D3 exhibits hormonal traits and is responsible for calcium (Ca) absorption. 1-α-Hydroxycholecalciferol (1α) is a synthetic form of D3 that has equal efficacy and is cheaper to synthesize than 1,25-dihydroxycholecalciferol (active form of D3), on broilers. However, 1α bypasses a critical regulatory point, the kidney, and may consequently lead to toxicity levels of Ca via Ca absorption. This study examined 1α supplementation in broiler diets with different Ca inclusion levels to determine if 1α at higher Ca levels caused Ca toxicity at starter and grower phases with Ross 708 male broiler chicks. In Experiment 1 (1–15 days of age), chicks were assigned to one of 10 treatment starter diets with five levels of Ca inclusion  with or without 1α supplementation (5 μg 1α/kg in feed) and eight replicate cages per treatment. In Experiment 2, chicks were fed common starter diet until 16 days of age, and then they were assigned to one of eight treatment diets with four levels of Ca inclusion  with or without 1α supplementation (5 μg 1α/kg in feed). At the end of both experiments, blood was collected from broilers to determine blood chemistry, including concentrations of vitamin D metabolites. Intestinal tissues were also collected to examine gene expression. In Experiment 1, broilers not fed 1α exhibited a quadratic effect in ionized blood Ca (iCa) as dietary Ca inclusion levels increased; 1α-fed broilers displayed an increase in iCa as Ca inclusion levels increased (p = 0.0002). For Experiment 2, 1α-fed broilers displayed a decrease in 25-hydroxycholecalciferol plasma concentration as dietary Ca inclusion levels increased (p = 0.035); also, increasing Ca inclusion in diets increased expression of duodenal sodium phosphate cotransporter type II b . Our findings imply that inclusion of 1α was beneficial because 1α enhanced Ca absorption during the starter phase; however, to avoid potential Ca toxicity or antagonism while using 1α during the grower phase, there should be consideration with reducing dietary Ca levels.Increasing biopotency of cholecalciferol (D However(OH)2-D3 , 13, andcalcemia . Althougcalcemia  fed fastP), because, together, they comprise a major part of bone structure  were approved by the Institutional Animal Care and Use Committee at North Carolina State University.3, Premex, Antioquia, Colombia) at 5 μg/kg of feed  by LC–MS/MS.Blood was collected into BD Vacutainer lithium heparin tubes  from two birds per cage (Experiment 1) or pen (Experiment 2). Blood chemistry parameters and iCa were determined using an i-STAT™ blood analyzer  using CG8+ cartridges , and the remaining blood was spun down, and plasma was collected and stored at −80°C to determine vitamin D metabolite levels. All plasma samples were sent to Heartland Assays  for measuring DTotal mRNA was extracted from duodenal tissue using Qiagen's RNeasy Mini Kit . Extracted RNA was diluted and normalized to ~200 ng/μl and reverse transcribed to complementary DNA (cDNA) using Applied Biosystems' High-Capacity cDNA Reverse Transcription Kit  and protocol to make a 20-μl working solution. Cycling procedure for reverse transcription started with 25°C for 10 min, 37°C for 120 min, and 85°C for 5 min and then held at 5°C indefinitely until storage or use.Genes expressed for qPCR were vitamin D receptor (VDR) mucin 2 (MUC2), calbindin D28k (CALB), sodium phosphate cotransporter type IIb (NPTIIb), and glyceraldehyde (GAPDH) as a housekeeping gene . qPCR wa2) and villus height/crypt depth were made per experimental unit (bird).Light microscopy (40 × magnification) was used for morphometric analysis of histological serial sections of jejuna prepared using standard hematoxylin and eosin staining to examine if dietary treatments affected gut morphology. Villus height, crypt depth, and villus width were measured using image analyzer AmScope version 3.7 . Ten measurements for villus surface area . Broilers from D3 treatments exhibited a quadratic effect with increasing iCa from 1.29 mmol/L to a peak of 1.47 mmol/L when dietary Ca increased from 0.80 to 1.10% Ca inclusion. Once dietary Ca went over 1.10%, iCa decreased (p = 0.006).In terms of blood chemistry, only iCa exhibited any differences between 0.80% and 1.10% Ca inclusion . For D3 2-D3 or 25-OH-D3. A statistical trend was observed with D3 + 1α broilers having a higher D3 concentration compared to D3 broilers .At 0.95% Ca inclusion, 1α supplementation did not affect plasma concentrations of 24,25-(OH)p = 0.003; 3 + 1α broilers had higher blood bicarbonate concentration (p = 0.041), and a trend was observed with bicarbonate concentration with increasing Ca inclusion levels abolites .p = 0.52; 3 + 1α treatments had decreased relative expression of MUC2 compared to control treatment  and Ca inclusion (p = 0.055) with a slight increase in expression with D3 broilers, but a larger sample size may be necessary to observe an effect . Similarly, Ca inclusion levels did not affect either parameter .Feeding 1α to broilers had no impact on surface area or height/crypt depth ratio of villi 2-D3 and excreted or used up before blood was collected.However, 1α's effects are more pronounced when broilers are older and fed a grower diet. An inverse relationship of vitamin Dnclusion . This rein urine . It is i 15 days . Conside3 in chickens is converted to 24,25-(OH)2-D3 by 24-hydroxylase in kidneys 2-D3 goes through a series of conversions and ends up becoming calcitroic acid, a water-soluble molecule that is readily excreted 2-D3. 1α would likely be converted to 1,25-(OH)2-D3 and then undergo multiple conversions to become calcitroic acid. We constructed a hypothetical model to compare how dietary D3 and 1α would be converted to calcitroic acid to be excreted 2-D3, which will reduce the amount of vitamin D3 being converted into 25-OH-D3 due to a negative feedback loop : this observation could signify that broilers were trying to bind dietary Ca for transport and absorption. Our results were similar to a different study by Li et al. ds to it . For VDRds to it .3 broilers, which was likely caused by P imbalance. Even though our study did not include citric acid, there was a study that noted how 1α, phytase, and citric acid increased phytate P utilization in broiler chicks (P can cause Ca to bind to P and form insoluble tricalcium phosphate (P, which triggers the increased expression of NPTIIb in the duodenum. A proposed model of the inverse relationship between CALB and NPTIIb expression relative to Ca inclusion levels is denoted for broilers not fed 1α (NPTIIb is an important cotransporter protein that requires sodium to move P in its phosphate form into cells from the intestinal lumen . Increasr chicks . Excess hosphate . Our reshosphate , in whict fed 1α . FurtherOur work highlights that 1α supplementation with certain levels of Ca inclusion can impact blood Ca concentration and affect vitamin D metabolite concentration. Our findings exhibit that 1α can improve Ca utilization in young broilers with an implication for reducing dietary Ca in their diets. We suggest 1α should be supplemented in broiler diets for the starter phase and either removed for the grower phase or provided with reduced dietary Ca levels. 1α's potential to reduce dietary Ca without any negative impacts on growth performance signifies the importance of synthetic nutrients for improving animal production while reducing potential environmental impacts from excreted excess nutrients. Future research should explore how much 1α supplementation will cause vitamin D toxicity in growing animals to characterize an animal's regulatory limits of removing vitamin D with consideration of 1α's bypassing of negative feedback regulation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Institutional Animal Care and Use Committee, North Carolina State University.MW, JF, and KL contributed to the conception and design of the study. MW and KL contributed to the acquisition, analysis, interpretation of data, wrote the first draft of the manuscript, and were responsible for the final editing of the manuscript. MW, TV, OT, and KL contributed to the manuscript. All authors read and approved the final manuscript.The authors declare that this study received funding from Premex. The funder had the following involvement with the study: conception and design of the study.


