoreactivity was revealed using the ECL chemiluminescence system  and imaged using the Amersham Imager 600 (GE Life Sciences). Optical densities of bands were quantified using the Gel Analyzer plugin in Fiji – ImageJ .3. MAP2 and tdTomato surfaces were also detected based on background subtraction using “local contrast” and automatic threshold. Automatic thresholds using the filter “absolute intensity” were then applied to find MAP2 and tdTomato surfaces. For the detection of EEA1 puncta on MAP2 and EEA1 puncta on MAP2+ /tdTomato+ cells, filters for the standard deviation of intensity were applied in the MAP2 and tdTomato channels respectively. Volume information for the EEA1 puncta were collated from each acquired image as CSV files. The volumes were sorted using Microsoft Excel. We used a cut-off maximum volume of 10µm3 for each field. The sorted volume data were then analyzed statistically using the GraphPad Prism software.The “surface” function on Imaris was used for detection of EEA1 puncta. Background subtraction was performed using “local contrast” and automatic threshold, warranting the same processing criteria for all images. Next, a manual filter cut-off was applied to detect all puncta volumes above 0.1 µmg. Organoids pellets were resuspended in 500 μl lysis buffer . Multiple mechanical resuspensions and wrecking steps in this buffer were perform for a total lysis time of 10 min, 500 μl of washing buffer was added  and the lysis suspension was centrifuged 8 min at 500g . Nuclei pellets were washed three times with one filtration step by MACS pre-separation filter 20 μm (Miltenyi Biotec). Nuclei pellets were resuspended in 100 μL of staining buffer , 10 μL of Fc blocking reagent HumanTruStainFc™  and incubated 5 min at 4 °C. 1 μl of antibody was added  and incubated 15 min at 4 °C. Nuclei pellets were washed three times in staining buffer with one filtration step by MACS pre-separation filter 20 μm (Miltenyi Biotec) to a final resuspension in 300 μl of staining buffer for Malassez cell counting with Trypan blue counterstaining . Isolated nuclei were loaded on a Chromium 10 × Genomics controller following the manufacturer protocol using the chromium single-cell v3 chemistry and single indexing and the adapted protocol by Biolegend for the HTO library preparation. The resulting libraries were pooled at equimolar proportions with a 9 for 1 ratio for Gene expression library and HTO library respectively. Finally, the pool was sequenced using 100 bp paired-end reads on the Illumina NovaSeq 6000 system following the manufacturer recommendations.Nuclei isolation and Hash-tagging with oligonucleotides steps were realized on ice with pre-cold buffers and centrifugations at 4 °C. BIN1 WT and KO organoids of 6 months (n=4 per genotype) were cut in 2 parts, washed twice with 1 ml of Deionized Phosphate Buffer Saline 1X  and centrifuged 5 min at 300UMI Count Matrices for gene expression and for HTO libraries were generated using the CellRanger software (10 × Genomics). After filtering for low quality cells according to the number of RNA, genes detected, and percentage of mitochondrial RNA, and normalizing the HTO matrix using centered log-ratio (CLR) transformation, 2,990 cells were assigned back to their sample of origin using HTODemux function of the SeuratV3 R Package , resulting to 1,794 and 1,196 cells for BIN1 KO and WT, respectively. Then, Seurat Workflow with SCTransform normalization was used to cluster the cells according to their transcriptome similarities. Each cluster was annotated using cell type specific markers. Finally, differential expression analysis between BIN1 KO and WT cells within each identified cell type was performed using DESeq2 package .https://www.R-project.org/), RStudio 1.2.1335 and GraphPad Prism. For boxplots, the bold segment, lower and upper hinges represent the median, first quartile and third quartile respectively. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge . The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge.Statistical information are available in the method section and in the figure legends. Two-tailed statistical tests were used. Hypothesis testing was carried out with Kruskal Wallis test followed by Mann–Whitney comparison and ANOVA with post-hoc Tukey. Statistical analyses were performed using R 3.6.0 (R Core Team (2019). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL BIN1 belongs to the amphiphysin family and in mammals, two genes compose this family: amphiphysin I (AMPH) and amphiphysin II (AMPH2) also named BIN1. Drosophila has only one ortholog called Amphiphysin (Amph) that is referred to as dAmph henceforth in this article. dAmph has 3 isoforms but only the BAR and SH3 domains are conserved in dAmph. Within this background, we generated 3 transgenic Drosophila lines expressing the human BIN1iso1, BIN1iso8 and BIN1iso9 isoforms. As a control, we also generated transgenic Drosophila lines expressing the longest dAmph isoform, dAmphA  had a normal number of outer photoreceptor neurons (6 per ommatidium) with normal morphology Fig. a, b. TheSince photoreceptor neurons are specialized neurons highly dependent on the phototransduction cascade , we wondWe asked why BIN1iso1 was more toxic for photoreceptor neurons than the other BIN1 isoforms. We wondered whether it could originate from the subcellular localization of BIN1 isoforms. We labelled retinas of 1-day old flies expressing either BIN1iso1, BIN1iso8 or BIN1iso9 for BIN1 Fig. f. All BITo further understand the cause of the BIN1iso1-induced degeneration, we analyzed the degenerating photoreceptor neurons by electron microscopy. While neurons in 15-day-old flies either expressing luciferase, BIN1iso9 or dAmphA did not show any abnormalities, the degenerating neurons in BIN1iso1 flies exhibited a strong accumulation of vesicles Fig. a and didWe next evaluated the nature of these vesicles by immunofluorescence using specific organelle GFP- or YFP-tagged markers for endoplasmic reticulum, Golgi, plasma membrane, early endosome, late endosome/multivesicular body, recycling endosome, lysosome and autophagosome  Fig. 3b: Fig. S5CA, Rab5DN, Rab11CA and Rab11DN. Surprisingly Rab5DN rescued BIN1iso1-expressing photoreceptor neurons, whereas Rab5CA had no effect  was removed or by knocking down Rab5 (Rab5HMS00145)  , recycling endosome (Rab11), late endosome Rab7 and Rab9) and lysosome (subunits of the V-ATPase) . We obsect Fig. c. This i and Rab9Appl or overexpression of human APP could modulate BIN1iso1-induced photoreceptor neuron degeneration. Loss of one copy of Appl had no effect suggesting that Appl is not required for BIN1iso1 toxicity , the product of APP cleavage by β-secretase and precursor of Aβ peptide through γ-secretase cleavage, induces endosome enlargement in neurons . Drosophity Fig. g. Overexppl Fig. g. OveralBIN1 wild type (WT) and knockout (KO) pluripotent stem cell (hiPSC) lines by CRISPR/Cas9 technology. Both hiPSC lines showed similar expression of pluripotency cell markers  expressed similar levels of NESTIN and SOX2 -expressing endosomes in microtubule-associated protein 2 (MAP2)-expressing cells in both 2D cultures and cerebral organoids  in BIN1 KO hiNs, BIN1iso1 expression rescued early endosome sizes but did not lead to enlargement of these vesicles; and (iii) in WT hiNs, BIN1iso1 overexpression induced early endosome enlargement. These results suggest that homeostatic BIN1iso1 expression levels need to be tightly regulated to allow proper endocytic trafficking in neurons. Of note, BIN1iso9 overexpression in WT hiNs induced a reduction of early endosome size. Since BIN1 forms dimers through its BAR domain [Our results indicate context- and isoform-specific functions of BIN1: (i) Expression of BIN1iso1, BIN1iso8 and BIN1iso9 altered photoreceptor