SLG2 (SLENDER GUY2) gene that specifically regulates grain width by affecting cell expansion in the spikelet hulls. SLG2 encodes a WD40 domain containing protein, and our biochemical analyses show that SLG2 acts as a transcription activator of its interacting WOX family protein WOX11. We demonstrate that the SLG2‐associated WOX11 binds directly to the promoter of OsEXPB7, one of the downstream cell expansion genes. We show that knockout of WOX11 results in plants with a slender grain phenotype similar to the slg2 mutant. We also present that finer grains with different widths could be produced by combining SLG2 with the grain width regulator GW8. Collectively, we uncover the crucial role of SLG2 in grain width control, and provide a promising route to design rice plants with better grain shape and quality.Grain size is specified by three dimensions of length, width and thickness, and slender grain is a desirable quality trait in rice. Up to now, many grain size regulators have been identified. However, most of these molecules show influence on multi‐dimensions of grain development, and only a few of them function specifically in grain width, a key factor determining grain yield and appearance quality. In this study, we identify the  Rice is one of the most important cereal crops and the stable food of nearly half of the world's people. With the increase of the world population, the deterioration of the environment and the reduction of the arable land, increasing grain yield remains the major challenge for most rice growing areas. Rice grain yield is composed of three major factors: panicle number, grain number per panicle, and grain weight. The grain weight is determined by grain size and grain filling. Because grain size is a key determinant of yield and appearance quality, it is one of the major targets of artificial selection during domestication, and is an important trait for rational design of high‐yield and high‐quality rice varieties.et al., SMALL GRAIN 1 (OsSMG1)/(OsMKK4), OsMAPK6 and OsMKKK10 control grain size through the mitogen‐activated protein kinase signalling pathway /(OsOTUB1), GW2 and OsUBP15 affect grain size by the ubiquitin‐proteasome pathway , we identified a mutant with obviously decreased grain width . Compared with the wild type (WT), the grain width of slg2 was reduced by about 10.5% , the caryopsis growth of the mutant lagged behind WT in the transversal direction  and G2‐to‐M transition  between slg2 and WT , six genes  showed suppressed transcription in slg2 relative to WT . This result suggests that the slg2 mutant phenotype is caused by the recessive mutation of a single locus. By using 20 bulked WT and slg2 mutant plants from the F2 population between slg2 and KY131 (japonica), the causal gene was initially mapped to the short arm of chromosome 2 between markers M1 and M2 , leading to the formation of a premature stop codon in the mutant  and 2‐bp (SLG2‐KO2) insertions for phenotypic analysis . Consistent with the results of the complementation test, overexpression of SLG2 fully rescued the overall phenotypes of the mutant . In rice, there are 231 OsWD40s in the genome . Because these two genes shared an identity of about 65% at the protein level . To understand whether HSLG2 also plays a role in grain size control, we knocked out this gene in the ZY66 background by specifically editing the target site in the second exon  to screen the interacting proteins of SLG2 by the yeast two‐hybrid system. Among the sequenced positive colonies, we were highly interested in the potential partner WOX11  and roots  was markedly suppressed  assay in the rice protoplast . We expressed the WOX11 protein in E. coli and purified it as a maltose binding protein (MBP)‐fusion protein. For the probes, we synthesized biotin‐labelled oligonucleotides containing the native (hot probe) and mutated (mutant probe) WOX binding site presented in the OsEXPB7 promoter  and ZY66 (GW8‐KOWT) respectively, by specifically editing a target site in the third exon . All the materials used in this study were cultivated in the experimental fields of the Institute of Genetics and Developmental Biology in Changping (40.2°N/116.2°E), Beijing during the summer or Lingshui , Hainan province during the winter.The 2/s photon flux density. Morphological investigation and RNA isolation were performed on 7‐day‐old seedlings.For seedling culture, healthy seeds were surface‐sterilized with 3% sodium hypochlorite for 30 min, soaked at 37 °C for 3 days. Germinating seeds were sowed into 96‐well PCR plates, and water‐cultured in the phytotron (SANYO) with 12 h light (28 °C)/12 h dark (28 °C), 65%–70% relative humidity, and 150 μM/mslg2 with KY131 (japonica). Whole genome polymorphic markers were designed based on resequencing data of the two parents (30×), and primers used for fine mapping are listed in Table 2 plants with WT and mutant phenotypes respectively, the candidate gene was first mapped to the short arm of chromosome 2 between the markers M1 and M2. Further analysis of the F2 mutant plants subsequently fine‐mapped the causal gene to the region between the markers M6 and M7, and sequence comparison was then performed between slg2 and WT based on the resequencing data (30×).The mapping population was generated by crossing Investigation of agronomic traits was performed on plant height, days to heading, panicle number, grain number per main panicle, 1000‐grain weight, seed setting rate and grain yield per plant at the maturation stage. Caryopsis development was observed on florets marked at the time of anthesis, and developing ovaries were collected at different developmental stages. The grain filling rate was calculated as the percentage of the dry grain weight at the two adjacent indicated days. The length, width and thickness of mature grains were measured using Olympus stream software under a light microscope .For histological analysis, florets before anthesis were sampled and fixed in formalin‐acetic acid alcohol (FAA) solution  overnight at 4 °C after 20 min vacuum treatment. After dehydration with a gradient of ethanol, replacement with xylene and embedding in paraffin , sections (8 μm thick) were cut with a rotary microtome , stained with 0.2% toluidine blue and observed under a microscope . The outer parenchyma cell number and cell area were measured with Olympus stream and Image J software, respectively. For SEM observation, the outer glumes of fully mature, dry grains were sprayed with gold particles and scanned with a scanning electron microscope . The outer glume cell number was counted and cell size was measured using the Olympus stream software . To observe the grain endosperm structure, mature grains were hand‐cut using the bladeless cutter, then the cutting surfaces were coated with gold in a vacuum with an ion sputtering