Xenopus early development with chromatin assays. Here we investigate the extent to which H3.3 mutated at residues that differ from H3.2 rescue developmental defects caused by H3.3 depletion. Regardless of the deposition pathway, only variants at residue 31—a serine that can become phosphorylated—failed to rescue endogenous H3.3 depletion. Although an alanine substitution fails to rescue H3.3 depletion, a phospho-mimic aspartate residue at position 31 rescues H3.3 function. To explore mechanisms involving H3.3 S31 phosphorylation, we identified factors attracted or repulsed by the presence of aspartate at position 31, along with modifications on neighboring residues. We propose that serine 31-phosphorylated H3.3 acts as a signaling module that stimulates the acetylation of K27, providing a chromatin state permissive to the embryonic development program.Vertebrates exhibit specific requirements for replicative H3 and non-replicative H3.3 variants during development. To disentangle whether this involves distinct modes of deposition or unique functions once incorporated into chromatin, we combined studies in  Xenopus gastrulation, independently of the pathways; and characterize H3.3 S31 phosphorylation importance for the underlying mechanisms.Replicative and non-replicative H3 variants have different cell cycle regulation and incorporation modes. Here, the authors identify H3.3 S31 deposition as key at  The basic unit of chromatin, the nucleosome core particle, is composed of a histone tetramer (H3-H4)2 flanked by two histone dimers H2A–H2B, around which 147 bp of DNA is wrapped3. Functional modulation of the nucleosome occurs though the choice of histone variants and reversible posttranslational modifications (PTMs) such as acetylation, methylation, and phosphorylation4. Three out of the four core histone families possess histone variants7. Within the H3 family of histones, the centromere-specific CENP-A is distinct, whereas the other well-characterized H3 variants are closely related and are thought to function similarly8. Those H3 variants exhibit similar structural features at the core particle level9; however, they display important differences in their cell cycle regulation and modes of incorporation into chromatin10.The organization of DNA into chromatin provides not only a means for compaction but also a versatile landscape contributing to cell fate and plasticity11. In humans, the two replicative variants H3.1 and H3.2 are incorporated into chromatin in a DSC manner via the histone chaperone complex CAF-118. The non-replicative form H3.3, which differs from H3.1 and H3.2 by five and four residues, respectively, is incorporated in a DSI manner21. DSI incorporation depends on the HIRA histone chaperone complex in euchromatin regions22, whereas the presence of H3.3 at telomeres and pericentric heterochromatin relies on the DAXX/ATRX histone chaperone complex24. Thus, the dynamics of the different histone variants in regards to their deposition is linked to dedicated histone chaperones26.To date, two distinct modes of incorporation have been described: DNA-synthesis independent (DSI) and DNA-synthesis coupled (DSC); the latter being used during both DNA replication and repair27,” we considered H3 variants in light of their conservation in different organisms. In Saccharomyces cerevisiae, there is only one non-centromeric histone H3 (which is most related to human H3.3) that provides both essential replicative and non-replicative variant functions30. Paradoxically, however, in humanized S. cerevisiae strains where all histones are exchanged for human orthologs, replacement with hH3.1 more readily produced colonies than with the hH3.3 variant31. In metazoans such as Drosophila melanogaster, the replicative variant can compensate for the loss of H3.3 during development in somatic tissues, although the adults are sterile36. As sterility could simply reflect a shortage of maternal H3.3 to replace protamine from sperm chromatin after fertilization, the most parsimonious hypothesis suggests that the nature of the variant itself might not be critical. Similarly, H3.3 is not essential in Caenorhabditis elegans, where its removal is not lethal but reduces fertility and viability in response to stress37. However, in Arabidopsis thaliana, replicative and non-replicative H3 variants are essential. The absence of H3.3 leads to embryonic lethality and also partial sterility due to defective male gametogenesis38.Inspired by the quote “nothing in biology makes sense except in the light of evolution41. In human, dominant effects of substitutions in H3.3, such as H3.3 K27M and H3.3 G34R/V, along with mutations affecting their chaperones such as DAXX/ATRX, have been implicated in different types of cancer49. Thus, the developmental defects observed mouse models and mutations associated with particular cancers underline the importance of individual histone H3 variant and their chaperones in vertebrates.In mouse, the deletion of one of the two copies of the H3.3 gene results in developmental defects at E12.5 and sterilityXenopus laevis model, as it represents an ideal system to tackle such issue. Indeed, extensively characterized both in developmental biology and chromatin studies52, its external development permits direct access to embryos for observation and manipulation53. With retention of H3 variants in sperm54 and only one replicative histone (H3.2), it provides an ideal situation while retaining amino acid sequence conservation with human variants for both H3.2 and H3.3. Following fertilization, X. laevis development starts with 12 rapid embryonic cell divisions, which include only S and M phases57. At the midblastula transition (MBT), zygotic activation occurs concomitantly with a progressive lengthening of the cell cycle, to reach a typical cell cycle with two gap phases at gastrulation. In addition, cells begin to differentiate with the acquisition of migration properties. Importantly, previous work in our laboratory revealed a specific requirement for H3.3 during X. laevis early development at the time of gastrulation58. That work demonstrated that depletion of endogenous H3.3 leads to severe gastrulation defects that cannot be rescued by providing the replicative counterpart H3.2. Interestingly, there are only four residues that differ between H3.2 and H3.3. A first region of divergence within the AIG motif—in the globular domain of H3.3—is involved in histone variant recognition by dedicated histone chaperones62. The other distinct residue, a serine only present in H3.3, is located on the histone N-terminal tail at position 31 and can be phosphorylated65.Given the high degree of sequence identity between H3 variants, whether the need for a particular histone variant could reflect either (i) a unique mode of incorporation and provision or (ii) a distinct identity once incorporated into chromatin to drive their functions remains puzzling. Although the first hypothesis has largely been favored based on previous work, including our own, the issue has not been formally addressed. To disentangle these two possibilities, we decided to use the Xenopus, Ser31—which is conserved in multicellular organisms including humans—is phosphorylated with a peak in mitosis by a network of mitotic kinases, including CHK1 and Aurora B. Remarkably, a phospho-mimic form of H3.3, S31D  still fully rescues gastrulation. Interestingly, analysis of protein interactions and repulsions on the phospho-mimic peptide, as shown by mass spectrometry, reveals attraction of transcription cofactors as those involved in the β-catenin pathway in interphase, but repulsion of factors involved in chromosome condensation and splicing in mitosis. We also find that H3.3 S31D exhibits an increase in H3.3K27ac and a loss of H3.3K27me3 in-cis. We discuss how this evolutionarily conserved residue conveys, in both interphase and mitosis, unique properties for the H3.3 variant in vertebrates during cell cycle and cell-fate commitment.Here we systematically mutate the H3.3 histone variant at each of its distinct residues, to assess their ability to rescue the gastrulation defects and examine their mode of chromatin incorporation. We find that mutations affecting the incorporation pathway are neutral in setting specific H3.3 functions at the time of gastrulation. In contrast, serine 31 is critical to rescue defects following endogenous H3.3 depletion. In Homo sapiens have two replicative H3 variants H3.1 and H3.2, X. laevis only possesses one replicative variant H3.2. Both H3.2 and H3.3 are conserved with their human orthologs . We found that this morpholino against H3.3 leads to defects during late gastrulation  and is mediated by the CAF-1 complex18. Importantly, structural studies enabled the identification of how histone chaperones discriminate the distinct histone variants through a motif located in the globular domain of histones61. Both DAXX and HIRA complexes bind to the AIG motif of H3.3, with a particular affinity for the glycine at position 90. Therefore, we tested the ability of mutated H3.3 in the histone chaperone recognition motif to rescue the loss of H3.3  to fulfill H3.3 dedicated functions in this time window during development. Interestingly, this particular residue has been found phosphorylated in human cells during mitosis63. Furthermore, a threonine substitutes for this serine in Arabidopsis, a