neuron rhabdomere morphogenesis during development in an isoform-independent and dose-dependent manner, whereas in the adult photoreceptor neurons BIN1iso1 expression induced neurodegeneration in an isoform-dependent and dose-independent manner; (ii) in human neurons, we observed a shift in BIN1 isoform expression from low molecular weight isoforms in hiPSCs and NPCs (likely mainly BIN1iso9) to high molecular weight isoforms in hiNs (likely mainly BIN1iso1); (iii) BIN1iso1 expression rescued the size of BIN1-deficient early endosomes contrary to BIN1iso9. Collectively, these results argue for an important role of BIN1iso1 in mature neuronal cells and support the fact that BIN1iso1 is a neuron-specific isoform with specific functions in this cell-type , 69. FurR domain , we postOur results support a direct role of BIN1iso1 in neurons at the early endosome crossroad as indicated (i) by the labeling of large vesicles with early endosome markers in Drosophila, (ii) by the Rab5 rescue experiment in Drosophila and (iii) by the early endosome size modulation in hiNs. Of note, the phenotype with very large vesicles in Drosophila likely results from Drosophila being a heterologous system for human BIN1iso1, and as a consequence, exhibiting stronger phenotypes. Additionally, in Drosophila, some of the vesicles also exhibited markers for late endosome/MVB and exceptionally, lysosome. However, regulation of late endosome/MVB or lysosomal function did not modulate photoreceptor degeneration and secretion of exosomes did not appear to be blocked, suggesting that BIN1iso1 defect do not occur at this level. BIN1 has been proposed to regulate endocytosis by interaction with clathrin, Adaptor Proteins (AP) and dynamins , 50, 65.BIN1, PICALM, EPHA1, CD2AP, SORL1 and RIN3) encode proteins that function predominantly in endocytic trafficking [Appl, the Drosophila ortholog of APP, did not rescue BIN1iso1-induced neurodegeneration, suggesting that APP β-CTF does not mediate BIN1iso1 toxicity. The involvement of APP β-CTF have also been questioned in SORL1 knocked-out hiNs with contradictory results in two studies showing APP β-CTF-dependent or -independent endosomal defects [Interestingly, a growing body of evidence supports a dysregulation of the endosomal-lysosomal system as a plausible underlying mechanism in AD pathogenesis , 58. Sevfficking . Endosomfficking , hiNs cafficking  and hiNsfficking , 32, recfficking  and overfficking . We havefficking . To do sfficking . However defects , 32. Ano defects . Upregul defects  and RIN3 defects , 57. Fur defects  and indi defects , 66. OveSince we observed endosome enlargement and neurodegeneration upon BIN1iso1 overexpression, our results also suggest that an increase in BIN1iso1 may be deleterious to neurons and contribute to early phases of AD pathology through early endosome alterations. It implies an increase in BIN1iso1 levels in neurons, which remains elusive. To date, most experiments showing altered BIN1 expression in the AD brain have focused in samples obtained at late stages of the pathology , 52, 60 BIN1 polymorphisms associated with an increased AD risk could affect the expression of BIN1iso1 in neurons. Interestingly, the variant rs59335482, an insertion allele associated with a higher AD risk, is able to increase transcriptional activity in a luciferase assay in vitro using HEK cells and SH-SY5Y neuroblastoma cells, and is also associated with an increase in BIN1 mRNA expression in the brain [BIN1 expression specifically in human induced microglia, but not in Neurog2-induced hiNs. Unfortunately, neither the transcriptional effect of the exact variants nor the expression of BIN1 in spontaneously differentiated hiNs have been tested yet. In addition, the possibility that the impact of functional variants may depend on specific AD pathophysiological process, e.g. Aβ exposure, has not been assessed. There is, thus, a high uncertainty about functional variants in the BIN1 locus. Even if our current study was not intended to directly assess functional consequences of AD risk-related BIN1 polymorphisms but to focus on BIN1 isoforms function in neurons, which are still insufficiently described and understood, it may provide a context for BIN1-associated risk, namely the dysregulation of early endosome size and function. Up to now and not exclusive from each other, BIN1 polymorphisms have been associated with Tau but not amyloid loads in post-mortem AD brain tissue [Future studies should also address the possibility that he brain , 46. Deln tissue , 19.In conclusion, an increase of BIN1iso1 in neurons could contribute to AD pathogenesis by increasing the size of early endosomes observed early in the pathogenic process and by inducing neurodegeneration. Other AD genetic determinants have also been shown to regulate early endosome size, supporting early endosome defects as a major event in the pathogenesis of AD.Additional file 1. Supplementary information and supplementary methods.Additional file 2. Figure S1. Creation of transgenic lines expressing human BIN1 isoforms.Additional file 3. Figure S2. Scheme of AmphMI08903-TG4.0 allele.Additional file 4. Figure S3. Generation and test of transgenic Drosophila expressing truncated human BIN1-1 forms for the Exon7 (BIN1-1 ΔEx7) and the CLAP domain (BIN1-1 ΔCLAP).Additional file 5. Figure S4. Electron microscopy images of BIN1-1-induced photoreceptor degeneration in Drosophila eyes.Additional file 6. Figure S5. Screening of organelle markers in BIN1-1-induced degenerating photoreceptor neurons.Additional file 7. Figure S6. Effect of Rab4 modulation on BIN1iso1 neurotoxicity in Drosophila photoreceptor neurons.Additional file 8. Figure S7. Graphic representation of proportions of cell types (in percentages) in BIN1 KO and WT cerebral organoids.Additional file 9. Table S1. Differential gene expression analysis for all different cell types identified in BIN1 WT and KO cerebral organoids.Additional file 10. Table S2. Gene ontology enrichment analysis for the 41 DEGs observed in glutamatergic neurons.Additional file 11. Figure S8. Decreased APP/β-CTF levels in BIN1 KO cerebral organoids.


IME1 expression. Further studies showed that the absence of H3K18ac impaired respiration, leading to the reduction of Rim101p, which further upregulated a negative regulator of IME1 transcription, Smp1p. Together, our studies reveal a novel meiosis initiation pathway mediated by histone H3 modifications.Meiosis is essential for genetic stability and diversity during sexual reproduction in most eukaryotes. Chromatin structure and gene expression are drastically changed during meiosis, and various histone modifications have been reported to participate in this unique process. However, the dynamic of histone modifications during meiosis is still not well investigated. Here, by using multiple reaction monitoring (MRM) based LC-MS/MS, we detected dynamic changes of histone H3 lysine post-translational modifications (PTMs). We firstly quantified the precise percentage of H3 modifications on different lysine sites during mouse and yeast meiosis, and found H3 acetylation and methylation were dramatically changed. To further study the potential functions of H3 acetylation and methylation in meiosis, we performed histone H3 lysine mutant screening in yeast, and found that yeast strains lacking H3K18 acetylation (H3K18ac) failed to initiate meiosis due to insufficient  To retain genetic stability and variety, most higher eukaryotes produce offspring through sexual reproduction, which includes the production and fusion of two haploid gametes . The proMeiosis is an integral part of gametogenesis . During Post-transcriptional modifications (PTMs) of histones are essential for regulating chromatin structure and gene expression, and extensively involved