device , and observed with a scanning electron microscope .OsActin was used as the internal control for normalization. The primers used are listed in Table Samples were taken from shoots and roots of 7‐day‐old seedlings, and from flag leaf blade, flag leaf sheath, culm, young panicle and mature floret before anthesis. Total RNA was extracted using RNAiso PLUS reagent , and 1 μg RNA was reverse‐transcribed by oligo (dT) primers using a reverse transcription kit  after digestion with RNase‐free DNaseI . The RT‐qPCR assay was performed in triplicate with SYBR Green I Master reagent and the Light Cycler Nano system . SLG2, the first exon of WOX11 and the third exon of GW8 by the CRISPR‐GE genome editing tool (http://skl.scau.edu.cn/) and ligated to the CRISPR/Cas9 vector pHUN4C12  was amplified into four fragments, and cloned into the pZH2B vector by a seamless cloning kit . For knockout vector, a 20‐bp carefully designed target was selected from the third exon of SLG2 and WOX11 were cloned into the pGADT7 or pGBKT7 vector , and the resulting constructs and the corresponding empty vectors were then co‐transformed into the yeast strain Golden Yeast with different combinations. Interactions were detected on SD/−Leu‐Trp‐His‐Ade medium. The transformation was conducted according to the Yeast Two‐Hybrid System User Manual (Clontech). The primers used in this assay are listed in Table The coding sequences of SLG2 was inserted into the pSAT6‐EYFP‐N1 vector (PSAT6) to construct the SLG2‐EYFP (SLG2‐PSAT6) vector. The empty vector was used as a negative control. For the BiFC assay, the coding sequences of SLG2 and WOX11 were ligated into the pUC19‐VYNE (R) or pUC19‐VYCE (R) vector fused with the N‐ or C‐terminus of the Venus YFP sequence, respectively. These plasmids and the corresponding empty vectors were co‐transformed in different combinations into rice protoplasts. The fluorescent signal was detected with a confocal laser scanning microscope (Leica TCS SP5) after 28 °C incubation for 16 h in the dark.For subcellular localization, the coding sequence of WOX11 was fused with the GAL4 DNA binding domain in the pRT‐BD vector, and the resulting construct (GAL4‐BD‐WOX11) was used as the effectors. The coding sequence of SLG2 was fused with pSAT6‐EYFP‐N1 vector to form SLG2‐PSAT6 fusion protein, and was taken as the cofactor of WOX11. The PSAT6 protein was used as the control. The LUC vector, which contains six copies of GAL4 binding motif and luciferase coding region, was used as the reporter, and the vector that expressed Renilla luciferase (pTRL) was employed as the internal control. The effectors and cofactors were then co‐transformed with the reporter and the internal control into rice protoplasts.For transactivation activity, the coding sequence of OsEXPB7 expression, the coding sequence of WOX11 was fused with PSAT6 (WOX11‐PSAT6) as the effector, and the promoter sequence of OsEXPB7 was cloned into the LUC vector by replacing the 35S promoter (pEXPB7‐LUC) and acted as the reporter. The above‐mentioned fused protein SLG2‐PSAT6 was used as the cofactor of WOX11, and the PSAT6 empty vector was used as the negative control. As the internal controls, the LUC and pTRL vectors were co‐transformed with the resulting constructs into rice protoplasts. All transformations were performed via the PEG (polyethylene glycol) mediated method. After 16 h incubation at 28 °C in the dark, the protoplast was centrifuged at 150 g and the pellet was used for the dual‐luciferase assay as described in the Dual‐Luciferase® Reporter Assay System Manual (Promega), and the relative LUC/REN ratio was measured with a luminometer . Primers used for these assays are listed in Table To detect the WOX11‐mediated activation of SLG2 was inserted into the pGEX‐4T‐1 vector to express GST‐SLG2, and the coding sequence of WOX11 was inserted into the pMAL‐c5X vector to express MBP‐WOX11. All plasmids were transformed into Escherichia coli strain BL21. Fusion proteins were induced with 1.0 mM IPTG at 37 °C for 5 h. Anti‐GST  and anti‐MBP  antibodies were used in immunoblotting analysis. GST pull‐down assay was performed as previously described , forming pGAD424‐WOX11. To generate pEXPB7‐pHISi reporter vector, the promoter fragment of OsEXPB7 was synthesized and cloned into the pHISi vector. The plasmids were co‐transformed into yeast strain YM4271, and DNA‐protein interactions were determined by the growth of the transformants on the nutrient‐deficient medium with 30 mM 3‐amino‐1,2,4‐triazole (3‐AT), following the manufacturer's manual (Clontech). Primers used in this assay are listed in Table The coding sequence of cis‐element. The biotin‐labelled DNA was synthesized by RuiBiotech . The WOX11‐MBP fusion protein was expressed in Escherichia coli strain BL21 and purified using amylose resin  affinity chromatography. The detailed procedure of EMSA follows the manufacturer's instructions. Photos were taken using Charge‐coupled device (CCD) camera. Primers and probe sequences used for EMSA are listed in Table The LightShift Chemiluminescent EMSA Kit  was used in this experiment. Biotin was labelled at the 5′ end of http://plants.ensembl.org/index.html), and alignments were performed using the online Clustal Omega program  with manual curation.The protein sequence of SLG2 and HSLG2 were downloaded from Ensembl Plants  and starch packaging (b) from the mature grains of WT, slg2, GW8‐KOWT and GW8‐KOslg2.Figure S2slg2 shows normal grain filling.Figure S3 Expression analysis of cell division and cell expansion genes (a) and grain width‐related genes (b) in slg2 and WT.Figure S4 Comparison of agronomic traits between slg2 and WT.Figure S5 The reduced grain width of slg2 is caused by decreased cell expansion.Figure S6 Knockout of SLG2 results in plants with the slg2 mutant phenotype.Figure S7 Overexpression of SLG2 fully rescues the slg2 mutant phenotype.Figure S8 Protein sequence comparison between SLG2 and HSLG2.Figure S9 The SLG2 homologue HSLG2 shows no regulatory roles in rice development.Figure S10 Verification of the WOX11 knockout lines (WOX11‐KO) by PCR‐based sequencing.Figure S11 The WOX11‐KO lines show reduced grain width similar to the slg2 mutant.Figure S12 Verification of the GW8 knockout lines in the background of WT (GW8‐KOWT) and slg2 (GW8‐KOslg2) by PCR‐based sequencing.Figure S13 Statistical analysis of plant height (a), panicle number (b), grain number per main panicle (c), 1000‐grain weight (d), seed setting rate (e), and grain yield per plant (f) of WT, slg2 and GW8‐KO.Figure S14 Expression analysis of cell division and cell expansion genes in WT, slg2, GW8‐KOWT and GW8‐KOslg2.Click here for additional data file.Table S1 Primers used in this study.Table S2 Effects of SLG2 and known grain width genes on four grain traits.Click here for additional data file.