residue that might possibly also undergo phosphorylation, something that has however not yet been documented. A key question is thus whether the actual need for H3.3 is linked to the capacity of H3.3 S31 to become phosphorylated. To this end, we first examined H3.3 S31 phosphorylation in the Xenopus system using a Xenopus A6 cell line derived from the kidney  with gap phases and acquisition of checkpoints56. Histones play a key role in regulating the start of the MBT, possibly through titration mechanisms87. It would therefore be of interest to explore the role of specific variants in this context, specially in the case of acetylation and histone variant crosstalk88. In addition, previous studies have suggested that the presence of H3.3 could affect chromosome segregation. Indeed, in double H3.3 knockout mESCs, an increase in anaphase bridges and lagging chromosomes has been observed89. Furthermore, H3.3 S31ph can coat lagging chromosomes, which is associated with p53 activation and the prevention of aneuploidy65. Considering H3.3 S31ph accumulation at centromeres in mitosis63, it is tempting to speculate that H3.3 S31ph has a crosstalk with CENP-A incorporation at late mitosis and the beginning of G1. Interestingly, CENP-A incorporation at the centromere has been shown to be dependent on both the presence of H3.3 as a placeholder90 and on transcription92. To reconcile both aspects, we envision that H3.3 S31ph could act as a phospho-switch in mitosis to regulate transcription at critical chromosomal landmarks and in interphase to control and maintain a transcription program. Alternatively, one could also consider that the alanine present on H3.2 could prevent particular interactions specific to H3.3 S31 or H3.3 S31ph, and permitted by an aspartic acid. Notably, in Arabidopsis, the replicative form cannot be rescued by H3.3, or more specifically the A31T mutation93. Indeed, ATXR5, a plant-specific H3K27 methyltransferase, specifically recognizes H3.1 and not H3.394. Therefore, the alanine at position 31 on replicative histone H3.1 prevents the heterochromatinization of H3.3-rich regions during replication. Thus, in plants, the replicative histone H3 variant avoids the presence of a negative charge at position 31. It also suggests that the specific residue at position 31 of the replicative and non-replicative H3 variants may exhibit different functions by promoting or excluding specific binding partners. Therefore, the mass spectrometry data we have generated in our Xenopus studies provides an important source of information to explore further. The fact that H3.3 S31D can attract factors in interphase involved in the β-catenin pathway or repulse proteins in metaphase involved in chromosome condensation and mRNA splicing are first insights, but we believe that there will be more to discover. For instance, H3.3 S31D may impeach on specific developmental programs, as the β-catenin pathway is important in the epithelial–mesenchymal transition that occurs following gastrulation and is also implicated in metastatic signaling in cancer95. Also, the impact of H3.3 S31D on the mitotic condensing machinery will be another challenge to consider for the future, opening new avenues on the control of mitosis and chromosome organization.The exact role of H3.3 S31 remains enigmatic. Although we and others76, further studies will be essential to better delineate the role of H3.3 S31ph in these contexts and the different proteins involved. The presence of H3.3 S31D also alters neighboring residues in-cis. Conversely, other modifications on the H3 tail could themselves impact H3.3 S31 modification and in this respect, the effects of mutations affecting neighboring residues will be interesting to explore. Notably, as H3.3 S31 is close to residues often mutated in aggressive cancers such as H3 K27M and G34R in pediatric glioblastoma96, it will be interesting to evaluate the impact of these onco-histone mutations on H3.3-specific phosphorylation as well. Altogether, we show that H3.3 S31 is the key residue that confers specific functions to H3.3 within chromatin, compared with its H3.2 counterpart. It also establishes the importance of a distinct histone variant residue for the proper development of a vertebrate during gastrulation. Future work should explore whether a similar requirement also occurs in mammals to provide a comprehensive view of the importance of the non-replicative variant H3.3 and its role during vertebrate development and in disease states.As H3.3 S31-negative charge has been linked to intron retention and pre-mRNA processing by preventing binding of the tumor suppressor ZMYND11X. laevis adults (2 years old) from the Centre de Ressource Biologie Xenope. We prepared embryos at 18 °C as in ref. 88 and staged them according to ref. 53. We acquired embryos during gastrulation with a MZFLIII magnifier (Leica) and the SPOT software. Animal care and use for this study were performed in accordance with the recommendations of the European Community (2010/63/UE) for the care and use of laboratory animals. Experimental procedures were specifically approved by the ethics committee of the Institut Curie CEEA-IC #118  in compliance with the international guidelines. D.S., E.B., and G.A. possess the authorization for vertebrates’ experimental use.We used 97. This vector stabilizes RNA and improves their translation efficiency, while injected into Xenopus eggs. In addition, an HA-tag has been inserted in the C-terminal of H3. We obtained mRNAs by in vitro transcription of PCR-amplified fragments of the different pβRN3P vectors (forward: 5′-gtaaaacgacggccagt-3′ and reverse: 5′-ggaaacagctatgaccatga-3′). We transcribed mRNAs starting with 5 ng of PCR-amplified fragment, 10 μL Buffer 5×, 5 μL of dithiothreitol (DTT) 100 mM, 0.25 μL of bovine serum albumin (BSA) 10 mg/mL (NEB), 5 μL of ATP, CTP, UTP 10 mM, 1.65 μL of GTP 10 mM (Sigma-Aldrich), 3.35 μL of Me7GTP 10 mM (NEB), 2 μL of RNasin Plus RNase Inhibitor (Promega), and 50 μL H2O qsp. After 10 min on ice, we added 2 μL of T3 RNA Polymerase (Promega) to each sample,  incubated for 30 min at 37 °C, then added 0.5 μL of  fresh T3 RNA Polymerase and incubated for another 10 min at 37 °C. After DNA digestion with 2 μL RQ1 DNase (Promega) for  20 min at at 37 °C, we extracted mRNAs with phenol–chloroform and purified them through Sephadex G-50 Quick Spin Column for radiolabeled RNA purification (Sigma-Aldrich), previously equilibrated six times with 1 mL of TE 10 mM.We cloned all H3 cDNAs in the pβRN3P vectorX. laevis H3.3 mRNAs from gene b as described in ref. 58. We selected this gene given its peak of expression specifically at gastrulation98. The morpholino only targets endogenous H3.3, as verified in details98. H3.3 morpholino sequence 5′-GGTCTGCTTTGTACGGGCCATTTCC-3′ targets 5′-GGAAATGGCCCGTACAAAGCAGACC-3′. Xenopus H3.3 sequence for the gene b is 5′-ATGGCCCGTACAAAGCAGACTGCCCGTAAA-3′, whereas Xenopus H3 sequence is 5′-ATGGCTCGTACTAAGCAGACCGCTCGCAAG-3′. eH3 sequences used in the study start as 5′-ATGGCCCGAACCAAGCAGACTGCTCGTAAG-3′.We microinjected two-cell embryos using a Brinkmann micromanipulator and a Drumond microinjector on two-cell stage eggs with an injection volume set to 9.2 nL, to deliver the appropriate quantity of morpholino and mRNAs (1× = 4.6 ng). Morpholino and mRNA concentrations have been optimized for efficient depletion and rescue at gastrulation. More than 30 embryos are injected per condition, with at least 3 biological replicates. We knocked down endogenous H3.3 with morpholino designed to bind to the initiation region of ATG corresponds to the first codon and mismatch DNA bases are in bold and underlined.X. laevis embryos using the CelLytic Express reagent (Sigma-Aldrich) and centrifuging at 20,000 × g for 20 min at 4 °C the extracts after 30 min incubation on ice. We analyzed protein samples by electrophoresis on 4–12% NuPAGE SDS-polyacrylamide electrophoresis gels with MES SDS Running buffer (Life Technologies) and corresponding LDS buffer NuPage (Invitrogen) with DTT. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch Laboratories) and SuperSignal West Dura Extended Duration Substrate (ThermoFisher). The signal was acquired using ChemiDoc Imager (Biorad).We prepared total protein extracts from 58. We lysed the embryos in 200 µL of Lysis Buffer 1  and centrifuged them at 1000 × g for 4 min at 4 °C. We collected supernatant (soluble fraction) and cleared it with ultracentrifugation at 210,000 × g for 30 min at 4 °C. We washed the chromatin fraction three times with Lysis buffer and once with Buffer A . We performed MNase (Sigma) digestion during 12 min with 2.5 U/mL final concentration, after addition of CaCl2 . We stopped the reaction by EDTA  and recovered solubilized chromatin fraction after a ultracentrifugation at 210,000 × g for 30 min at 4 °C.We collected 30 embryos at stage 12 and performed fractionation as in ref. g for 30 min at 4 °C. We used 100 µg of protein for each condition for immunoprecipitation with anti-HA magnetic beads (Thermo Scientific) overnight at 4 °C in 400 µL of IP buffer . After three washes, we eluted proteins with LDS buffer NuPage (Invitrogen) with the reducing agent and analyzed samples by electrophoresis as described above.We collected embryos at stage 12 and prepared either total embryo extracts or soluble fractions as above. We ultracentrifuged total extracts at 210,000 × X. laevis sperm nuclei and low-speed extracts arrested by cytostatic factor (CSF) of X. laevis eggs as previously described99. Briefly, we collected eggs freshly and centrifuged them at low speed  to conserve the mitotic phase, below lipids. We induced interphase by the addition of CaCl2 at the final concentration 0.06 mM to CSF-arrested egg extracts. We added sperm chromatin at a concentration 1000–4000 nuclei/μl. After DNA replication, a 2/3 volume of the CSF-arrested extract was added to induce mitosis. For experiments in Fig. 2 at a final concentration of 0.06 mM induced transition to the second interphase. For the experiments of chromatin assembly in the presence of kinase or phosphatase inhibitors, we supplemented CSF-arrested extracts with the indicated concentration of Hesperadin hydrochloride , ZM 447439 , SB 218078 , PF 477736 , and Calyculin A  prior to the addition of 5000 sperm nuclei/μl. Incubation lasted for 50 min at room temperature. For treatment with okadaic acid, similarly using sperm nuclei, we first allowed chromatin assembly in CSF-arrested extracts treated with 20 µg/ml nocodazole (Sigma) for 30 min at room temperature. Next, we treated the reaction with either dimethyl sulfoxide or 1 µM okadaic acid  for another 20 min. We isolated the assembled chromatin and analyzed by western blotting as described above.We prepared 2, 5 mM EGTA) containing 5% glycerol, 0.5% Triton X-100, and protease and phosphatase inhibitors. We eluted proteins with LDS buffer NuPage (Invitrogen) with the reducing agent and analyzed samples by electrophoresis as described above.Briefly, we produced recombinant H3 mutant proteins from mRNAs using rabbit reticulocyte lysate (Promega L4600). After 3 h of incubation at 4 °C in interphase extracts followed by another 3 h incubation with anti-HA beads, we pulled down and washed the proteins in 0.8× CSF-XB buffer . We then supplied 15 µL of the rabbit reticulocyte lysate used to produce recombinant H3 mutant proteins from mRNAs. After 40 min of incubation at room temperature, we purified chromatin and we analyzed protein samples by electrophoresis as described above.g for 5 min at 4 °C. We resuspended the pellets in the same buffer and repeated the centrifugation step. For purification of interphase chromatin, we diluted 100 μl aliquots of extract with 0.8× CSF-XB buffer containing 20 mM β-glycerophosphate and 5% glycerol, which we incubated for 1 min at room temperature, followed by centrifugation through the cushion at 10,000 × g for 5 min at 4 °C. We resuspended purified chromatin directly in LDS buffer NuPage (Invitrogen) with NuPage reducing agent (Invitrogen). We analyzed samples by electrophoresis as described above.We diluted fivefold 100 μl aliquots of each reaction with 0.8× CSF-XB buffer containing 20 mM β-glycerophosphate, 5% glycerol, and 0.5% Triton X-100, which we incubated for 1 min at room temperature. We then layered the samples onto a 35% glycerol-containing CSF-XB cushion and centrifuged them at 10,000 × 100, we displayed the alignments using WebLogo3101 with probability units.After performing multiple sequence alignment using MUSCLESee Supplementary Data Xenopus H3.3 WT, H3.3 S31A, and H3.3 S31D cDNAs into pcDNA5/FRT/TO (Invitrogen) using BamHI/NotI restriction enzymes. We co-transfected these plasmids into Flp-In T-Rex 293 cells (Invitrogen), together with Flp-recombinase expression vector pOG44 (Invitrogen) using JetPRIME (Polyplus). We selected stably transfected cells with 150 mg/mL Hygromycin B (Gibco). We induced histone expression by adding 1 µg/ml doxycycline at least 120 h before analysis. For total-extract western blotting analysis, an equal number of cells were resuspended directly in LDS buffer NuPage (Invitrogen) with NuPage reducing agent (Invitrogen) and Universal Nuclease (Pierce).We have inserted fragments corresponding to HA-tagged 2, 10 μM CaCl2, 5 mM EGTA-KOH pH 8.0, 1 mM DTT, and protease inhibitors) and incubated these suspensions for 30 min at 4 °C. We then supplemented these samples with CaCl2 up to 1 mM and treated with 2.5 U/ml MNase (Sigma) for 12 min at 37 °C. The reaction was stopped by addition of EDTA-NaOH, pH 8.0 up to 4 mM. We ultracentrifuged the extracts at 100,000 × g for 30 min and diluted supernatant with an equal volume of dilution buffer . We used 500 µg total protein for IP with anti-HA magnetic beads (Thermo Scientific) for an overnight incubation at 4 °C in 500 µL of IP buffer . After three washes with IP buffer, we eluted proteins with LDS buffer NuPage (Invitrogen) and reducing agent (Invitrogen) to process samples for electrophoresis analysis as described above.We resuspended dry cell pellets in an equal volume of the lysis buffer  and further incubated at 37 °C for 30 min. Cells were finally filtered with Cell Strainer Snap Caps (Corning) and processed for analysis on an Accuri C6 flow cytometer (BD). Data were analyzed using FlowJo.Flp-In T-Rex 293 cells were washed twice in phosphate-buffered saline (PBS) and trypsinized. They were recovered in PBS and centrifuged at 300 × 4HCO3, to eliminate the remaining detergents. Beads were resuspended and digested by adding 0.2 μg of trypsine/LysC (Promega) in 100 µL of 25 mM NH4HCO3 for 1 h at 37 °C. Peptides were desalted and concentrated using homemade C18 StageTips. After elution, peptides were analyzed using an RSLCnano system  coupled online to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) as in ref. 102. For identification, the data were searched against the X. laevis Database (June 2019) from Xenbase.org and a database containing the common contaminants using SequestHF through proteome discoverer (version 2.2). Enzyme specificity was set to trypsin and a maximum of two-missed cleavage site have been allowed. Oxidized methionine, N-terminal acetylation, and carbamidomethyl cysteine were set as variable modifications. Maximum allowed mass deviation was set to 10 p.p.m. for monoisotopic precursor ions and 0.6 Da for tandem mass spetrometry peaks. The resulting files were further processed using myProMS v3.6103 (Supplementary Data n = 3), were analyzed further.Streptavidin magnetic beads  were washed with PBS and incubated with 60 pmol of the different peptides (GeneCust) corresponding to H3 N-terminal tails  in 300 µL of PBS. After 2 h of incubation and a 30 min block with BSA at room temperature, beads coupled to peptides were incubated 3 h with either mitotic or interphase eggs extracts diluted 5× in 400 µL total CSF-XB buffer, supplemented with 10% glycerol, 0.1% Triton, 0.1% Tween-20, and 1 mM DTT. Beads coupled to peptides were finally washed three times in PBS before digestion. Proteins on magnetic beads were washed two additional times with 100 μL of 25 mM NHWe fixed A6 cells on coverslips for 20 min in 4% paraformaldehyde, Flp-In T-Rex 293 and HeLa B cells in 2% paraformaldehyde before permeabilization with 0.2% Triton X-100. We blocked them for 45 min with 5% BSA. We then incubated coverslips with primary and secondary antibodies, and stained them with 4′,6-diamidino-2-phenylindole. We mounted the coverslips in Vectashield medium. We used a Confocal Zeiss LSM780 and we acquired images using 63×/1.4 numerical aperture under Zen blue software (Zeiss Germany) and analyzed the data using ImageJ.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Data 1Supplementary Data 2Reporting Summary


The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward 1N-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.AlkB is a DNA/RNA repair enzyme that removes base alkylations such as  It isbstrates .E. coli AlkB enzyme to remove modifications from tRNA to facilitate efficient and quantitative tRNA sequencing  is heavily chemically modified which hinders its direct sequencing as modifications frequently block the reverse transcription, reducing the formation of long cDNA products in sequencing. Recently, two studies utilized the quencing ,6. As a e by m1A .E. coli AlkB are m1A and m3C; however, targeted AlkB mutants can have new substrate preferences (1G). AlkB mutant D135S/L118V is able to demethylate N2,N2-dimethylguanosine (m22G) which is a poor substrate for the wild type enzyme  demethylase activity on DNA. We identified positive hits which serve as a starting point for further engineering of AlkB.Here, we introduce a Broccoli RNA-based fluorescence assay for high throughput screening of AlkB demethylation activity on RNA and DNA substrates. Using our assay, we systematically evaluated two functionally important residues in the E. coli crude lysate was purchased from Sigma Aldrich (P8465–5ML); Pierce™ Protease Inhibitor Mini Tablets EDTA free used for protein purification was purchased from Thermo Fisher Scientific (88666). SUPERase• In™ RNase Inhibitor (AM2696), lysozyme Solution (90082), DNase I (EN0521) and M-MLV RT (28025013) were purchased from Thermo Fisher Scientific. T7 RNA polymerase (M0251) and Taq DNA (M0273) polymerase were purchased from New England Biolabs Inc. ZR-96 Oligo Clean & Concentrator was purchased from Zymo Research (D4062). The 4x Laemmli sample buffer was purchased from Bio-Rad (161-0747). Nuclease P1 from Penicillium citrinum and shrimp alkaline phosphatase used for digesting yeast tRNA for LC–MS/MS analysis were purchased from Sigma Aldrich (N8630) and New England Biolabs Inc. (M0371), respectively.The 96-deep square well plate was purchased from Thomas scientific (1159Q92). Protease Inhibitor Cocktail powder for preparing in vitro transcription and fluorescently labeled oligonucleotides for characterizing reverse transcription were purchased from Integrated DNA technologies (IDT) with standard desalt purification. The RNA oligonucleotides containing m1G and the DNA oligonucleotide containing O6mG were synthesized in-house on an Expedite DNA synthesizer using commercial m1G-phosphoramidite and O6-methyl-G phosphoramidite from Glen Research on 1 μmol scale with the same coupling times as the commercial unmodified phosphoramidites. Based on DMTr cation calculations, both modified phosphoramidites were coupled as efficiently as unmodified phosphoramidites. After synthesis, regular deprotection procedures recommended by Glen Research were followed. After deprotection, the RNA oligonucleotides were purified through HPLC with a C18 column and were eluted with 0–20% acetonitrile in 0.1 M triethylammonium acetate. The desired peak was collected and dried by lyophilization. Synthesized RNA and DNA were dissolved in 10 mM Tris–HCl, pH 7.5, and the quality was examined on a 15% PAGE-urea gel. All oligonucleotides looked pure on the gel and were used directly. Sequences of oligonucleotides used are listed in Oligonucleotides for mutagenesis, reverse transcription, PCR and AlkB D135S gene with deletion of amino (N) terminal 11 amino acids was cloned into a PET28a (+) vector to generate a 6x His-tag fusion construct. Dead mutant of AlkB was made following the QuikChange II XL Site-Directed Mutagenesis Kit  using the AlkB D135S plasmid as the template. To make D135X and R210X libraries, saturation mutagenesis was introduced at positions 135 or 210 following the QuikChange II XL Site-Directed Mutagenesis Kit using primer oligonucleotides containing the degenerative codons NNS at the target positions and AlkB D135S as the template. The resulting mutagenesis products were transformed into XL 10 Gold E. coli cells following manufacturer's protocol. For each library, around 200 E. coli colonies were pooled to ensure coverage at least 10 times greater than the library diversity. The libraries were confirmed with Sanger sequencing.The Escherichia coli cells (2 × 107) expressing AlkB plasmids were pelleted and supernatant was removed. The cell pellet was resuspended in 20 μl lysis buffer containing 10 mM Tris–HCl pH 7.4, 300 mM NaCl, 5% glycerol, 2 mM CaCl2 and10 mM MgCl2, boiled at 93°C for 3 min and then chilled on ice. Two microliter DNase I was added to each sample and the digestion was incubated at RT for 2 min. Two microliter of the digested sample was added with 15 μl lysis buffer and 6 μl 4× Laemmli sample buffer and then subjected to electrophoresis to visualize target proteins on a 4–12% Bis–Tris gel.E. coli crude lysate with and without the addition of RNase inhibitor. Following the manufacturer's protocol, 6 μl cell lysate and 0 or 3 μl SUPERase• In™ RNase Inhibitor were added in a standard 60 μl reaction. The fluorescence signals were recorded on a plate reader , with excitation wavelength of 472 nm and emission wavelength of 507 nm.RNase Activity Detection/Quantification Assay Kit  was used to measure the RNase activity in E. coli cells (New England Biolabs) following the manufacturer's protocol. Single colonies were picked from culture plates and grown in 0.5 mL LB media supplemented with 50 μM kanamycin in a 96 deep square well plate (Thomas Scientific). Cultures were incubated at 37°C overnight with shaking at 250 rpm. The next day , 80 μl of culture was used to inoculate 1.2 ml fresh media in a second 96 deep-square well plate and the culture was continued for 3–4 h at 37°C with shaking at 250 rpm (until OD600 was around 1). Subsequently, IPTG and freshly made FeSO4 solution were added to the culture to 1 mM and 5 μM, respectively, and the cultures were incubated for additional 4 h at 30°C with shaking at 250 rpm to induce AlkB protein expression from the plasmids. Cells were then pelleted to the bottom of plate wells by centrifugation at 4000 rpm for 30 min at room temperature. The pellets were each resuspended in 160 μL lysis buffer containing 10 mM Tris–HCl pH 7.4, 300 mM NaCl, 5% glycerol, 2 mM CaCl2, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 1 mg/ml lysozyme and protease inhibitor (Sigma Aldrich) (5 μl was added per ml lysis buffer). Cell lysis was performed at 37°C for 1 h, with shaking at 250 rpm. Cell debris were pelleted from the lysate by centrifuging the plate at 4000 rpm for 30 min at 4°C and the supernatant was used for downstream experiments. The first overnight cultures were pelleted and saved in –80°C freezer for later usage as described below.Plasmids encoding AlkB enzymes were transformed to T7 express competent 2, 50 μM (NH4)2Fe(SO4)2•6H2O, 2 mM l-ascorbic acid, 300 μM α-ketoglutarate, 50 μg/ml BSA, 270 mM KCl, 1 U/μl SUPERase•In RNase inhibitor (Invitrogen), 4 pmol RNA with the m1G modification and 2 μl lysate supernatant. The reaction was incubated at 25°C for 1h and then quenched by adding EDTA to 2.5 mM and heating at 65°C for 10 min to denature the protein.The demethylation reaction was conducted in a 50 μl mixture containing 25 mM MES buffer (pH 6.0), 2 mM MgClin vitro transcription reaction  containing 2 mM rNTP, 25 mM Mg2+, 10 mM DTT, 50 μM DFHBI-IT and 25 U T7 polymerase in 1× RNAPol buffer. The K+ ion needed for the Broccoli RNA G-quartet structure is supplied from the PCR reaction mixture at a final concentration of 17 mM. Soon after the T7 polymerase was added to the reaction, the fluorescence signal was recorded in a 384-well plate on a plate reader , with settings of 37°C incubation for the in vitro transcription reaction, excitation wavelength of 472 nm and emission wavelength of 507 nm. The in vitro transcription reaction was conducted for up to 3 h and fluorescence was recorded at an interval of one read per min.The product (2.4 μl) from the demethylation reaction was used directly in a reverse transcription reaction in a 20 μl scale containing 0.5 μM primer, 5 μM dNTP, 10 μM DTT, 2U/μl RNase inhibitor and 10 U/μl M-MLV RT in 1× RT buffer. The reverse transcription was performed following the manufacturer's protocol except that 5 μM dNTP was used. The reaction mixture containing the RNA template, the primer and dNTP was first heated at 65 °C for 5 min, followed by quick chill on ice. After the addition of the other components, the reaction was incubated at 37°C for 50 min, followed by denaturing the M-MLV RT by heating at 70°C for 15 min. Two μl of the reverse transcription product was used in a 20 μl scale PCR reaction and amplified using the Q5 high fidelity DNA polymerase (New England Biolabs Inc.) following the manufacturer's protocol. PCR followed the program of 98°C for 30 s;  × 20 cycles; 72°C for 2 min and 4°C hold. The PCR products (7 μl) were then used in the E. coli culture based on corresponding locations in the 96-well plate. The genotypes of the AlkB mutants were determined by Sanger sequencing the E. coli cell cultures. For small-scale confirmation of the positive hits with the fluorescence assay, the same procedure as the screening assay was followed except that E. coli was grown and lysed in 1.5 ml eppendorf tubes.Positive hits identified from the screening were tracked back to the first overnight 2O. We have found that purifying DNA substrates after the demethylation reaction substantially improved the fluorescence signals in the assay. Seven μl of each sample was then used in a 15 μl scale PCR reaction with Taq DNA polymerase following the manufacturer's protocol. A nested PCR program was used: 95°C for 30 s;  × 10 cycles; 68°C for 2 min;  × 20 cycles; 68°C for 5 min; 4°C hold. The PCR products (7 μl) were then used in the in vitro transcription reaction while fluorescence was detected as described above.The screening assay for the DNA substrate followed procedures for the RNA substrate described above with following modifications. In the demethylation reaction with DNA, 0.432 pmol DNA substrate was added in a 50 μl scale reaction, and the SUPERase•In RNase inhibitor was not used. Following the demethylation reaction, DNA substrates were purified with ZR-96 Oligo Clean & Concentrator with each sample eluted with 15 μl ddHE. coli cells (New England Biolabs Inc.). Cells were grown in a 2 l scale LB medium at 37°C until the OD600 reached 0.4–0.8. Isopropyl β-d-1-thiogalactopyranoside (IPTG) and freshly made FeSO4 solution were added to the culture to 1 mM and 5 μM, respectively, and the culture was grown at 30°C for additional 4 h to induce the AlkB expression. Cells were pelleted and resuspended in lysis buffer containing 10 mM Tris–HCl pH 7.4, 300 mM NaCl, 5% glycerol, 2 mM CaCl2, 10 mM MgCl2, 2 mM 2-mercaptoethanol and 1× protease inhibitor (Thermo Fisher Scientific). Cells were then lysed with sonication and centrifuged at 13 000 rpm for 45 min. The supernatant was first purified with a manual Ni-NTA column and eluted with a buffer containing 10 mM Tris–HCl pH 7.4, 300 mM NaCl and 250 mM imidazole. The protein was then further purified using Mono S chromatography. The protein was eluted with gradient buffers composed of different ratios of buffer A (10 mM Tris–HCl pH 7.4 and 100 mM NaCl) and buffer B (10 mM Tris–HCl pH 7.4 and 1.5 M NaCl) and then buffer exchanged to the storage buffer . Glycerol was then added to 30%. The protein was aliquoted, snap frozen