in many vital cellular processes . Until nSMP1, an inhibitor of IME1 transcription, which finally led to the failure of meiosis initiation. Therefore, our studies uncover a new relationship between histone H3 modifications and meiosis initiation.Here, we performed multiple reaction monitoring (MRM) based LC-MS/MS to examine the dynamic changes of histone H3 lysine post-translational modifications during meiosis in both mice and yeast. We found H3 acetylation and methylation were dramatically changed during meiosis. To further study their potential functions, we performed a systematic mutational screen on H3 lysine in yeast and found that H3K18 acetylation (H3K18ac) was critical for meiosis initiation. Further studies showed the absence of H3K18ac influenced yeast respiration, which further downregulated the expression of Rim101p. In addition, deficiency of Rim101p activated the expression of HHT1-HHF1 in plasmid pYES2 and then this plasmid was replaced with the pRS303 carrying mutant HHT1-HHF1. All deletion strains were homozygous, generated using a PCR-mediated gene replacement method as previously described , spermatocytes (n = 4) and round spermatids (n = 3)] and yeast . Histone proteins were extracted and depurated as previously described  at 4°C for 40 min. Samples were then centrifuged for 10 min at 10,000 × g to deposit nuclei. Nuclei were digested with 0.4 M H2SO4, deposited with 100% (w/v) trichloroacetic acid (TCA), and washed twice with ice-cold acetone to retrieve core histones. Histone proteins were further dephlegmated on a C8 column , using an Agilent series 1200 system . Finally, histone H3 fractions were mixed together and exsiccated using a SpeedVac for further experiments.Samples for MRM-based LC-MS/MS were collected from mouse . To achieve the relative quantitation of a specific kind of modification on the lysine site in H3, the concentration of the targeted modification divided by the concentrations of all peptides on this targeted site. Using H3K4me1 as an example, the amount of H3K4me1 was calculated as below: Percentage H3K4me1 = Σ H3K4me1containing peptides/[Σ H3K4containing peptides + Σ H3K4me1containing peptides + Σ H3K4me2containing peptides + Σ H3K4me3containing peptides] × 100%. As some peptides were isobaric and difficult to be resolved by HPLC or precursor peptide ions, the b3+ fragment ions were selected in the MRM transitions to distinguish these peptides.The quantification of the histone modifications was performed as previously described . The cong for 5 min at 4°C. After washed once by PBS, the sediments were re-suspended in 15 ml of PBS with 1 mg/ml DNase I and 0.25% Trypsin. Thereafter, the samples were shaking gently in 37°C water bath for 5 min. Then, the cells were assembled and washed by PBS with 0.5% BSA before filtrated through a 40 μm Nylon Cell Strainer. The cells were carefully loaded on a 2–4% BSA gradient in PBS and separated through sedimentation by gravity. The separate cells were collected into different tubes. The cells from each tube were examined with light microscopy to confirm cell-type and purity. The samples containing same cell type  with proper purity (≥90%) were collected together.Testes from 7-day, 17-day, and 8-week old C57BL/6 mice were used to obtain spermatogonia, spermatocytes and round spermatids, respectively, following a method previously described . Briefly600 = 0.3. Following growth for 10 h at 30°C, cells were gathered, washed, and resuspended in SPM  to OD600 = 1.9 and cultured at 30°C for sporulation. In addition, another method was used for WT and respiratory defective cells for sporulation, which was induced by rapamycin as previously depicted  or SD medium  for 24 h, yeast was deliquesced with YPA medium  to ODdepicted . The str7 cells were fixed overnight with 1 mL cold 70% ethanol, and then resuspended in 1 mL 50 mM sodium citrate for DNA content analysis. The cells were centrifuged at 845 × g for 5 min, and the sediments were digested with RNase A  for 2 h at 37°C. After sonication for 2 s at 20% power, the samples were stained with 1 mM Sytox Green . A BD FACS vantage SE Flow Cytometry System  was used to analysis the results.About 1 × 10600 of 1.0 at 30°C. The cells were gathered, washed, and serially diluted. Each dilution was then spotted onto an auxotrophic plate containing glucose or glycerol and then cultured for 2 days at 30°C.Yeast strains were cultured in YPD or SD medium to ODTrans-Blot SD Semi-Dry Transfer Cell system. After incubation overnight at 4°C using a primary antibody, the blots were incubated with a secondary antibody . Then, an Odyssey infrared 740 imager  was used to scan the blots. H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K18me1, H3K23ac, H3K79me2, and H3K79me3 antibodies were obtained from EASYBIO . MYC and HA antibodies were purchased from Abmart . FLAG antibodies were purchased from Bioregent Bio company  and Abmart . The Pgk1p polyclonal antibody was generated in rabbits.After mild alkaline treatment, cells were boiled in a standard electrophoresis loading buffer similar as previously described . The proThe Seahorse XF96 Extracellular Flux Analyzer  was used to detect respirations of WT and mutant yeast cells. The methods were similar as previous description . 25 μL 5600 = 0.2. After 4 h culture, the exponential phase cells were harvested and resuspended in YPD, YPA, YPG , and YPE , respectively. 175 μL of each kind of cultures were added into the treated Seahorse plate that each well contained about 1 × 105 cells. Then the plate was centrifuged for 3 min at 500 × g and incubated for 30 min at 30°C before loaded into the analyzer. The measure times of basal oxygen consumption rates were 2.5 min, 30 s, and 2.5 min. And 10 cycles were detected.Yeast cells were cultured in YPD overnight at 30°C. On the day of measurement, cells were diluted to ODg for 2 min, the supernatant was transferred to a new RNase free tube. The samples were then mixed with phenol and chloroform (1:1) and held at room temperature for 5 min, and the supernatant was again transferred to a new tube after centrifuging at 13,523 × g for 5 min. 2.5 × volume of ethanol and 40 μL 3M sodium acetate were added to the samples, which was finally centrifuged at 13,523 × g for 10 min to collect the RNA. 1 mL 80% ethanol was used to wash the RNA pellet and the supernatant was removed again after centrifuging at 13,523 × g for 10 min. Finally, the RNA pellet was dried, dissolved in 20 μL diethyl pyrocarbonate-treated water, and stored at −80°C.Yeast total RNA extraction was accomplished using previously described methods . BrieflyTAF10. A Light Cycle 480 software 1.5.1 was used to analyzed the results. All primers used are shown in A PrimeScript RT Reagent Kit  was used to synthesize complementary DNA (cDNA). Amplification was performed in a 10 μl reaction containing 5 μL 2 × EvaGreen mix , 0.5 μL each primer (10 nmol/L), 2 μL sample cDNA, and 2.5 μL ddH2O. A Roche Light Cycler 480II System  was used to perform real-time PCR. The PCR program was initiated at 95°C for 10 min, followed by 40 cycles of denaturation for 5 s at 95°C, annealing for 30 s at 60°C, and elongation for 60 s at 72°C. Fluorescence signals were detected at 72°C in the time of the elongation step. Three biological replicates were taken for each sample and normalized to housekeeping gene t-test. The data were considered significant when the P-value was less than 0.05 (∗), 0.01 (∗∗), or 0.001 (∗∗∗).All data were shown as the mean ± SD and statistic differences were calculated using a two-tailed Student’s To assess the role of chromatin changes during gametogenesis, we detected the acetylation and methylation of H3 and its variants by using multiple reaction monitoring (MRM)-based LC-MS/MS of three crucial germ cell stages of spermatogenesis