Route A indicated that with WDR4 as the scaffold, METTL1 catalyzed G46 methylation modification after binding of the tRNA variable loop via its αC and α6 helices, as reported by Richard I. Gregory's group. Route B showed the METTL1–WDR4‐mediated tRNA methylation modification model and the effect of binding of the S‐adenosylmethionine on the N‐terminus of METTL1, as reported by Yunsun Nam's group.The molecular mechanism of N Gregory's group revealed mechanism by which METTL1–WDR4 specifically recognized and combined tRNA substrates, emphasized the critical function of METTL1 N‐terminus in tRNA modification, and expounded the effect of METTL1 N‐terminus phosphorylation on methyltransferase activity.Phe and METTL1–WDR4–tRNAVal complexes, the authors observed that the overall structure of both ternary complexes was similar to a sail‐boat‐like arrangement. Furthermore, by superimposing the structures of METTL1–WDR4–tRNAPhe and METTL1–WDR4, the authors discovered that the only region of METTL1–WDR4 that underwent a structural rearrangement after binding to tRNA was the αC loop, which indicated that αC loop formation of αC helix promoted the recognition of tRNA by METTL1–WDR4. In addition, the authors found that WDR4 acted as a scaffold during METTL1–WDR4 binding to tRNAs. B4 of WDR4 not only mediated the interaction with METTL1 but also participated in the binding of tRNAs to B3, suggesting that B3–B4 was essential for the methylation activity of METTL1–WDR4. In the presence of METTL1–WDR4, tRNA would partially unravel and bend to bring its variable loop closer to the METTL1 catalytic pocket, where it would flip its G46 site into the catalytic pocket for methylation modification. In short, using WDR4 as the stable scaffold, METTL1 bound to the tRNA variable loop via αC and α6 helices, catalyzed its binding to the flipped G46 site, thereby achieving m7G46 modification of tRNA  was disordered by expressing isotope‐labeled METTL1. Then, the authors found that residues 18−27 were related to the central region of METTL1 through nuclear magnetic resonance (NMR) titration experiments. Truncation of the METTL1 N‐terminus and mutagenesis of the METTL1 α2 helix showed that residues 16−27 were crucial to METTL1 activity. In addition, after NMR analysis of the S27D mutant at the N‐terminal of METTL1, the authors pointed out that the phosphorylation of S27 would cause steric hindrance and would introduce negative charges to repel tRNA as well as destroy the catalytic center of METTL1, thus inhibiting its methylation activity.Nature by Yunsun Nam's group clarified how METTL1 and WDR4 cooperated with each other to recognize tRNA substrates and catalyze m7G46 modification.Lys and catalyze its methylation modification only when it formed METTL1–WDR4 complex with WDR4. In addition, the authors found that the molecular interactions between WDR4 and METTL1 as well as between S‐adenosylmethionine (SAM) or S‐adenosylhomocysteine (SAH) and METTL1 were critical to the activity of METTL1–WDR4 complexes, through the crystal structure and site‐directed mutagenesis studies of METTL1–SAM, METTL1–SAH, and METTL1–WDR4 complexes. Then, the cryo‐EM structure of METTL1–WDR4–tRNA revealed that METTL1–WDR4 complex specifically bound tRNA through shape and charge complementary form. Meanwhile, by comparing the cryo‐EM structures of METTL1–WDR4–tRNA, METTL1–WDR4–tRNA–SAM, and METTL1–WDR4–tRNA–SAH, the authors observed that the conformation of the methyl donor cofactor SAM binding state was an intermediate state, whereas there was a significant difference in the conformation of the apo and cofactor demethylation product SAH binding states. The C‐terminal helix of WDR4 becomes more stable and orderly in the SAH binding state, which is crucial to the formation of optimal enzyme activity. However, the helix is flexible in the apo and SAM binding states. Besides, the truncation and induced mutation analysis of the C‐terminal helix of WDR4 showed that its truncation and induced mutation would reduce the methylation ability of the complex. Using the structural and biochemical experimental data, the authors found that the truncation of the METTL1 N‐terminus or the phosphorylation of S27 could inhibit the methylation activity of METTL1–WDR4. Moreover, they observed that the N‐terminal of METTL1 was disordered without cofactor binding. However, after binding with SAH, the N‐terminal of METTL1 would cross the narrow channel between SAH and the D arm of tRNA orderly  to gain a deeper understanding of the relationship between the dysregulation of m7G tRNA modification and disease occurrence, further researches should be conducted in animal models to investigate the mechanism of METTL1–WDR4‐mediated m7G tRNA modification in cancer development; (3) based on the new finding that the N‐terminus of METTL1 and the C‐terminus of WDR4 play important roles in the methylation activity of METTL1–WDR4 in this study, it is possible to design and develop METTL1–WDR4 methyltransferase inhibitors targeting these two regions, which will provide new directions for the early diagnosis and treatment of cancer.In summary, two studies have jointly expounded the molecular mechanism of how METTL1 and WDR4 cooperated to recognize tRNA and catalyze mQ. Z. conceptualized the manuscript. Q. W. and Q. Z. wrote the manuscript and prepared the figure, and F. Z. proofread and edited the manuscript. All authors have read and approved the article.The authors declare no conflict of interest.


O‐based directing groups. The logical design of C2‐fluorinated mannose building blocks, and their application in the fully (α‐)stereocontrolled automated assembly of linear and branched fluorinated oligomannosides, is disclosed. This operationally simple strategy can be integrated into existing AGA and post‐AGA protocols to augment the scope of programmed carbohydrate synthesis.Automated glycan assembly (AGA) on solid support has become invaluable in reconciling the biological importance of complex carbohydrates with the persistent challenges associated with reproducible synthesis. Whilst AGA platforms have transformed the construction of many natural sugars, validation in the construction of well‐defined (site‐selectively modified) glycomimetics is in its infancy. Motivated by the importance of fluorination in drug discovery, the biomedical prominence of 2‐fluoro sugars and the remarkable selectivities observed in fluorine‐directed glycosylation, fluorine‐directed automated glycan assembly (FDAGA) is disclosed. This strategy leverages the fluorine atom for stereocontrolled glycosylation on solid support, thereby eliminating the reliance on  3)−F bond at C2 to enable α‐stereocontrolled glycosylation that eliminates the reliance on O‐based directing groups. Reactions display extremely high levels of efficiency and selectivity, and the method has been demonstrated in the synthesis of linear and branched fluorinated oligomannoses.Motivated by the importance of fluorination in drug discovery, fluorine‐directed automated glycan assembly (AFGA) is disclosed. This strategy leverages the C(sp Successful protocols are therefore contingent on being efficient and highly diastereoselective (α/β) to prevent mixtures being amplified across synthesis algorithms  regimes.anti to the C−F bond (low‐lying σC−F*), see top insert Figure As the glycoscience revolution continues to build momentum, the ubiquity and diversity of carbohydrates in all biological kingdoms creates opportunities for creative endeavor in the stereocontrolled synthesis of complex glycans.s Figure A. EmulatN‐glycans, decorate around 50 % of all proteinsN‐glycans on the surfaces of HIV[19F NMR probe,The selection of target structure was informed by the importance of bacterial mannan repeat units, which are structurally distinct from human glycans,es of HIV18 and S1 and 2, bearing orthogonal PGs commonly employed in AGA, were identified as the most promising candidates . This difference in selectivity is a consequence of competing conformational preferences in the transient oxocarbenium ion.