in liquid nitrogen and stored at –80°C.AlkB enzymes  were expressed in T7 express competent 1G-broccoli RNA in a 50 μl mixture containing 25 mM MES buffer (pH 6.0), 2 mM MgCl2, 50 μM (NH4)2Fe(SO4)2•6H2O, 2 mM l-ascorbic acid, 300 μM α-ketoglutarate, 50 μg/ml BSA, 270 mM KCl, and 1 U/μl SUPERase•In RNase inhibitor (Invitrogen). In the control experiment, EDTA was added to 4 mM to chelate Fe2+ and Mg2+ ions required for the enzymatic reaction. The demethylation reaction was conducted at 25°C for 8 min and quenched by adding EDTA to 4 mM and heating at 65°C for 10 min. Then the demethylation product (2.4 μl) was reverse transcribed, followed by PCR and in vitro transcription as described above. The PCR was performed for 15 cycles.Purified D135T or D135S (3.375 pmol) was mixed with 4 pmol m1G)UUAG-3′) was used in the assay to measure enzyme kinetics for D135T and D135S mutants. Purified D135T or D135S protein (80 pmol) was mixed with various amounts of the oligonucleotide  in a 20 μl mixture containing 25 mM MES (pH 6.0), 2 mM MgCl2, 50 μM (NH4)2Fe(SO4)2•6H2O, 2 mM l-ascorbic acid, 300 μM α-ketoglutarate, 50 μg/ml BSA, 270 mM KCl, and 1 U/μl SUPERase•In RNase inhibitor (Invitrogen). The demethylation reaction was conducted at 25°C for varying times and quenched by adding EDTA to 4 mM and heating at 65°C for 10 min. Demethylated RNA was purified by phenol–chloroform extraction and ethanol precipitation. The purified RNA was dissolved in 5 μl water and deionized through incubation with 20 μl cation exchange resin  for 20 min. The RNA was then analyzed by MALDI-TOF/TOF MS using 2′,4′,6′-Trihydroxyacetophenone (THAP) as matrix and negative ion reflector mode. The MALDI spectrum was analyzed using flexAnalysis software with areas of target peaks integrated to quantify the demethylation fraction. The resulting data was fitted to the equation y = A*(1 – exp(–k*t)), where y is the demethylation fraction at time t, A is the fitted reaction saturation point and k is the fitted rate constant (min−1). The values of KM, kcat and kcat/KM were obtained by fitting the data to the equation: ([S]/[E])*A*k = kcat[S]/(KM + [S]), where [S] is the substrate concentration and [E] is the enzyme concentration.A 9-mer RNA oligonucleotide 2Fe(SO4)2•6H2O, 0.1 M KCl, 50 ng/μl BSA and 4 U/μl SUPERase•In RNase inhibitor. In the control experiment, EDTA was added to 4 mM to chelate the Fe2+ ion required for the enzymatic reaction. The demethylation reaction was conducted at 37°C for 10 or 30 min and quenched by adding EDTA to 4 mM and heating at 65°C for 10 min.In the demethylation assay with yeast tRNA , a modified protocol was used that contained no magnesium but still worked efficiently. Twenty pmol of purified D135T or D135S was mixed with 8 pmol (∼200 ng) yeast tRNA in a 25 μl mixture containing 50 mM MES pH 6.0, 2 mM l-ascorbic acid, α-ketoglutarate and (NH4)2Fe(SO4)2•6H2O were made fresh right before usage.In all demethylation reactions described in this paper, 4HCO3 pH 5.3 at 42°C for 2 h, followed by digestion with shrimp alkaline phosphatase (0.5 U) in 1× CutSmart buffer (New England Biolabs Inc.) at 37°C for 2 h. The sample was diluted to 90 μl with RNase free water and filtered , and the solution was injected into LC–MS/MS. Nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column with on-line mass spectrometry detection using an Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside to base ion mass transitions of 268 to 136 (A), 284 to 152 (G), 282 to 150 (m1A) with retention time of 0.8 min, 298.1–166.1 (m1G), and 258.2–126.1 (m3C). Quantification was performed in comparison with calibration curve obtained from pure nucleoside standards running on the same batch of samples. The ratios of m1G to G, m1A to A and m3C to G were calculated based on the calibrated concentrations.Yeast tRNA (∼200 ng) from the demethylation reaction was purified using oligo clean & concentrator kit (Zymo Research). The tRNA was digested by nuclease P1 (1 U) in a 30 μl scale reaction containing 10 mM NHsourceforge.net/projects/bbmap/) with the script ‘merge.sh’. Sequencing adaptors were trimmed off automatically during the step of merging reads. By design, reads contain 6-nt UMI sequences on the 5′ ends for labeling of unique sequences. Reads were first de-duplicated to remove PCR duplicates using the script ‘clumpify.sh’, followed by trimming the UMI sequences with the script ‘trim_UMI.sh’. Reads also contain sample-specific 3-nt internal barcode sequences on the 3′ ends which were introduced in early steps of the library constructing process to decrease the sample handling complexity. Based on the 3-nt barcode, data were decomplexed by searching sequences ending with the barcode sequences using the grep command with options of ‘-A 2 -B 1 ‘XXX$’’, where ‘XXX’ means the 3-nt barcode. After this, the 7-nt adaptor sequences (including the 3-nt barcodes) on the 3′ end of reads were further trimmed. The script used for these two steps is ‘sort_and_trim.sh’. The trimmed reads underwent quality control with options of ‘-t 20 -l 22′ using the script ‘quality_control_trim.sh’ to filter off reads with quality lower than 20 or length shorter than 22 nt. Genome was built using the script ‘build_genome.sh’ on the basis of the human tRNA genome sequence file named ‘hg19CCA+mito+spike-in.fasta’, which is modified from the hg19-mature-tRNA file downloaded from http://gtrnadb.ucsc.edu/Hsapi19/Hsapi19-seq.html, by removing identical genes, adding ‘CCA’ to the ends, and adding in the mitochondrial tRNAs. Reads were then mapped to the reference genome using bowtie2, and the resulting SAM files were converted to the BAM files, sorted and indexed using the script ‘map_hg19-CCA.sh’.Paired-end reads (150 bp) from Illumina sequencing Hiseq 4000 platform were first merged using BBMap tools   ; however1G modification  as a negative control and used RNase inhibitor in our assay. The AlkB D135S and the dead mutant were confirmed to express at the same level in E. coli, whereas the protein in the uninduced lanes may represent both endogenous AlkB and leaky expression of the expression plasmid  than recommended by the manufacturer  library. Each library was screened in a 96-well plate, ensuring coverage of the theoretical library diversity. The genotypes of mutants with various levels of fluorescence signals (adjusted relative fluorescent unit or RFU) are summarized in 1G and confirming the capability of the assay to distinguish different levels of demethylation activities  exhibited around 7-fold higher catalytic efficiency than D135S (kcat/KM = 2.2 M−1min−1)  Figure . Neverth1G in our system, we applied D135T in tRNA-seq experiments. We first tested D135S and D135T in removing modifications from total yeast tRNA through LC-MS/MS. Under our condition (see Methods), both D135T and D135S efficiently removed > 90% of m1A (located in the T loop), 40–60% of m1G (located in both anticodon loop and position 9 in the tRNA tertiary structure) and around 50% of m3C from yeast tRNA  and m1G37 in tRNALeu(CAA), whereas D135T exhibited better results for sites including m1A58 in tRNAGly(TCC), and m1G9 in tRNAArg(CCG)  Figure .E. coli AlkB has long been known as a DNA repair enzyme which can catalyze the repair of m1A and m3C in single stranded DNA (E. coli); however, the active form of MGMT cannot be regenerated after one round of catalysis, limiting its repair capacity especially in the case of excessive damage levels  an error-prone polymerase (Taq polymerase) was used in the PCR reaction to introduce a G-to-A mutation at the modification site  a 51 mer DNA Broccoli sequence bearing an O6mG modification at a specific G position was used as the substrate; (ii) following the demethylation reaction, the DNA substrate was directly used in PCR followed by an e Figure –C. Presu41, G46, G53 and G59) in the Broccoli sequence and found that G59 is highly sensitive to the G-to-A mutation , with an average adjusted RFU value of 800, consistent with the screening result for the D135X library (Figure We noticed that there was variability in fluorescence readout for the same variant in the 96-well screening Figure , D. Thisy Figure . This su1G and m22G, additional mutants with better activity will still be desired for RNA-seq applications. Comprehensive screenings at position L118 and other positions proximal to N2 of guanosine such as M61 may help identify better variants towards m22G (1G9. Second, it is desirable to screen for AlkB mutants with better specificity. Examples include mutants capable of more stringent discrimination between m1G and m22G to facilitate the identification of either modifications in the mRNA transcriptome. Third, the assay can be used to screen for small molecule inhibitors of AlkB and homologs. Human ALKBH2 and ALKBH3 are involved in diseases and studies identifying selective inhibitors are of therapeutic potential (We envision several other immediate applications of our screening system. First, the assay can be used to study other important residues in AlkB or other demethylases against various types of modifications . Althougrds m22G . In addiotential . Lastly,otential , our scrhttps://github.com/yuruwang26/tRNA_seq_scripts.RNA-seq data has been deposited to GEO under accession number: GSE152694. The scripts for data analysis have been deposited in GitHub: gkaa1213_Supplemental_FileClick here for additional data file.