in mouse: spermatogonia, spermatocytes and round spermatids . ProteinThese dynamically changed H3 modifications were clustered into three groups using Cluster 3.0 software . ClusterAs MRM-based LC-MS/MS could determine the concentration of each histone modification, we attempted to quantify the precise percentage of H3 modifications at different lysine sites during spermatogenesis and found that modifications on different lysine sites in H3 or its variants were dramatically different from each other . For exaBecause we lack a direct way to manipulate histone modification sites in mice, it is difficult to directly assess the function of histone modification in mammals. Given yeast’s genetic operability and conservatism with mammals, budding yeast is a powerful model to study the functions of histone modifications in meiosis . To explDuring yeast sporulation, four types of H3 modifications were identified using Cluster 3.0 software . ClusterNext, we quantified the precise percentage of H3 modifications at different lysine sites during yeast sporulation. Similar to our MRM-based LC-MS/MS results in mice, some H3 lysine sites showed rare modification , whereas other H3 lysine sites showed that more than 50% of the regions could be modified by acetylation or methylation . At thesTo examine the conservation of H3 acetylation and methylation in mouse and yeast meiosis, we compared MRM-based LC-MS/MS results from mouse and yeast. Although some H3 modifications showed variable tendencies in mice and yeast, the dynamics of some H3 modifications during meiosis were conserved, such as H3K4me1, H3K4me2, and H3K18me1 . In summHHF1, HHF2, HHT1, and HHT2), and stabilized histone H3 and H4 protein levels by transforming a plasmid that contained HHT1, HHF1, and a URA3 selectable marker (URA3 with 5-Fluoroorotic Acid (5-FOA)  . Examina (5-FOA) . Importa (5-FOA) .Next, we performed sporulation of these H3 mutant strains and found that the sporulation efficiency of H3K4A, H3K18A, H3K27A, H3K42A, and H3K79A mutants showed significant decreases compared with that of the WT and shuffle strain , indicatGiven that H3K18A absolutely blocked the sporulation progress , 4A and IME1 expression. To test our speculation, we examined mRNA levels of IME1 in WT, H3K18A and H3K18Q strains using quantitative real-time PCR and found that levels of IME1 mRNA in WT, H3K18A, and H3K18Q strains correlated with their protein levels , homologous recombination, meiotic sex chromosome inactivation (MSCI) and crossover formation . AlthougH3f3b-null male mice are complete infertile and show disrupted spermatogenesis-related genes expression in germ cells , which supports a series of physiological function . In budd pathway . Because pathway , respira pathway . Our cur meiosis .SIRTUIN 7 (SIRT7) is known to be responsible for H3K18 deacetylation in mammals .The data presented in the study are deposited in the PeptideAtlas repository, accession number .WL, HY, and CL conceived and designed the experiments. JS, YM, and HH performed most of the experiments. YL performed some of the experiments. HY performed LC-MS/MS. JS, YM, CL, and WL wrote the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5’ splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5’ exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution. 6A. Here the authors show that m6A modification on U6 snRNA promotes efficient recognition of the splice site through cooperating with U5 snRNA, indicating that U6 snRNA m6A allows the 5’ exon sequence variation.Spliceosomal U6 snRNA is modified by m Recent studies using deep-sequencing technologies successfully mapped several species of RNA modifications in eukaryotic mRNAs and non-coding RNAs in a transcriptome-wide manner6. These findings raise the concept of the “epitranscriptome” and highlight the importance of RNA modification as a previously unrecognized layer of regulatory gene expression.Post-transcriptional modification is a characteristic structural feature of RNAs. About 150 types of chemical modifications have been found in various RNAs from all domains of lifeN6-methyladenosine (m6A), an abundant modification in eukaryotic mRNAs and long non-coding RNAs, plays a critical role in various biological processes, including meiosis8, circadian rhythm9, sex determination11, cell proliferation12, differentiation13, reprogramming14, and stress responses15. The biogenesis and dynamics of m6A have been studied extensively. The METTL3/METTL14 writer complex introduces m6A in the RRACH motif 23. Internal m6As are abundant in the last exon of mRNAs, near the stop codon25, and are decoded by several reader proteins, including YTH proteins, thereby contributing to the diverse fates of mRNAs33. m6A also has the ability to destabilize double-stranded regions of structured RNA, thereby facilitating recognition by hnRNP C and hnRNP G35. It has been proposed that m6A is a reversible modification that can be modified back to adenosine by erasers FTO and ALKBH537.6A, N6, 2′-O-dimethyladenosine (m6Am) is present at the transcription start site of mRNAs in vertebrates39. Recently, our group reported a cap-specific m6A writer, CAPAM/PCIF1, which catalyzes N6-methylation of m6Am40. Structural studies of CAPAM revealed the molecular basis of cap-specific m6Am formation, and ribosome profiling analysis revealed that CAPAM-mediated N6-methylation promotes translation of mRNAs starting from m6Am.In addition to internal m6A writer for U6 snRNA and MAT2A mRNA43, which encodes S-adenosylmethionine (SAM) synthetase. METTL16 targets a specific nonamer sequence in structured RNA (UACAGAGAA) for m6A formation45. At high SAM concentrations, METTL16 introduces m6A into six hairpin structures in the 3′ UTR of MAT2A mRNA, leading to degradation of the mRNA mediated by YTHDC1. At low SAM concentrations, METTL16 is bound to the hairpin structures and acts as a splicing enhancer for the 3′ terminal intron, thereby increasing production of mature MAT2A mRNA. Thus, METTL16 mediates a feedback mechanism that controls SAM homeostasis. This regulatory mechanism is critical for cell viability, as shown by the essentiality of METTL16 gene in human culture cells46 and Mettl16 in mouse embryo44. Mettl16 null mutations are embryonically lethal, and homozygotes die around the implantation stage (E3.5). Early embryos lacking Mettl16 contain reduced amounts of Mat2a mRNA, highlighting the importance of Mettl16 for production of this message.METTL16 is an m54. U snRNAs contain a number of modified internal nucleosides, including pseudouridines, ribose 2′-O-methylations, and several base methylations58. 2′-O-methylation and pseudouridylation of U2 snRNA are essential for snRNP biogenesis and efficient splicing65. In U6 snRNA from mammals66, S. pombe57, and broad bean67, an m6A is present in the middle position of the ACAGA box that recognizes the 5′ splice site (5′SS)67 67 Fig. . In huma6A in U6 snRNA, we performed RNA-seq analyses of an S. pombe mtl16 knockout strain. We found that a subset of introns was retained in mRNAs, indicating a splicing defect caused by loss of m6A in U6 snRNA. The most significantly impacted introns were enriched for adenosine at the fourth position from the 5′SS, which base-pairs with m6A of U6 snRNA. In addition, the retained introns tended to have sequences that are weakly recognized by U5 snRNA at the 3′ termini of their 5′ exons, suggesting that U6 and U5 snRNPs cooperatively recognize the 5′SS, and that m6A in U6 snRNA facilitates efficient splicing of a subset of pre-mRNAs.To elucidate the functional role of m6A in U6 snRNA + RNA fraction  and mtl16 knockout (mtl16Δ) strains by reciprocal circulating chromatography (RCC)70. The isolated U6 snRNA was digested by RNase T1 and subjected to capillary LC-nano-ESI-MS to analyze the RNA fragment