[iPrOH), with the characteristic signal of the thiodonor (ca. −190 ppm) remained clearly visible in both scenarios (Scheme 2 until full conversion was achieved in a timeframe that was suitable for AGA (see the Supporting Information for details). Glycosylation of 2 with acceptor 5, under modified conditions, proceeded smoothly to furnish disaccharide 7 with complete α‐selectivity and 99 % yield (Scheme 2S2O3), the generality of the Fmoc/Bn/ester‐design was evaluated. BB 8, to permit subsequent construction of 1,4‐glycosidic linkages, and orthogonally protected BB 9, to enable the construction of 1,3‐ and 1,6‐glycosidic linkages such as those found in N‐glycans, both reacted smoothly with acceptor 6 to furnish the α‐glycosylated products 10 , the all α‐1,6‐linked tetramannoside 13 was selected as a first target structure , NIS (1.5 equiv relative to donor), TfOH (0.4 equiv relative to donor), 0 °C for 5 min, 20 °C for 20 min). Compound 13 was successfully assembled on photocleavable Merrifield resin R1 and the overall efficiency was evaluated by MALDI‐MS and 19F{1H} NMR analysis of the crude glycan mixture after photocleavage using a procedure previously reported by this group.13 as the major reaction product. The satisfactory performance of C2‐fluorinated BB 2 in AGA was also confirmed by 19F{1H} NMR analysis, showing a single resonance of the major fluorinated species  corresponding to 13. Importantly, no resonance was observed in the β‐region (−215 to −225 ppm) of the spectrum, allowing a diastereoselectivity of >88 : 1 to be established based on the 19F{1H} NMR spectrum. Collectively these data indicate that the fluorinated BB 2 and the parent BB 12 show comparable levels of diastereoselectivity and reactivity as both donors and acceptors in AGA. This is an encouraging validation of fluorine‐directed glycosylation on solid support. Similar results were obtained for BB 8 in the construction of tetramannoside 15, thereby extending the generality of the concept to use C2‐fluorinated BBs for fluorine‐directed AGA , the un‐desired β‐diastereomer (around −220 ppm) was never detected, and it was possible to isolate 15 in 58 % yield over ten steps.These conditions were then translated to an automation paradigm using a Glyconeer® 2.1 synthesizer. Following a brief process of optimization to integrate the well‐established, non‐fluorinated BB 16 was synthesized in a single operation. MALDI‐MS and 19F{1H} NMR analysis again revealed the smooth reaction of BB 2, enabling the target trimannoside to be generated in 87 % yield after cleavage from the solid support and with complete (α‐)diastereocontrol.As a proof of concept for the AGA of multiply fluorinated oligosaccharides, homotrimer 13 (from 2+12) in 62 % yield (15.2 mg). NMR structural characterization confirmed the α‐1,6‐connectivity of 13, and subsequent solvolysis under Zemplén conditions  proceeded smoothly to afford semi‐protected tetrasaccharide 17 in 67 % yield  of fluorinated tetramannoside 18 after HPLC purification. These results compare well to the previously reported yields (16 %)15 and 16 , thereby demonstrating the utility of fluorine‐directed glycosylation in the automated preparation of site‐selectively modified glycans.To further demonstrate the synthetic utility of C2‐fluorinated BBs in AGA, standard downstream manipulations  were integrated into the sequence Scheme . Purific% yield 1.2 mg. NMN‐glycans,Having validated C2‐fluorinated building blocks for the synthesis of linear glycans by FDAGA, preliminary studies towards the synthesis of branched bioactive oligomannosides were initiated. Cognizant of the ubiquity of oligomannosides in human10, 152 and 9 in accessing both linear and branched targets by FDAGA, the doubly fluorinated pentamannoside 20 was conceived as a target: this F‐glycan contains the core trimannoside −F bond has been validated as a steering group to regulate the anomeric configuration for the synthesis of linear and branched targets. Through judicious building block design, C‐2 fluorinated mannose modules can be integrated into established automation protocols to expand the scope of this enabling technology. Reactions are characterized by extremely high levels of efficiency and selectivity (>88 : 1 α:β), and a comparative analysis demonstrates that the C−F bond is an excellent substitute for the C−OBz motif (8 and 12) in directing the nucleophile to the α‐face. Given the prominence of fluorine in molecular editing, and the ubiquity of oligomannoses in human biomedicine, it is envisaged that this enabling advance will be effortlessly integrated into automated synthesis algorithms.In addressing the persistent challenge of stereocontrol in automated glycan assembly, the C should be addressed to the authors.Supporting InformationClick here for additional data file.


Whether MSTO2p variant contributes to the observed decrease in MSTO1 levels in patients remains unclear.Gal  The minor allele frequency of the MSTO2p pseudogenic alteration is 0.326 compared to the reported mutation which is a rare variant of MSTO1 with a 0.000202 allele frequency.The correspondence article by Gerber MSTO1 and MSTO2p, and also with the MSTO1 and MSTO2p‐specific primers introduced by Gerber et al  and 2 (II/1) samples with our original primer that does not discriminate between er et al . Using thttps://www.ncbi.nlm.nih.gov/snp/rs11264409) and results by Gerber et al  and 2 (II/1) in our original paper Gal et al (MSTO1 in HeLa cells recapitulated the fusion defects observed in the patient cells (fig 5). Thus, changes in MSTO1 protein levels seem to contribute to the mitochondrial dynamics phenotype of the family.Database information  is decreased in our patients, the diminished band in our western blots is reflective of loss of MSTO1 protein in the patient cells . Based on this, the question arises as to whether the interaction of MSTO2p with another gene affects MSTO1 or mutations in a yet unidentified gene that can regulate MSTO1 levels is responsible for the phenotype in the family we reported previously (Gal et al, The new analysis suggests that in our patients, mutations in MSTO2p mutation contributes or not to the decrease in MSTO1 mRNA and protein abundance identified in our patients. For example, quantification of MSTO1 and MSTO2p mRNA levels using primers specific for each isoform can define if the decrease in MSTO1 mRNA in patient fibroblasts reflects changes in one or both isoforms. Similarly, selective perturbations of MSTO1 and MSTO2p expression using siRNA or CRISPR can determine if MSTO2p might exert an effect on MSTO1 RNA expression. Finally, characterization of the mitochondrial phenotype in patient cells upon decreases of MSTO2p or overexpression of wild‐type or the homozygous mutant will further shed light onto the role of this pseudogene in patients carrying MSTO1 defects.Further studies will be necessary to validate if the homozygous MSTO2p from MSTO1 made it possible to reinterpret our original WES results as causative to the disease phenotypes of our patient population. However, the pathway we described – from decreased MSTO1 to mitochondrial dynamics and function in the patient fibroblasts – remains well supported and, as in our original conclusion, likely contributes to the patients' disease. Effectively, these new data now allows to extend our original results in trying to understand whether and how changes in MSTO2p might contribute to disease phenotypes associated with MSTO1 loss.In summary, recent progress in discriminating of