Owing to the synergetic contribution of the multielement chemical substitution strategy, this novel cathode not only shows excellent air stability and thermal stability as well as a simple phase-transition process but also delivers outstanding battery performance in half-cell and full-cell systems. Meanwhile, various advanced characterization techniques are utilized to accurately decipher the crystalline formation process, atomic arrangement, structural evolution, and inherent effect mechanisms. Surprisingly, apart from restraining the unfavorable multiphase transformation and enhancing air stability, the accurate multielement chemical substitution engineering also shows a pinning effect to alleviate the lattice strains for the high structural reversibility and enlarges the interlayer spacing reasonably to enhance Na+ diffusion, resulting in excellent comprehensive performance. Overall, this study explores the fundamental scientific understandings of multielement chemical substitution strategy and opens up a new field for increasing the practicality to commercialization.The O3-type layered oxide cathodes for sodium-ion batteries (SIBs) are considered as one of the most promising systems to fully meet the requirement for future practical application. However, fatal issues in several respects such as poor air stability, irreversible complex multiphase evolution, inferior cycling lifespan, and poor industrial feasibility are restricting their commercialization development. Here, a stable Co-free O3-type NaNi Owing to the environmental pollution and political unrest with regard to fossil fuel production, electrochemical energy storage and conversion (EESC) technology with low cost, high Tefficiency, long lifespan, and adequate safety is important –8. Sodiu2  have aroused considerable interest due to their high initial Coulombic efficiency, excellent ionic conductivity, and good environmental benignity [0.5Mn0.5O2 cathodes have the advantage of being cobalt-free, they normally show poor air stability and exhibit notable performance losses, which originate from the formation of electrochemically inactive NaOH or Na2CO3 [0.5Mn0.5O2 cathodes accompanied by multiple voltage plateaus in the electrochemical curves, such as O3hex.-O3′mon.-P3hex.-P3′mon.-P3′′hex., are inclined to result in rapid capacity decline and the sluggish kinetics as well as poor rate performance [0.5Mn0.5O2 cathode materials through simultaneously enhancing the air stability, reducing or suppressing the irreversible phase transition, and alleviating the sluggish kinetics is of great importance to realize sodium-ion battery commercialization for market applications. According to the reported works, chemical substitution strategy could modulate the physical as well as chemical properties and indeed enhance comprehensive performance [As cathode materials are the key determinant for SIBs, a large variety of cathode materials have been mostly studied so far –20. Amonenignity –23. Howeenignity , 25. (2)r Na2CO3 , 27. Mear Na2CO3 , 29. (3)formance . Therefoformance –35. Howe0.4Cu0.05Mg0.05Mn0.4Ti0.1O2 cathode material for practical SIBs was synthesized by a facile high-temperature solid-state method with large-scale production. By virtue of partially substituting copper and magnesium into the nickel sites and titanium into the manganese sites, this novel layered oxide cathode not only shows excellent physical and chemical properties but also delivers outstanding cycle performance (76.4% after 1000 cycles at 5C) and excellent capacity retention of 73.6% (10C compared to 0.2C) in half-cell system and superior battery performance in full-cell system. Meanwhile, the inherent synergetic contributions of rationally selected multielement chemical substitution engineering strategy are clearly articulated and confirmed by various in situ X-ray diffraction (XRD) and multiscale scanning transmission electron microscopy as well as other advanced characterization techniques. All these results indicate that this novel compound promises to be a very competitive cathode material for future large-scale practical application.Hence, a stable Co-free O3-type NaNi0.4Cu0.05Mg0.05Mn0.4Ti0.1O2 cathode material (hereafter denoted as O3-NaNCMMT) is ascribed to the α-NaFeO2 layered structure with rhombohedral RRwp 6.84% and Rp 4.77%) are displayed in Table 2+ = 0.69 Å, Cu2+ = 0.73 Å, Mg2+ = 0.72 Å, Mn4+ = 0.53 Å, and Ti4+ = 0.605 Å), the uniform incorporation of multielement chemical substitution into the TM layers could effectively modulate the crystal structure and alleviate the lattice strains driven by the large Ni ionic radius change during Na+ intercalation/deintercalation, resulting in better structural stability [c = 16.0301 Å) of Cu and Ti cosubstituted NaNi0.45Cu0.05Mn0.4Ti0.1O2 sample, the substitution of Mg2+ in O3-NaNCMMT cathode material not only shows a pinning effect but also enlarges the interlayer spacing reasonably (c = 16.0812 Å), which could improve the Na+ diffusion coefficient and contribute to the high rate capability, as discussed later [2 layers, where TMO2 layers duplicate in the manner of αβγαβγ with the ABCABC-stacking mode of oxygen columns (in situ high-energy XRD (HEXRD) was executed to accurately decipher the formation process and the structure change as well as thermal stability of O3-NaNCMMT cathode material in air atmosphere from room temperature to 1000°C  STEM images and the white dots in the HAADF-STEM images reveal the transition metal atom positions [0.4Cu0.05Mg0.05Mn0.4Ti0.1O2 cathode material has been successfully fabricated by high-temperature solid-state reaction with large-scale production.The contrast of the high-angle annular dark-field (HAADF) image of scanning transmission electron microscopy (STEM) exhibits an approximately Zositions –46. In aositions , 48. It −1 and 397.9 Wh kg−1, respectively, at 0.2C within 2.0–4.0 V, and the first three cyclic voltammetry (CV) curves overlap well  after the rate comes back to 1C  of O3-NaNCMMT electrode is calculated from the CV  peaks shift to a lower angle and then split into two, revealing that the new P3 phase starts to form and the intensity of (104)O3 peak is clearly reduced via an O3–P3 two-phase reaction [To test the air stability of O3-NaNCMMT cathode material, ried out . Accordireaction , 54. Whereaction –57.−1 at 0.2C corresponding to energy density of 367.4 Wh kg−1 based on the mass of cathode  was synthesized by a high-temperature solid-state reaction process with a large-scale production line. The stoichiometric mixtures of Na2CO3 , NiO , CuO , MgO , Mn2O3 , and TiO2  were ground and calcinated at 1000°C in air for 12 hours. The product was stored in an argon-filled glove box for further use (H2O and O2 < 0.1 parts per million).O3-type NaNiIn situ XRD test experiment was supported by a special Swagelok cell. Detailed morphological and crystalline structural information were obtained through both field-emission scanning electron microscopy  and transmission electron microscopy . The atomic arrangement of cathode material was elucidated by scanning transmission electron microscopy .Powder XRD pattern was obtained by D8 Advance  Diffractometer at a scan rate of 1°/min over the 10°-80°. 4 in the mixed solvent of propylene carbonate and fluoroethylene carbonate . The cathode film of the half-cell system was prepared by mixing the active material (70 wt%), Super P carbon (20 wt%), and polyvinylidene fluoride (10 wt%) binder in N-methyl-2-pyrrolidone (NMP) followed by casting slurry onto a clean Al foil and drying at 80°C in a vacuum oven overnight. Electrochemical measurements were performed on a Land BT2000 battery test system (1C = 120 mA g−1). The hard carbon anode of the full-cell system was prepared by a uniform mixture of 80 wt% active material, 10 wt% Super P carbon, and 10 wt% polyvinylidene difluoride (PVDF). The hard carbon anode was activated by an electrochemical presodiated process using the metal sodium foil as the counter electrode (1C = 300 mA g−1). The weight ratio of cathode and anode was balanced by referring to their reversible capacities. The electrochemical performance test of the full cell was also evaluated in the coin cell (CR2032), and the corresponding average loading of active material was about 2~3 mg cm−2. The current density was based on the mass of positive electrodes (1C = 120 mA g−1) with the voltage window of 1.9-3.9 V at room temperature.The electrochemical properties were evaluated in coin cells (CR2032) with Na disks and porous glass fiber as the counter electrode and separator, respectively. The electrolyte was a solution of 1 M NaClO


The CeO2 NPs were synthesized using the E. globulus leaf extract-mediated hydrothermal technique. The synthesized NPs were characterized by various analytical instruments including powder X-ray diffractometer (PXRD), scanning electron microscope (SEM), transmission electron microscope (TEM) and dynamic light scattering (DLS) analysis. The XRD results showed an average NPs sizes of 13.7 nm. Cytotoxic study results showed an IC50 value of 45.5 µg/L for A549 and 58.2 µg/L for HCT 116, indicating that CeO2 NPs are more toxic to A549 compared to HCT116 cell lines. The generation of ROS was responsible for its cytotoxic activity against cancer cell lines. Specific surface area (40.96 m2/g) and pore diameter (7.8 nm) were measured using Brunauer–Emmett–Teller (BET) nitrogen adsorption–desorption isotherms. CeO2 NPs with a high surface area were used as photocatalyst in degrading sunset yellow (SY) dye under UV-irradiation and 97.3% of the dye was degraded within 90 min. These results suggest that the synthesized CeO2 NPs could be used as a good photocatalyst as well as a cytotoxic agent against human cancer cell lines. Ceria nanoparticles (CeO Human cell lines A549 and HCT-116 were procured from ATCC  for the calculated cytotoxic study. Milli-Q water was used in all experiments.Fresh E. globulus leaves were washed thrice with Millipore water and dried at ambient temperature in a dust-free chamber without sunlight exposure until constant weight was obtained. Ten grams of dried powder of the leaves mixed with 100 mL Milli-Q water was heated at exposure to 80 °C for 2 h, and the resulting light yellow solution was filtered, centrifuged and stored at 4 °C for further studies.Required amount of cerous nitrate hexahydrate (0.1N) was dissolved in 100 mL water, and then 100 mL aqueous plant extract was added dropwise through a peristaltic pump with stirring over 2–3 h. The pH of the mixture  was adjusted to 8.2 by the addition of NaOH. After 24 h, pH of the mixture increased to 7.5 due to the formation of cerium hydroxide in the solution. The brownish yellow colored solution formed was centrifuged at 10,000 rpm for 15 min followed by ethanolic wash to remove impurities from solution. The obtained brownish yellow colored residue was dried at 80 °C for 2 h in a hot air oven and the dried powder was properly pulverized and then annealed at 400 °C for 3 h to obtain crystalline