containing m6A37. m6A was clearly present in U6 snRNA isolated from the WT strain  and thiabendazole (TBZ) ; these sensitivities were exacerbated when the cells were cultured at 37 °C  Fig. . In addi °C Fig. . The mtlrce Fig. , implyin6A loss affects stability of U6 snRNA, we analyzed steady-state levels of U snRNAs by Northern blotting + RNAs from WT and mtl16Δ strains and compared the splicing properties of the two samples. Based on S. pombe genome annotations, we first took reads that mapped to each intron and its neighboring exons and classified them into five groups  of mtl1644 confirmed that the alterations in splicing were dependent on the methyltransferase activity of Mtl16  and icp55 (intermediate cleaving peptidase 55) are required for mitochondrial function74. The slow-growth phenotype of the mtl16Δ strain under nonfermentable condition 75 and pot1 (shelterin complex subunit)76 is associated with sensitivity against hydroxyurea (HU) and/or thiabendazole (TBZ)  Fig. .Z-score of a two-sample Z-test with a null hypothesis of an equal IRS, as IRS values follow normal distribution .To characterize the 5′ and 3′SSs of the introns that exhibited a splicing defect, we chose a subset of introns showing large differences in IRS. To this end, we gave each intron a les Fig. . Sequenco77 Fig. , then th 78 Fig. . Notablyres Fig.  than in ion Fig. . By contion Fig. . Given tn = 2539), T4 (n = 1846), C4 (n = 514), or G4 (n = 157). We then plotted the IRS values for each group of introns in mtl16Δ versus WT strains  into two groups based on 5′ exonic sequence, AAG-A4 introns (n = 289) and non-AAG-A4 introns (n = 2250); we then plotted their IRS values in mtl16Δ versus WT strains . Furthermore, when the ACC was mutated to the AAG consensus sequence, splicing of this construct reached maximum efficiency even in the absence of m6A in U6 snRNA   Fig. . Then, axon Fig. . The A4 ted Fig. . When U5IRS Fig. , becauseion Fig. . The A4 ion Fig. . As a neant Fig. . The IRSion Fig. , because82, genetic90, and structural studies94 reveal that the ACAGA box of U6 snRNA and loop I of U5 snRNA recognize 5′SS at B or Bact complexes, and maintain this interaction until the intron lariat–spliceosome dissociation  helicase pulls the intron to bring the branch site to the active site to form the B* complex. During these dynamic conformational changes, the 5′SS needs to be anchored to the active site, which enables the branch site 2′-OH to attack the splice site. We speculate that the stability of this anchoring interaction between pre-mRNA and U5/U6 snRNA decides the overall efficiency of the first transesterification . Among fungi, S. pombe has over 5000 introns and S. cerevisiae has 296; the consensus sequences of the 5′SS are GUAAGU (A4 intron) and GUAUGU (T4 intron), respectively100. Given that m6A in U6 snRNA facilitates efficient splicing of A4 introns, S. cerevisiae may preferentially use T4 introns rather than A4 introns to achieve efficient splicing in the absence of this modification. C. albicans also has a 5′SS consensus of T4100. On the other hand, C. merolae has only 27 introns, and its 5′SS consensus sequence is GUAAGU (A4 intron)101. Curiously, in C. merolae U6 snRNA, the ACAGA box is ACUGA, and the central U base pairs with the A4 position of introns with Watson–Crick geometry102. This is an alternative means of stabilizing 5′SS recognition by U6 snRNA without having m6A in the ACAGA box. Based on these cases, m6A in U6 snRNA has likely contributed to expansion of 5′SS sequence variation in organisms with large numbers of introns as it facilitates efficient splicing of non-AAG-A4 introns.The evolutionary conservation of METTL16 implies that m43. METTL16 introduces m6A into the hairpin structures in the 3′ UTR of MAT2A mRNA by sensing cellular SAM concentrations, thereby controlling mRNA stability. In addition, METTL16 acts as a splicing enhancer to recognize the same hairpins and produce functional MAT2A protein. In fact, Mettl16 null mice die during early embryogenesis with a reduced steady-state level of Mat2a mRNA44, highlighting the importance of Mettl16 function in regulation of Mat2a levels. The 3′ UTR hairpins of MAT2A mRNA are conserved in vertebrates, but not in other organisms103. In fact, S. pombe sam1 mRNA, an ortholog of MAT2A, is not affected in the mtl16Δ strain . Thus, the observed phenotypes of this strain can be explained as a function of the splicing defect caused by loss of m6A in U6 snRNA. Nematode and plant homologs of METTL16 also have physiological functions, and strains harboring knockouts of these genes exhibit severe phenotypes. mett-10, a Caenorhabditis elegans ortholog, is required for embryonic development and germ cell differentiation104. FIONA1, an Arabidopsis thaliana homolog, plays an essential role in regulating the circadian clock105. These phenotypes might be explained by splicing alterations caused by loss of m6A from U6 snRNA. Because little change in global splicing is observed in early embryos (E3.5) of Mettl16 null mice44, the functional significance of m6A in U6 snRNA in mammals remains elusive. Presumably, however, maternal U6 snRNA bearing m6A is still present at this stage. Further studies will be necessary to determine whether m6A loss of U6 snRNA affects global splicing in vertebrate systems.METTL16 is a key regulator of SAM synthetase (MAT2A), which maintains cellular SAM homeostasis6A in U6 snRNA is nearly 100%  (WT) and SPAC27D7.08cΔ (mtl16Δ) strains were obtained from the Bioneer deletion collection74. The deletion mutation was verified by PCR. Strains were grown at 30 °C in YE5S media  or EMM media . For spot analyses, yeast cells were first grown to mid-log phase, serially diluted (1:5) from a starting sample of 104 cells, and spotted onto YE5S agar plates supplemented with inducers of cellular stress: 15 μg/ml thiabendazole (TBZ), 7.5 mM hydroxyurea (HU), 1 M KCl, or 100 mM CaCl2. The plates were photographed after 3–4 days of incubation at 30 °C or 37 °C. To measure non-fermentable growth, the yeast strains were cultured in medium containing 0.5% yeast extract, 0.1% glucose, and 3% glycerol supplemented with adenine, uracil, lysine, histidine, and leucine (225 mg/mL each). OD600 was measured every hour for 12 h.The ED668 (106. Cell pellets were resuspended in extraction buffer , mixed with an equal volume of neutral phenol, incubated at 95 °C for 15 min, and centrifuged at 8000 × g for 30 min at 4 °C. The supernatant was collected and mixed with 1/4th volume of chloroform, followed by centrifugation at 8000 × g for 15 min at 4 °C. The supernatant was subjected to isopropanol precipitation to obtain an RNA pellet. The RNA pellet was dissolved in 10 mL buffer consisting of 20 mM HEPES-KOH (pH 7.5) and 80 mM NaCl, and then mixed with 4 mL of 2-butoxyethanol. The mixture was placed on ice for 30 min. The precipitate containing polysaccharide was removed by centrifugation at 8000 × g for 30 min at 4 °C. The supernatant was subjected to ethanol precipitation to obtain total RNA.Total RNA from yeast cells for snRNA isolation was prepared by hot phenol extractionS. pombe WT cells underwent four rounds of enrichment of poly(A)+ RNA using Dynabeads oligo [dT]25 (Invitrogen). Eluate RNAs from each round of enrichment (1 μg each) were digested into nucleosides with 0.07 units of nuclease P1 , 0.127 units of phosphodiesterase I , and 0.08 units of bacterial alkaline phosphatase . The digests were subjected to LC/MS analysis using a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) equipped with a Dionex UltiMate 3000 LC System (Thermo Fisher Scientific) and an InertSustain C18 column . The mass chromatogram peak area of adenosine , N6-methyladenosine , N6-dimethyladenosine , and 2′-O-methyladenosine  was measured. Then, the peak area of m6A, m6,6A, and