A single 1-h session (or 7 d of daily 1-h sessions) of noninvasive visual stimulation with LED light flickering at 40 Hz, but not at 20 or 80 Hz, was reported to increase microglial size and decrease amyloid β (Aβ) load in the 5xFAD mouse model of Alzheimer’s disease. To achieve better therapeutic benefits, we explored the effects of daily 1-h sessions of visual stimulation with continuous light or LED light flickering at 24, 40, or 80 Hz for a period of five weeks in 5xFAD mice. As expected, 33-week-old 5xFAD mice but not control wild-type mice of the same age exhibited an abundance of swollen microglia and Aβ plaques in the visual cortex and hippocampus. Unexpectedly, however, compared with similar session of stimulation with continuous light or a light flickering at 24 or 80 Hz, daily sessions of stimulation with LED light flickering at 40 Hz for five weeks failed to further increase the microglial size and could not noticeably decrease the Aβ load in the visual cortex and hippocampus of the 5xFAD mice. In conclusion, contrary to previous findings based on shorter treatment periods, our data showed that daily noninvasive exposure to a light flickering at 40 Hz for a period of five weeks is not effective in reducing Aβ load in the 5xFAD mouse model of Alzheimer’s disease. The recent discovery that a short session of noninvasive visual stimulation with a light flickering at 40 Hz can decrease amyloid β (Aβ) load in a mouse model of Alzheimer’s disease gave hope to the idea that this chronic debilitating disease can be managed by daily sessions of staring at a flickering light. However, we found in this study that daily 1-h sessions involving exposure to a light flickering at 40 Hz for five weeks had no effect on Aβ load in the same mouse model. Our data suggest that visual stimulation alone may not be sufficient for long-term management of Alzheimer’s disease.1–40 in the visual cortex in mouse models of Alzheimer’s disease  disease . Importa for 7 d . In addineration . Likewisneration . Howeverneration .Similar to visual stimulation at 40 Hz, auditory stimulus at 40 Hz has been shown to reduce amyloid load in a mouse model of Alzheimer’s disease, and impressively, combined visual and auditory stimulation at 40 Hz was found to provide more widespread benefit than either visual or auditory stimulation alone . ImportaGiven that video playbacks from cathode ray tube (CRT) TV displays can entrain the primary visual cortices of macaques and humans , we wereMale hemizygous 5xFAD 6799Vas/Mmjax, stock #006554, MMRRC #34 840-JAX; For each 1-h visual stimulation session, a mouse was placed in a box with bedding; the front-facing wall of the box was transparent to allow light input from a 38-bulb LED light source, and the other three walls were black and opaque to minimize diversion of visual attention from the light source. The ceiling was also transparent to allow for video recording and monitoring of mouse behavior during each visual stimulation session. The light source was preprogrammed by an Arduino single-board microcontroller and generated either continuous light or light flickering at 24, 40, or 80 Hz. The frequency of light flickering was confirmed by manually counting flickers from slow-motion video recordings of selected sessions. The 5xFAD mice subjected to visual light stimulation were 28 weeks of age at the first session of visual stimulation and were euthanized at 33 weeks of age after five weeks of daily 1-h visual stimulation sessions.Immunofluorescence staining of microglia and Aβ was performed with primary antibodies against Iba1  and Aβ , respectively, followed by an anti-rabbit secondary antibody , as described previously . The aret test. The effects, or lack thereof, of daily 1-h visual stimulation with continuous light or light flickering at 24, 40, or 80 Hz for five weeks were analyzed by one-way ANOVA.The data are presented as the mean ± SEM. The area occupied by microglia and Aβ plaques in the visual cortex and hippocampus was compared between control C57BL/6 mice and 5xFAD mice by unpaired n = 4 mice per group; p = 0.0235 for the difference in the area occupied by microglia in the visual cortex and p = 0.0104 for the difference in the area occupied by microglia in the hippocampus, unpaired t tests; n = 4 mice per group; p = 0.6999 for the difference in the area occupied by microglia in the visual cortex and p = 0.9194 for the difference in the area occupied by microglia in the hippocampus, one-way ANOVA; A hallmark of Alzheimer’s disease in mice is neuroinflammation associated with enlarged and activated microglia in the brain; notably, it has been reported that visual stimulation by light flickering at 40 Hz can further increase the microglial cell body diameter, indicating enhanced Aβ engulfment, in the visual cortex of 5xFAD mice . To expln = 4 mice per group; p = 0.0118 for the difference in the area occupied by Aβ in the visual cortex and p = 0.0273 for the difference in the area occupied by Aβ in the hippocampus, unpaired t tests; n = 4 mice per group; p = 0.7682 for the difference in the area occupied by Aβ in the visual cortex and p = 0.9415 for the difference in the area occupied by Aβ in the hippocampus, one-way ANOVA; To explore whether chronic exposure to light flickering at 40 Hz can significantly decrease the Aβ load in 5xFAD mice, we immunostained brain slices from the abovementioned mice for Aβ. Consistent with the observation of amyloidosis in mice with Alzheimer’s disease but not in control mice, we found a large amount of Aβ in the visual cortex and hippocampus of 5xFAD mice at 33 weeks of age but not in control C57BL/6 mice of the same age A,B, 4A,BA single session of 1-h noninvasive visual stimulation with light flickering at 40 Hz was shown to be sufficient to increase the average microglia size and decrease the Aβ load in 5xFAD mice, while similar sessions of stimulation with continuous light or light flickering at 20 or 80 Hz was found to have no effect on the Aβ load . TherefoThe explanation for why the effect of light flickering at 40 Hz on microglia and Aβ load in 5xFAD mice could not be replicated by our laboratory and another laboratory may be multifactorial see . In addiIn conclusion, although we cannot rule out the possibility that the failure to replicate the effects of flickering light in 5xFAD mice could be because of the very large variability in Aβ load between individual 5xFAD mice, lack of visual attention to and focus on the light source during treatment sessions, or both, our study highlights the need to further explore this subject and question whether the lack of changes in Aβ load in mice could explain the lack of clinical benefit in early clinical trials. Moreover, in comparison to visual stimulation alone, combined visual and auditory stimulation at 40 Hz have shown greater therapeutic efficacy in mouse models and promising results in a recent phase 2A clinical trial . Therefo