particles.2 NPs were prepared in two steps. In the first step, cerium nitrate was mixed with E. globulus leaf extract, which formed a three-dimensional bridge complex due to interaction of phytochemicals with cerium ions. In this step, E. globulus extract containing 9, 12 octadeca trienoic acid chains reacted with tetravalent Ce+4 via hydroxyl groups from two different chains to form network complex bridge with Ce+4 which conjugates to all the compounds present in the extract caused synergistic effect resulting in a complex structure. In the second step, the polymeric network chains suffered slow decomposition during calcination. The mechanism of CeO2 NPs formation is shown schematically in CeO2 NPs powder after CuKα radiation (λ = 1.54 Å). Crystallite size of CeO2 NPs was calculated by Scherrer’s formula as D = Kλ/(β cosθ), where λ = wavelength of X-ray radiation source (1.5406 Å), K = Scherrer constant as 0.9 for spherical particles, β = the width of the XRD peak at half height. Powder X-ray diffractometer (Bruker D8 Advance X-ray Diffractometer) confirmed the crystalline texture of CeO2 NPs which was compared with XRD and HR-TEM results. Zeta potential was measured to predict stability using Horiba Scientific Nano Particii (SZ-100). CeO2 NPs dispersion was prepared by sonicating 1 mg NPs in 100 mL water at pH 7.5 for DLS and zeta potential measurement. Thermal analysis of the synthesized CeO2 NPs was carried out to determine weight loss with temperature. ATR-FTIR (Jasco-4100) at wave number range of 4000–400 cm−1 was used to trace functional groups attached to NPs as well as the extracts. UV-Vis-DRS study measured absorption intensity and band gap of the NPs in the wavelength ranges of 200–800 nm. The band gap of CeO2 NPs was calculated by using the following equation as bulk is bulk ‘band gap’.Surface morphology was checked by FE-SEM and elemental composition was monitored by energy dispersive X-ray spectroscopy (EDAX). In addition, XRD was used to check phase purity which was cross-checked with selected area electron diffraction (SAED) pattern. SAED patterns were recorded using high resolution TEM (HR-TEM) equipped with Gatan CCD camera. DLS experiment identified particle size distribution of CeO2 NPs was used to degrade SY dye from aqueous solution using photoreactor  under UV irradiation (λ 254 nm). The photodegradation efficiency (%) was assessed using 50 mL of SY dye aqueous solution (10 mg·L−1) mixed with varying amounts of photocatalyst  by UV-Visible spectrophotometer at different time intervals.The synthesized photocatalyst CeO2 one day prior to the exposure studies with CeO2 NPs.Cell culture study was carried out using conventional method where Dulbecco’s modified Eagle’s medium (DMEM) containing 100 µg penicillin/mL, 100 U streptomycin/mL) and 10% heat-inactivated FBS was used in cell culture T25 flasks. Human A549 and HCT116 cell lines were cultured at 37 °C and 5% COoxidoreductase enzymes. Briefly, cells were seeded at a density of 1 × 104 cells/well into a 96 well plate and incubated overnight at 37 °C with 5% CO2. Then the cells were washed with PBS and media was discarded. The cells were then exposed to test chemical for 24 h at 37 °C with 5% CO2 and RH more than 80%. After removal of medium, 100 µL PBS was added to each well followed by addition of 100 µL MTT solution (0.5 mg/mL MTT in DMEM without FBS) and incubated for 1 h at 37 °C and 5% CO2. The formed formazan crystals were dissolved in 10% SDS (sodium dodecyl sulfate in 0.01 N HCl). MTT solution was then removed and 100 µL DMSO was added to each well and absorbance was measured using ELISA reader at 570 nm . Additionally, one blank was used without CeO2 NPs for comparison.Cell viability was determined using MTT assay which is based on the conversion of the tetrazolium salt [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium] to formazan crystals by mitochondrial NAD(P)H-dependent 50 values were obtained from the graph plotted using concentrations of CeO2 NPs along X-axis vs. cell viability (%) along Y-axis.Cell viability calculation was used to check the cytotoxic effect of the toxicants. MTT assay was performed in 96 well plate for negative control (only cell culture medium and cells were added), positive control , experimental wells (having cells with CeO NPs), and blank samples (having only medium). All the experiments were carried out in triplicate. Cell viability was calculated as the ratio of the mean absorbance of replicated wells compared to that of the negative control wells. The experimental wells were compared to negative control with 100% growth. The IC2 NPs dispersion which indicates the absorbance peak at 310 nm. UV-DRS (diffuse reflectance spectroscopy) spectrum of CeO2 NPs shows bandgap value as 2.91 eV. g) is found by the extrapolation of a Kubelka-Munk function fitted in a Tauc plot along the X-axis, as shown in 2 NPs. The optical band gap  and weight percentage (43.30% and 46.56%), respectively. A very less intense carbon peak is seen due to the contribution of carbon tape attached on the stub surface.2 NPs exhibiting the peaks at 2θ values with hkl indexing of 28.54° (111), 33.07° (200), 47.47° (220), 56.33° (311), 59.07° (222), 69.40° (400), 76.68° (331), 79.06° (420), and 88.41° (422). This pattern indicates crystal system of cubic phase structure having space group number Fm3m (225), unit cell volume (158.55 Å) and lattice parameters (a = b = c = 5.4124 Å) [2 NPs is 20.72 nm as per calculation by the Scherrer’s formula [, 33.07° 0, 47.47° formula .2 NPs is stated in terms of zeta potential. It is defined as the potential difference around surface charge groups associated on the NPs surfaces, and dispersed solvent medium containing groups of opposite charge. Basically, higher negative value of zeta potential indicates more repulsion between the particles which minimizes aggregation and results in higher stability of bio-fabricated NPs [2 NPs as 20.9 nm. 2 NPs as −35.5 mV at pH 7.5, which indicates higher dispersibility of the NPs with high stability.The stability of CeOated NPs . Figure 2 particles having fringe space of 3.1 Å The results are in close agreement with XRD data with (111) plane (3.24 Å) of CeO2 NPs and the low crystalline nature of particles resembles its high reactivity to radicals [radicals . Figure E. globulus extract (−1), and carboxyl (C=O) group (absorption band at 1690 cm−1) which reduces Ce4+ ions into CeO2 NPs [−1 correspond to the stretching vibrations of alkanes, 1390 cm−1 for the CH2 bending, 1110.8 cm−1 for C–O bending vibrations. Additionally, lower shift functional groups correspond to 940.3 cm−1 for C–O and 640.3 cm−1 for Ce–O bond respectively. It resembles the formation of CeO2 NPs and the polyphenol groups present in the E. globulus extract behave both as reducing agent to cerium ions and stabilizing agent of CeO2 NPs.FTIR analysis is carried out to find out the functional groups attached to NPs surface as well as in phytochemicals present in  extract a. FTIR sCeO2 NPs . Other a2 NPs are calculated using nitrogen adsorption–desorption isotherms. 2 NPs is 40.96 m2/g, which is much higher than commercial CeO2 NPs (8.50 m2/g) [Specific surface area and pore size distribution of the synthesized CeO50 m2/g) . Additio50 m2/g) .2 NPs against temperature (@ 5 °C/min) under nitrogen gas atmosphere, with two-step weight losses within the temp range of 0–800 °C. The first step is associated with 11.6% weight loss in the temperature range of 20–200 °C due to elimination of water molecules and absorbed moisture. Second major weight loss of 18.6% occurred in 300–400 °C due to oxidative decarboxylation of Ce(NO3)3 to CeO2 [Thermogravimetric analysis shows weight loss of the compound with respect to temperature. 2 NPs was studied during the degradation of SY dye under UV-irradiation. It was observed that the intensity of SY dye colour did not change in the dark, during a time period of 1–2 h, with or without CeO2 NPs. SY dye was found to degrade only under UV-irradiation, which was confirmed by UV-Visible spectroscopy (no appearance of dye peak)  versus irradiation time (min), and a linear relationship is obtained based on the following equation as Ln(C/C0) = −kt, where C0 is the concentration of SY at time t “0 min”, C is the concentration of SY at time “t min” and k is the slope constant. Slope value obtained for SY with optimized catalyst dosage (35 mg) is k = 0.0250 min−1, which follows pseudo first-order kinetic rate. The recyclability of the catalyst is worked out and up to three cycles, whereby no significant change in percentage of degradation is seen, as shown in The photocatalytic activity of the synthesized CeOye peak) . Figure 2 NPs is performed against A549 and HCT 116 cancer cell lines. The cell viability is analyzed by MTT assay and IC50  value obtained is 45.5 µg/L for A549 cell line and 58.2 µg/L for HCT 116, which clearly suggests that the biosynthesized CeO2 NPs are more toxic to A549 cell lines compared to HCT 116 cell lines. It can be noted that it is toxic to both the cell lines suggesting its potential as anticancer agent. The cytotoxicity of CeO2 NPs is compared with standard drug cisplatin (CDDP), which is mostly similar at high dose of CeO2 NPs (80 µg/mL) (Cytotoxicity study of CeO0 µg/mL) a.2 NPs due to dose-dependent production of ROS species [2 NPs at different concentrations  at an emission wavelength of 523 nm. It clearly exhibits ROS generation by CeO2 NPs with increasing intensities of DCF dye, which is directly proportional to the dose. Compared to the control, bio-synthesized CeO2 NPs show higher emission intensity due to more generation of ROS species or more cell deaths. ROS is generated from reduced nicotinamide adenine dinucleotide dehydrogenase II in the respiratory chain by the auto-oxidation process [50 concentration of CeO2 NPs dispersion shows the ROS emission with green colour spotted images, which indicate both cell lines generated ROS in the presence of CeO2 NPs  is used, which can be de-esterified intracellularly to highly fluorescent 2-dichloro fluorescein (DCF+) upon oxidation . It is c species . Basical species . Figure  process ,30,31,32CeO2 NPs a,b.2 NPs were synthesized using E. globulus leaves extract via a green method. Synthesized NPs show photoluminescence, cubic structure, and spherical shape with particle size ranging from 8–20 nm, which are confirmed by various analytical techniques like XRD, SEM and TEM. The bio-synthesized CeO2 NPs are a superior photocatalyst compared to other photocatalysts due to oxygen vacancies, decreased band gap, surface defects and lower recombination tendency of the electron–hole pair. The synthesized nanoparticles remain stable up to four recycles without change in photocatalytic efficiency. Additionally, CeO2 NPs are cytotoxic to human lung A549 and colon HCT 116 carcinoma cell lines, with IC50 values of 45.5 and 58.2 µg/L respectively. The current results suggest the promising potential of CeO2 NPs for potential anticancer therapy. In summary, CeO