Am was divided by the peak area of A, and these ratios at each round of enrichment were normalized by the ratio from the first eluate and then plotted for each modification. Data were analyzed on Xcalibur 4.1 and Excel 2016, and visualized by R 4.0.3 and Canvas 15.Total RNA extracted from exponentially growing 70 using a DNA probe  was used for assignment of the product ions in CID spectra108.Isolation of yeast U6 snRNA was performed by RCC109. Briefly, DNA template was mixed with in-house made T7 RNA polymerase, NTPs, and a transcription buffer consisting of 40 mM Tris-HCl (pH 7.5), 24 mM MgCl2, 5 mM DTT, 2.5 mM Spermidine, and 0.01% Triton X-100, and was incubated at 37 °C overnight. The reaction was stopped by addition of an equal volume of phenol/chloroform/isoamyl alcohol . The supernatant was desalted by PD-10. Desalted RNA was collected by ethanol precipitation. The transcript was resolved by 10% denaturing PAGE and purified by excision from the gel.DNA templates for in vitro transcription were prepared using a series of DNA oligos  of mtl16 ORF in the database was misannotated inside an unannotated intron , we constructed a recombinant Mtl16 starting from an AUG codon  upstream of the unannotated intron. The PCR product was cloned into the NheI and XhoI sites of pET-28a(+) (Novagen) to yield pET-Spmtl16, which produces N-terminal hexahistidine-tagged Mtl16 (His6-Mtl16). The E. coli Rosetta2 (DE3) strain was transformed with pET-Spmtl16 and cultured in LB medium containing 50 μg/mL kanamycin and 30 μg/mL chloramphenicol. When the bacteria reached OD600 of 0.5, protein expression was induced by addition of 10 μM IPTG and the cells were grown for an additional 3 h at 37 °C. Cells were harvested and suspended in buffer A  supplied with 0.5 mM PMSF, followed by sonication on ice. Cell lysates were cleared by ultracentrifugation at 100,000 × g for 60 min. The supernatant was loaded onto a nickel-charged HiTrap chelating column  and unbound proteins were washed out with buffer A. The recombinant proteins were eluted with a 50-mL linear gradient comprising 0–500 mM imidazole in buffer A. A fraction containing the recombinant protein was dialyzed overnight against dialysis buffer . The concentration of the purified protein was determined in a Bradford assay  using bovine serum albumin as a standard. To obtain recombinant human METTL16, the METTL16 cDNA was amplified using the primers listed in Supplementary Data 111 and cloned into pET-28a(+) to yield pET-hMETTL16, which produces C-terminal hexahistidine-tagged METTL16 (METTL16-His6). Expression and purification were carried out as described for S. pombe Mtl16. To obtain recombinant human METTL16 MTD, cDNA coding the methyltransferase domain of METTL16 was amplified using the primers listed in Supplementary Data S. pombe Mtl16. Following expression, cells were harvested and suspended in buffer A  supplied with 0.5 mM PMSF, followed by sonication on ice. Cell lysates were cleared by ultracentrifugation at 100,000 × g for 60 min. The supernatant was loaded onto a nickel-charged HiTrap chelating column  and unbound proteins were washed out with buffer A. The recombinant proteins were eluted with a 50-mL linear gradient comprising 0–500 mM imidazole in buffer A. A fraction containing the recombinant protein was dialyzed overnight against dialysis buffer  containing SUMO protease Ulp1. Dialyzed protein was applied to Ni Sepharose 6 Fast Flow  and the flow-through was collected. The cleaved protein was further purified on a HiScreen Capto SP ImpRes  column. The concentration of the purified protein was determined in a Pierce BCA Protein Assay (Thermo Fisher Scientific) using bovine serum albumin as a standard.To obtain recombinant 6-Mtl16, and 1 mM SAM. The reaction was stopped by addition of an equal volume of phenol/chloroform/isoamyl alcohol . RNA was collected by ethanol precipitation, digested with RNase T1, and subjected to RNA-MS analysis as described above.In vitro methylation of U6 snRNA was performed at 37 °C for 1 h in 50 µL of reaction mixture consisting of 10 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM DTT, 2 μM U6 snRNA transcript, 1 μM His2; CL-1000, UVP) to cross-link the blotted RNA. Hybridization was performed using PerfectHyb (TOYOBO) at 52 °C with 4 pmol of 5′-32P-labeled oligonucleotides specific to each U snRNA  from yeast cells was resolved by 10% denaturing PAGE and either stained with SYBR Gold (Invitrogen) or blotted onto a nylon membrane  in 1 × TBE using a Transblot Turbo (Bio-Rad). Fluorescence was visualized on an FLA-7000 imaging analyzer (Fujifilm). The membrane was air-dried and irradiated twice with UV light  and were sequenced on an Illumina NovaSeq sequencer .Yeast were harvested, frozen in liquid nitrogen, and homogenized in TriPure Isolation Reagent (Roche Life Science) using SK-100 (Funakoshi). Total RNA was extracted with chloroform, recovered from the aqueous phase by isopropanol precipitation, ethanol precipitated, and dissolved in water. The total RNAs underwent two rounds of enrichment of poly(A)112 to remove adaptor sequences and low-quality bases. The processed reads were first aligned to S. pombe rRNA and tRNA sequences, then the unmapped reads were aligned to S. pombe genome assembly ASM294v2.34 using STAR 2.7.3a113. The following parameters were used–twopassMode Basic–outSJfilterOverhangMin 30 8 8 8–alignIntronMax 2000–outFilterMultimapNmax 30–outFilterScoreMinOverLread 0.4–outFilterMatchNminOverLread 0.44–outFilterScoreMin 10. The statistics of trimming and mapping is listed in Supplementary Data 114 using the libraries Biopython115 and Pysam116, and the plots were written by R 4.0.3. For each segment comprising a pair of consecutive annotated exons (exon1 and exon2), reads that mapped to the annotated SS junction (CSR), exon 1–intron junction (EIJR), intron–exon 2 junction (IEJR), any alternative 5′SS to exon 2 (A5R), and exon 1 to any alternative 3′SS (A3R), were counted.Raw Illumina sequencing reads were trimmed using fastp softwaremtl16Δ strains, as the IRS values mostly follow normal distribution  > 10] and an average CSR over 10 in either strain were applied for this calculation so that the number of introns picked up is maximized and the variance in read counts among the replicates is minimized. This process picked up 5056 GY-AG introns with sufficient read counts out of 5364 annotated introns. The 5′ and 3′SS sequences of the introns with splicing deficiencies were characterized by choosing a subset of introns with large and small IRS differences. Upon sorting the introns by 117. The splicing-adjusted read counts of each annotated gene in each sample were calculated by Eq.  digested by NdeI and BamHI using the primers listed in Supplementary Data 121 to yield the WT plasmid p-mtl16WT. To construct an active-site mutant, both P169 and P170 were mutated to Ala residues by site-directed mutagenesis using the primers listed in Supplementary Data mut. S. pombe mtl16Δ strain was transformed with the WT or mutant plasmid by the lithium acetate method122, cultivated in EMM medium devoid of thiamine until the cell density reached OD600 = 1.0, and harvested. Total RNA was prepared using the TriPure Isolation Reagent (Roche Life Science). One microgram of total RNA from WT, KO, and transformed strains were treated with RQ1 RNase-free DNase (Promega) and subjected to reverse transcription with random hexamer and oligo dT primers using the Transcriptor First Strand cDNA Synthesis Kit (Roche), followed by PCR amplification with intron-specific primers  was extracted from WT or the mtl16Δ strains transformed with pDblet, pU5WT or pU5AGA grown to mid-log phase in EMM media without uracil. cDNA was synthesized by Superscript III (Thermo Fisher Scientific) using random N6 primers, according