The proposed structure is realized by putting a defective layer of material Al2O3 in the middle of a structure consisting of alternating layers of GaP and SiO2. The transfer matrix method has been employed to examine the transmission characteristics of the proposed defective one-dimensional photonic crystal in addition to MATLAB software. An external application of the hydrostatic pressure on the proposed structure is responsible for the change in the position and intensity of defect mode inside the photonic band gap of the structure due to pressure-dependent refractive index properties of the materials being used in the design of the sructure. Additionally, the dependence of the transmission properties of the structure on other parameters like incident angle and defect layer thickness has also studied. The theoretical obtained numeric values of the quality factor and sensitivity are 17,870 and 72 nm/GPa respectively. These results are enough to support our claim that the present design can be used as an ultra-sensitive pressure sensor.Present research work deals with the extremely sensitive pressure-sensing capabilities of defective one-dimensional photonic crystal structure (GaP/SiO The extensive research work on PhCs has been done so far. The present involvement of PhCs into variety of modern applications makes them suitable to be used as a promising candidate in research fields governing optical, chemical and medical engineering. PhCs have tremendous ability of restricting the propagation of electromagnetic waves of specific frequencies passing through them. This specific frequency range is called as photonic bandgap (PBG)3. The introduction of defects like cavities either by changing the thickness or material of the structure may bring sharp transmission peak inside PBG. Such modified structures are called as defective photonic crystals (DPhCs)4. One of the most suitable application of DPhCs is to use them as a chemical, biofluid or optical sensing structures. The easier fabrication techniques associated with 1d photonic structures allow them to be used as a most effective sensing structures due to their minute sensing and detection capabilities5. Many peculiar applications of 1d DPhCs related to biochemistry6, medical science14, petroleum engineering19, and other branches of sciences28 have been explored by the researchers recently. The photonic biosensors made up of 1d DPhCs have revolutionized the designing, development and testing methodology of biosensors due to significantly improved performance of such structures in terms of sensitivity (S), figure-of-merit (FoM), quality factor (Q) and detection limit (DL)36. Actually, 1d DPhCs based photonic biosensors improve the interaction time between incident photon and the target matter precured inside sensing zone of the structure for accomplishment of high-field localization. The effects of interference, phase and group birefringence associated with the biosensors composed of 2d DPHCs restrict their fabrication whereas the fascinating properties like tunable dispersion and birefringence, homogeneous and isotropic behavior of the structure and the devise compatibility makes the fabrication of 1d DPhCs biosensor easier. Such structures can be easily fabricated by adopting suitable thin film deposition techniques depending upon the choice of the materials used in the design.Pioneering research work on the photonic crystals (PhCs) was first carried out by Yablonovitch and John in 1987. They have articulated their architecture by considering a periodic arrangement of dielectric materials which may be in one-dimensional (1d), two-dimensional (2d), and three-dimensional (3d) as a 1d, 2d and 3d PhCs respectivelyN/Al2O3/(GaP/SiO2)N. The application of stress over the structure disturbs the isotropic and homogeneous nature of the refractive indices of the contitutent materials which induces the ultra high pressure sensing capabilities into 1d DPhC composed of (GaP/SiO2)N/Al2O3/(GaP/SiO2)N. The architectural details along with the theoretical formulation of the structure is discussed in Section “The present research work is based on the photo-elastic phenomenon under the influence of the stress applied over 1d DPhC (GaP/SiO2)2)N/Al2O3/(GaP/SiO2)N as shown in Fig. 2) of period number N on either side of an additional defect layer of alluminium oxide (Al2O3) located at the middle of the structure over glass substarte. The transfer matrix approach connencting the electric and magnetic fileds of electromagnetic wave propagating into the structure near to the interface conneting every two adjesesnt layers of the structure as defined in Eq. (28The transfer matrix method (TMM) has been employed to investigate the theoritical findings of the 1d DPhC composed of  of any nth layer of the structure can be defined asHere M2)N/Al2O3/(GaP/SiO2)N are defined byThe reflection (r) and transmission (t) coefficients of the proposed structure (GaP/SiOR) and transmittance (T) of the structure can be with the help of following relation (The reflectance (relation 4\documen0 = n0 cos(θ0) and ps = ns cos(θs) are admittances of input and output ends of the structure respectively, corresponding to s-polarized wave. For p-polarized wave, input and output admittances would be p0 = cos(θ0)/n0 and ps = cos(θs)/ns respectively. The symbols θ0 and θs are denoting angles of incidence and emergence at input and output ends of the structure respectively.Here p2)N/Al2O3/(GaP/SiO2)N made up of acousto-optic materials can be easily measured due to change in the refractive indices of the constituent materials used in the designing of the present structure. The change in characteristics properties of the acousto-optic materials employed in the proposed pressure sensing design can be observed with the help of stress–strain formulas under the influence of externally applied pressure on the structure. This pressure over the structure is enough to affect the refractive index of the material with stress due to photo-elastic phenomenon which in turn affects the optical performance of the structure. The stress (σ) is like a pressure which determines the force acting per unit area and can be decomposed into nine components forming a secon-rank stress tensor (σij). Here combination of the subscripts i and j is used to represesnt different combinations of x, y and z axes depending upon the direction of the force applied over the respective areas of the structure. The relation between the direction dependent refractive index (nij) and the stress tensor (σij) for a cubic media can be expressed asThe hydrostatic pressure applied externally over the structure  used in the above relation  and strain-optic (pij) constants associated with the materials GaP, SiO2 and Al2O3 are given in Table This section deals with the performance of the suggested pressure sensing photonic structure which is the main objective of the work. The thickness of alternating periodic layers of materials GaP and SiO2O3 of thickness 255 nm which disturbs the periodicity of the structure. Next efforts have been given to analyze the effect of pressure applied externally on the structure. This analysis makes us aware about the pressure sensing capabilities of our structure due to presence of elasto-optic materials in the design.First transmission spectra under normal incidence in absence of an external pressure has been computed by using MATLAB programing in addition to TMM as shown in Fig. 2)N /Al2O3/(GaP/SiO2)N is affacted under the influence of an external pressure which varies from 0 GPa to 2.0 GPa in steps of 0.5 GPa. This purpose is acommplished by applying the pressure 0.5, 1.0, 1.5 and 2.0 GPa seperalely on the structure along the priodicity of the structure and obserbed the respective changes on the transmission spectra of structure as shown in Fig. 2O3 due to elasto-optic effect  of Al2O3 dependent upon external pressure (P) is fitted with following curve fitting asThe linear curve fitting has been applied on to data presented in Table R2 is the corellation factor which high as expected.Here λ) inside PBG of the structure with respect to change in the external pressure (ΔP) applied over the structure. For our structure sensitivity can be defined asNext, efforts have been further extended to examine the pressure sensing capabilities of different pressure sensors of defect layer thicknesses dd = 100 nm, dd = 200 nm, dd = 300 nm and dd = 400 nm, each under the influence of external pressure 0.5 GPa, 1.0 GPa, 1.5 GPa and 2.0 GPa with respect to absence of an external pressure applied over the structures. The purpose of this additional work is to identify the most sensitive structure amongst all the pressure sensing 