to the manufacturer’s instructions. PCR was performed in a 10 μL mixture containing a 0.2-μL aliquot of the cDNA solution, 0.2 μM of each PCR primers, and 1 × KAPA SYBR FAST Master Mix optimized for LightCycler480 (Kapa biosystems). The thermal cycling conditions included 45 cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 1 s. Amplification of DNA was monitored by a LightCycler480 (Roche), according to the manufacturer’s instructions. To write a standard curve of Cp (crossing point) value against the amount of the template, depending on each set of primers, a dilution series of the S. pombe WT genomic DNA was amplified by the primers. To measure the IRS value of a single intron, primers spanning intron and exon were used to obtain the Cp value of the retained isoform, and primers constructed inside an adjacent single exon was used to obtain the Cp value of the total of both retained and spliced isoforms. Then, the abundance of cDNA derived from each isoform was calculated based on the standard curve. IRS is the binary logarithm of the value obtained by dividing the abundance of the retained isoform by the abundance of the spliced isoform.The 68. The annealed mixture was applied for methylation by incubating at 37 °C overnight in a mixture consisting of 200 mM NaCl, 50 mM HEPES-KOH (pH 7.5), 1 mM DTT, 2.5 mM SAM, 0.2 units/uL SUPERase-In RNase inhibitor (Thermo Fisher Scientific), 30 μM human recombinant METTL16 MTD, and 30 μM RNA. The reaction was stopped by mixing with an equal volume of phenol/chloroform/isoamyl alcohol . The RNA was collected by ethanol precipitation, then U6A4 RNA or U6U4 RNA was resolved by 10% denaturing PAGE and excised from the gel piece. The methylation efficiency was verified by LC/MS as described. The methylated or unmodified transcripts were dissolved at a final concentration of 3 μM in degassed buffer consisting of 20 mM sodium phosphate (pH 7.0) and 1 M NaCl, followed by incubation at 95 °C for 5 min and ramped at −0.1 °C/sec to 10 °C for annealing. A UV–Vis spectrophotometer  equipped with an 8 multiquartz microcell array  was used to monitor the hyperchromicity by monitoring absorbance at 260 nm. The temperature gradient was as follows: 25 °C for 30 sec, ramped at 5 °C/min to 40 °C and held for 5 min, and after that ramped at 0.5 °C/min to 105 °C. The measurement was performed in triplicate for each sample.In vitro transcripts U6A4, U6U4, and U6guide were synthesized from templates U6modelA4_T7R, U6modelU4_T7R, and U6modelGuide_T7R, respectively (Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Description of Additional Supplementary FilesReporting Summary


After injection, all groups experienced a reactive hyperaemia. With A200, it took about three min for StO2 to drop below baseline. For Aw/o and EPI200, perfusion reduction when compared to baseline was seen at 30 min with vasoconstriction >120 min. A100 caused vasodilation with hyperaemia >60 min. After three minutes, the perfusion pattern differed significantly (p < 0.001) between all groups except Aw/o and EPI200. The vasoactive effect of epinephrine-containing local anaesthetics can be visualised and dynamically quantified via StO2 using HSI. Aw/o + epinephrine 1:100,000 and 1:200,000 leads to perfusion reduction and tissue ischaemia after 30 min, which lasts over 120 min with no significant difference between both formulations. When using Aw/o containing epinephrine in terms of haemostasis for surgical procedures, a prolonged waiting time before incision of 30 or more min can be recommended.This study aimed to investigate the dynamic skin perfusion via hyperspectral imaging (HSI) after application of Articaine-4% ± epinephrine as well as epinephrine only. After the subcutaneous injection of (A100) Articaine-4% with epinephrine 1:100,000, (A200) Articaine-4% with epinephrine 1:200,000, (Aw/o) Articaine-4% without epinephrine, and (EPI200) epinephrine 1:200,000, into the flexor side of the forearm in a split-arm design, dynamic skin perfusion measurement was performed over 120 min by determining tissue oxygen saturation (StO Howevn (StO2) . This stA total of 50 healthy volunteer adults participated in this methodological basic research study. The study included males and females between 18 and 65 years, without known cardiovascular diseases, allergies, or intolerances to the ingredients of the LA solutions used, history of intolerance to LA, or other epinephrine-containing drugs. All patients who did not meet the inclusion criteria were not included.®D-S, Sanofi-aventis, Paris, France), (Aw/o) plain Articaine-4%  and (EPI200) epinephrine hydrochloride 1:200,000  1:1000, INFECTOPHARM®, Heppenheim, Germany). Each drug was applied subcutaneously at a puncture angle of 30° to the skin surface in a “split-arm design” with two substances per forearm and side (15 cm from each other to avoid overlapping). The measuring points were marked with a black rubber ring (6.7 mm inner diameter)  Articaine-4% +epinephrine 1:100,000 (40 mg/mL articaine + 0.012 mg/mL epinephrine hydrochloride), (A200) Articaine-4% +epinephrine 1:200,000  A,B.®, Diaspective Vision GmbH, Am Salhaff, Germany), specially developed for extracorporeal perfusion diagnostics, was used as described by our group beforehand [2 (0–100%)), near-infrared-perfusion index/deep perfusion (NIR as arbitrary units (0–100)) and distribution of haemoglobin (Tissue-Haemoglobin-Index (0–100)) and water (Tissue-Water-Index (0–100)) [2; 2) in 90° position (ROI-2) to the application direction vector through the centre of ROI-1 was performed ) . The meaerformed . Here, nn = 39 (40)) [n = 50 per solution was chosen for the present study. Raw datasets were saved in Excel® sheets  and subsequently transferred into SPSS-Statistics® . Data were expressed as mean (m), standard deviation (SD±), minimum, and maximum. Normal distribution was checked using a non-parametric Shapiro–Wilk-test(+), and the results were analysed for statistical significance by the use of analysis of variance (ANOVA(#)), unpaired non-parametric Mann–Whitney U-tests($), Wilcoxon Signed Ranks test(§), and Student’s t-test(*). To investigate whether the means of several dependent samples differ, we performed a one-factor analysis of variance with repeated measures. In the absence of sphericity (Mauchly W-test <0.05) and an epsilon correction factor Greenhouse–Geisser test of >0.75, the epsilon correction factor and significance according to Huynh–Feldt (HF) was chosen (§*). The partial Eta square (ηp2) shows how much percent of the variation of StO2 can be explained by the time of measurement. p-values of ≤0.05 were termed significant. Line charts were used for illustration purposes.In this feasibility study, the null hypothesis was that the vasoconstrictive effect of epinephrine (with and without LA) does not differ with regard to its concentration. In addition, it was hypothesised that the onset and duration of this effect on perfusion is not influenced by the epinephrine concentration. The parameters to be measured were the dynamic blood flow in superficial layers of the skin that can be objectively and reproducibly quantified and visualised by means of hyperspectral imaging. With an average assumed effect size (dz = 0.6/mean effect size according to Cohen (1988)), an α error = 0.05 and a power = 95%, the necessary sample size was (39 (40)) . TherefoROI−1Central2) did not differ significantly between substance groups/measurement points (mean: 40.7315% ± 5.62) (p = 0.827(#)).Before injection, baseline tissue oxygen saturation ).There was no significant difference between all groups at baseline (mean: 41.36 ± 5.4) ) and EPI200 (p ≤ 0.001).Thirty seconds p.i., blood flow was significantly increased with Aw/o compared to A100 and A200 ), and EPI200 (p = 0.011($)), whereby values revealed