1d DPhC structure considered in this study. This time we have calculated the sensitivity of each structures of defect layer thickness dd = 100 nm, dd = 200 nm, dd = 300 nm and dd = 400 nm under the influence of external pressure 0.5, 1.0, 1.5 and 2.0 GPa with respect to absence of an external pressure applied over the structures. The sensitivity (S) of the pressure sensor is one of the most significant parameters which determines how minutely and accurately our sensor can sense the change in the defect mode position  from normal incidence (θ0 = 0°) to oblique incidence (θ0 ≠ 0°) and observe the respective change on the senstivity of the 1d DPhC with optimized defect layer thickness dd = 400 nm. For this purpose the transmittance spectra of the photonic pressure sesning structure (GaP/SiO2)N/Al2O3/(GaP/SiO2)N with dd = 400 nm have been recorded at different incident angles θ0 = 0°, 20°, 40° and 60° as shown in Fig. θ0 changes from 0 to 60° respectively under external pressure of 0.5 GPa as evident from Fig. After getting optimized thickness of the defect layer dd = 400 nm to design a pressure sesnor of maximum sesntivity of 50 nm/GPa under normal incidence, we have focused ourself to study the effect of change in angle of incidence N/Al2O3/(GaP/SiO2)N with optimized defect layer thickness dd = 400 nm at θ0 = 60°. This perofrmance evaluation has been done on the basis of three additional parameterts like quality factor (Qf), figure of merit (FoM) and signal-to-noise ratio (SNR) as defined in refences [X–Y]. The numeric values of Qf, FoM and SNR in addition to the sensitivity have been summarized in Table Finally, we have examined the performance of our 1D photonic pressure sesning structure (GaP/SiOFrom the data of Table 2)5/Al2O3/(GaP/SiO2)5 with dd = 400 nm and θ0 = 60° shows extremely high sensitivity value of 72 nm/PGa when the externally applied pressure on the design is 0.5 (GPa). The sensitivity remains almost constant under the application of extremely high pressure of 2.0 GPa which makes our design special and useful as compared to their counterparts.The performance of proposed pressure sensor composed of 1d DPhC has been compared with the similar kind of work carried out by various eminent photonic worker between 2012 and 2020. The comparison based on sensitivity amongst the present and past reported works has been listed in Table In conclusion, we have theoretically examined a very sensitive pressure sensor based on a 1d photonic crystal with a defect. The suggested 1d photonic structure can be used as a sensitive hydrostatic pressure sensor due to their capability of detecting a very little change in the refractive index of the acousto-optic materials used in the design under the influence of an external pressure. In this work effect of various internal and external parameters on the device performance have been studied. Our design possesses the high-quality factor value of 17,870, and maximum sensitivity of 72 nm/GPa under the externally applied pressure of 0.5 GPa. The results of this study can be utilized as a framework of constructing a top-notch pressure sensor.


The X‐ray crystal structure of AbyV has been determined, which in combination with molecular dynamics simulations provides a structural framework for our functional data. This work demonstrates the power of combining selective carbon‐13 labelling with NMR spectroscopy as a sensitive tool to interrogate enzyme‐catalysed reactions in vitro with no need for purification.Abyssomicin C and its atropisomer are potent inhibitors of bacterial folate metabolism. They possess complex polycyclic structures, and their biosynthesis has been shown to involve several unusual enzymatic transformations. Using a combination of synthesis and in vitro assays we reveal that AbyV, a cytochrome P450 enzyme from the  13C labelling strategy was found to be a powerful approach to interrogate the biotransformation and determine the precise function of the enzyme.The cytochrome P450 enzyme AbyV catalyses a key epoxidation in the final stages of the biosynthesis of the spirotetronate antibiotic abyssomicin C. Combining structural and computational data with a  Micromonospora maris  responsible for abyssomicin C production, comprising a multi‐modular polyketide synthase and associated tailoring and regulatory proteins.2 via a stereospecific acetate elimination, catalysed by AbyA5,3 with the spirocyclic framework of abyssomicin C  biosynthesis by inhibiting the enzyme aminodeoxychorismate synthase, with the oxabicyclo[2.2.2]octane core closely resembling a solution state conformation of the enzyme's substrate chorismate.3 to give 4, followed by regioselective attack of the tetronate enol to give the natural product  cells. The resulting protein was purified to homogeneity , giving an orange‐brown enzyme solution that was characterised by UV/Visible spectroscopy. The purified protein showed an absorbance maximum at 420 nm, which upon reduction with sodium dithionite shifted to 410 nm concomitant with a modest reduction in peak intensity. Purging the dithionite reduced solution with CO gave the characteristic FeII‐CO complex absorbance spectrum with a maximum at 450 nm, thus providing physical evidence that AbyV is a cytochrome P450 .The gene encoding AbyV was amplified from 3 was synthesised by adapting the approach described in our earlier studies of the Diels–Alderase, AbyU .Kd) of 3 for AbyV, which was found to be 34±7 μM . Bacterial P450 catalysis relies on electrons from NAD(P)H co‐factors, shuttled to the heme centre by reductase and ferredoxin reductase partner proteins. However, as the native ferredoxin had not been identified in the aby biosynthetic gene cluster (BGC), the widely used heterologous spinach reductase system (FdR/FdX) was selected to reconstitute AbyV activity in vitro.+=347) consistent with the expected addition of one oxygen atom  were undertaken. After 4 hours, LC–MS revealed conversion of the substrate 3 to the previously observed more polar species (tR=9.0), but with no apparent change in the ratio of substrate to product , again with the same mass as the initially observed product ([M+H]+=347). These observations were consistent with an AbyV catalysed oxidation of substrate 3 giving one major product (epoxide 4), and the retention time of the new peak apparent after 4 hours was in accord with abyssomicin C 5 or atrop‐abyssomicin C 6.Spirotetronate m Figure B. In contR=9.0) the mixture was purified by reverse‐phase HPLC . 1H NMR analysis of the purified product  confirmed the presence of epoxide 4 with characteristic signals at δ 3.03 and δ 3.17 assigned to 12‐H and 11‐H respectively. Interestingly, atrop‐abyssomicin C 6 was also evident as a minor component in the sample. This observation was consistent with the oxirane ring in 4 having undergone in situ intramolecular ring opening by attack from the OH of the tetronic acid to form the ether bridge, presumably promoted by formic acid from the HPLC solvent system. This apparent sensitivity to the acidic HPLC solvent would also account for the appearance of the minor product in the 4 hour assay extract . The authors concluded that the product was abyssomicin 2 (9) and that broadening of the signal might be due to a tautomer of 9. Hence, it was proposed that AbmV is responsible for catalysing both epoxidation and subsequent etherification via epoxide opening (Scheme 7 and 3 respectively) is that 7 has a trisubstituted alkene in the 6‐membered ring whereas 3 has the disubstituted 11,12‐alkene. The structural differences in the resultant epoxides (8 from AbmV and 4 from AbyV) could then influence the rate of attack to form the cyclic ethers (9 and 5/6).The LC–MS and NMR data were in accord with the proposal that AbyV catalyses oxidation of alkene 4 was the major product from the AbyV assay  would be well‐resolved .