significantly increased with EPI200 when compared to A100 and A200 (each p < 0.001($)) (StO.001($)) .ROI−1Central vs. ROI−2Peripheral2 was higher in ROI-2 with Aw/o (p = 0.003($)) and EPI200 (p < 0.001($)) compared to ROI-1. No difference occurred for A100 and A200 ) and A200 (p ≤ 0.001(#)) and EPI200 (p ≤ 0.001($)). No significant difference was found between A100 and A200 and between EPI200 and A100 ), which revealed a significant lower StO2 (p < 0.001($)) when compared to EPI200 ). No difference occurred for A100 and A200.After 1 and 2 min, StOROI−1Central2 decreased significantly with EPI200 in comparison to Aw/o, A100, and A200 (each p ≤ 0.001($)). Simultaneously, StO2 significantly increased with Aw/o compared to A100 and A200 (each p ≤ 0.001($)); 2 dropped below baseline between 2 and 3 min p.i. ) between the agents in the same proportion as centrally (ROI-1). In contrast, StO2 after 4 min was not significantly different between Aw/o + 1:100,000 and A200 and EPI200, but it was significantly higher (p < 0.001) with Aw/o compared to the other groups, which persisted until five minutes after injection ) except A100. Centrally, StO2 was higher for the vasoconstrictor-containing LA formulations and lower for EPI200 (2 differed significantly with A100 between ROI-1 and 2 (p = 0.029($); p < 0.001($)) (StOr EPI200 . After 4.001($)) .ROI−1Central2 was significantly different when comparing all test substances, with the highest values for Aw/o, followed by A100 and A200 and EPI200 ; p = 0.041($)) between all groups  remained significantly higher in ROI-1 for Aw/o and A100 and A200 (p < 0.001($)) and significantly lower for epinephrine (p < 0.001($)), compared to ROI-2 ), and significantly higher when compared to EPI200 (p ≤ 0.001($)).StOand A200 . StO2 reROI−2Peripheral2 was similar to ROI-1 with no difference between the vasoconstrictor-containing LA-solutions A100 and A200 (p = 0.238($)). In all other groups, the blood flow was significantly (each: p < 0.001($)) different. In summary, between 15 and 30 min after injection, there was a decrease in the previously increasing blood flow with Aw/o and a further decrease with A100 and A200, as well as with EPI200 ), whereby the latter two peripherally dropped below baseline into the ischemic zone. Although not significant, there was an increase in StO2 in ROI-1 with EPI200 ). While a pronounced increase in StO2 in the sense of hyperaemia was still evident with Aw/o, StO2 was significantly lower with EPI200 (p ≤ 0.001($)).After 45 min, a clear drop of StOand A200 ; howeverROI−2Peripheral2 was significantly higher with Aw/o compared to EPI200 and A100 and A200 (each: p < 0.001($)). Values were also different between A100 and A200 (p = 0.046($)).StOROI−1Central vs. ROI−2Peripheralp < 0.001($); p = 0.003($); p < 0.001($)), whereas EPI200 revealed almost the same ).There were significant differences in StOROI−2Peripheral2 was significantly different (p < 0.01($)) between all substances except for EPI200 and A200 ) and A200 (p = 0.002($)) comparing ROI-1 and ROI-2 . There was also a significant difference in StO2 between Aw/o and all other groups (p ≤ 0.001($)).It revealed a significant difference in StOROI−2Peripheral2 dropped below baseline with Aw/o, which differed significantly from A100 and A200, and from EPI200 (p < 0.001($)) but not from baseline. In contrast, StO2 was significantly higher with EPI200 compared to A100 (p = 0.005($)) and A200 (p < 0.001($)).StOROI−1Central vs. ROI−2Peripheral2 was significantly different (p < 0.001($)) between ROI-1 and ROI-2 for all agents ; ηp2 ≥ 0.96). With A100 and A200, it revealed that StO2 was significantly different (p < 0.001(§)) from baseline at all measurement time points except after 30 min (p = 0.996(§) and p = 0.388(§)). With Aw/o, StO2 was found to be significantly (p < 0.001(§)) different from baseline at all measurement time points. With EPI200, StO2 was significantly different from baseline at all time points (p < 0.01(§)) except for 3 min p.i. (0.218(§)) ) .ROI−2Peripheralp < 0.001(§*); ηp2 ≥ 0.93). With A100 and A200, it revealed that StO2 was significantly different (p < 0.001(§)) from baseline at all measurement time points except after 15 min (p = 0.057(§) and p = 0.851(§)). With Aw/o, StO2 was found to be significantly (p < 0.001(§)) different from baseline at all measurement time points. EPI200 revealed that StO2 was significantly different from baseline at all time points (p < 0.01(§)) except for 3 min p.i. (0.218(§)) ) .2. As a major result, this study revealed that skin perfusion drops below baseline after 30 min following injection of A100 and A200. Furthermore, there was no difference between A100 and A200. In addition, the duration of vasoconstriction was sustained over a period of >120 min. Finally, a spatial pattern of extending hypoperfusion with epinephrine-containing LA with a primarily marginal circular onset of hypoperfusion with simultaneous hyperaemia around the injection site (centre) was found (This is the first study to investigate the dynamic vasoactive/perfusion effect after the subcutaneous injection of Articain-4% with (A100 and A200) and without (Aw/o) epinephrine in different concentrations as well as pure epinephrine (EPI200) using hyperspectral imaging of the highly sensitive blood flow surrogate parameter StOas found .Clinically, the maximum reduction of peripheral blood flow must be set against the risk of local and systemic drug side effects. Nonetheless, appropriate concentrations of epinephrine should be combined with the LA to guarantee sufficient depth and duration of anaesthesia ,11,21. R2) is detected at a depth of up to 1 mm, as it reflects the percentage of haemoglobin oxygen saturation in the capillary area, records arterial and venous blood, and displays changes in oxygen supply and consumption directly on site in the tissue. Uniform standard or limit values do not yet exist, although scientific studies are still underway. The tissue-oxygen-saturation values of healthy volunteers are typically 50%–70% [2 was much higher centrally (ROI-1) than in the peripheral region (ROI-2). This pattern was not seen with plain Aw/o and EPI200. The authors are not aware of any literature that has already described this effect. A plausible cause for this could be a more dynamic spread of epinephrine within the tissue, which is responsible for the marginally observed ischaemia and explains the long-lasting hyperaemic effect around the application site (ROI-1). From our point of view, the frequently described diffusion potential of the local anaesthetic solution towards the nerve (area) must be critically evaluated, which means that, for example, in the case of IANB, the technically correct implementation with application as close to the nerve as possible is of primary importance. Limitations of the study are the lack of association of the measured values to the actual reduction of blood flow after a skin incision as well as the transferability to other body regions such as the head and neck region with a stronger blood supply or the oral mucosa.In the present study, epinephrine was used in combination with Articaine-4% at a concentration of 1:100,000 (A100) and +1:200,000 (A200), as well as plain epinephrine 1:200,000 (EPI200). The lack of difference in perfusion reduction between A100 and A200 is consistent with other studies using lidocaine instead of articaine ,27. Gess 50%–70% . In the To take advantage of local hypoperfusion for vasoconstriction in the surgical field, it seems advisable to wait at least 30 min after subcutaneous application of Articaine-4% with epinephrine 1:100,000 or 1:200,000 until skin incision. Therefore, the use of epinephrine at or even below 1:100,000 must be discussed in the absence of benefits for anaesthesia and blood flow reduction.