[13C)Alkene 3 was prepared as shown in Scheme 13C)malonic acid 10 as the source of isotopic label. Knoevenagel–Doebner condensation of (2‐13C)malonic acid 10 and acetaldehyde followed by esterification gave ethyl (2‐13C)crotonate 11 in 80 % yield over the 2 steps. The subsequent steps were conducted without purification of the intermediates due to the challenge of working with volatile materials. Thus, sequential reduction of 11 to alcohol 12 using DIBAL‐H followed by oxidation with MnO2 gave (2‐13C)crotonal 13 which was used directly in a Horner–Wadsworth–Emmons (HWE) olefination with triethyl phosphonoacetate to give unsaturated ester (4‐13C)14 in 24 % yield over the three steps. A similar two‐step reduction/oxidation protocol converted ester 14 to (4‐13C)aldehyde 15 in 72 % yield. Freshly prepared (4‐13C)15 was then chain extended via a HWE olefination with phosphonate 17 giving triene (10‐13C)18 in 32 % yield from lactone 16. (10‐13C)18 was then oxidised to the corresponding aldehyde, which was reacted with tetronate 22 in a Dip‐Cl promoted vinylogous aldol reaction. Treatment with methyl iodide and subsequent oxidation with DMP gave linear ketone (10‐13C)20 with the exo‐methylene tetronate ring installed. A biomimetic thermal Diels–Alder reaction converted the linear compound (10‐13C)20 to spirotetronate (12‐13C)21 and finally demethylation with LiCl/DMSO gave (12‐13C)3, the required substrate for the in vitro assays with AbyV.Thus, we turned our attention to a selective carbon‐13 labelling based NMR assay to monitor the catalytic activity of AbyV since this would obviate the need for purification of the product mixture prior to analysis. The ideal site for incorporation of the isotopic label was C‐12 as the chemical shifts of the signals in the ctively).8, 10 1H NMR spectrum of methyl tetronate (12‐13C)21 was in accord with that of unlabelled 21, with characteristic signals assigned to the enone  and 8‐H δ 6.97 ). These signals closely align to the corresponding signals for atrop‐abyssomicin C indicating that the enone in both compounds adopts the same conformation. As expected, the main difference compared with the spectrum of unlabelled material was the signal assigned to 12‐H which showed characteristic 1H‐13C coupling: δ 5.85 . The enhanced signal at δ 136.7 assigned to C‐12 in the 13C NMR spectrum of (12‐13C)21 was accompanied by a minor slightly downfield signal in a ratio of approximately 5 : 1, as observed in previous syntheses.1H NMR spectrum of (12‐13C)3 were broad due to tautomerism of the free tetronic acid. The 13C NMR spectrum revealed a 3 : 1 mixture of major to minor isomers, clearly visible in the enhanced C‐12 signals shown in Figure 13C NMR spectrum was attributed to the presence of atropisomers, as reported for the natural product, and this material was used in in vitro AbyV assays.The tronate 1‐13C21 wa13C)3 with AbyV, spinach ferredoxin‐NADP+ reductase, spinach ferredoxin and NADPH. After two hours the assay mixture was extracted with ethyl acetate and the crude mixture analysed by 13C NMR  and the other at δ 59.0 ppm, in accord with the chemical shift of the C‐12 position of a synthetic sample of epoxide 4 . No trace of abyssomicin C or atrop‐abyssomicin C was detected in the 13C NMR spectrum of the crude mixture .[3 in which the reaction did not reach completion and with epoxide 4 as the major product . To investigate if the AbyV catalysed reaction could be taken to completion, the crude mixture from the initial assay containing both unreacted substrate (12‐13C)3 and epoxide 4 .8, 10 4, the sample of (12‐13C)epoxide 4 obtained from the dual enzyme assays  as the search model (Table S1).All the results described above were reproducible and fully consistent in providing unambiguous evidence to support the proposal that AbyV catalyses solely epoxidation of 3, as calculated using CASTp.Streptomyces laurentii, the multifunctional P450 monooxygenase GfsF from Streptomyces graminofaciens and the filipin tailoring hydroxylase CYP105D6 from Streptomyces avermitilis. Mapping explicitly conserved residues from the ten most structurally related AbyV homologues reveals that the majority of these residues are located in either the conserved heme coordinating loop, common to each of the identified polypeptides, or within tight turns required to achieve appropriate placement of secondary structure elements in the overall protein fold . Our crystal structure provides no evidence for the presence of atypical catalytic features in AbyV such as those reported in other P450 enzymes.Each molecule of AbyV contains a single heme group situated within a large solvent exposed active site pocket of ≈1600 Å3, we used molecular docking to obtain initial positions of 3 near the heme group in chain A of the AbyV crystal structure. We then performed multiple short molecular dynamics simulations for both atropisomers with heme modelled in its compound I state  should be addressed to the authors.Supporting InformationClick here for additional data file.


The water iso-activity lines of these three systems were obtained which have positive deviations from the semi-ideal solutions. This suggests that betaine-polymer and betaine-K3PO4 or betaine-K2HPO4 interactions are unfavorable; and these mixtures may form aqueous two-phase systems (ATPSs) at certain concentrations. Indeed the formation of ATPSs was observed experimentally. Then, osmotic coefficient values were calculated using the obtained water activity data; and, using the polynomial method the solute activity coefficients were determined. Using these activity coefficients, the transfer Gibbs energy (3PO4 and K2HPO4). The obtained positive In this work, to evaluate solute–solute, solute–solvent and phase separation in aqueous systems containing {betaine + poly ethylene glycol dimethyl ether with molar mass 250 g mol To replace or at least reduce the use of hazardous materials with benign ones in production or extraction processes is considered to be a vital goal to sustainable development3. Betaine is known as a non-toxic, biodegradable natural material with the obvious name for 2-(trimethylazaniumyl) acetate which is the derivative of the amino acid glycine. It is extracted as a by-product from sugar beet; and widely used as a feed additive and as an osmolyte4. The osmolyte property of betaine is related to its dipolar zwitterions structure which dissolves easily in water. In addition to the mentioned properties, betaine is a methyl group donor and can be taken part in transemethylation reactions and it acts as an organic osmolyte in protecting cells under stress. Finally, due to the methylating and amino acid properties it participates in energy and protein metabolism5.Recently, betaine as an environmentally safe material has received more attention by some research workers6. Recently, using these kinds of ternary systems partitioning studies has been made on some proteins6. However, further studies regarding the solute–solvent interactions, two-phase formation behavior and partitioning of biomaterials is needed.Design and development of new green processes requires the study of thermodynamic properties of the system. To contribute to a better knowledge of thermodynamic behavior of the system under investigation experimental phase equilibrium data is needed. These data enable us to calculate osmotic and activity coefficients in mixtures which can be used for obtaining some information regarding the solute–solvent interactions and development of reliable thermodynamic models. The obtained results are important in regard with possible two-phase formation of these kinds of ternary systems which may have applications in partitioning and extraction of drugs or proteins−1 (PEGDME250) + water}, {betaine + K3PO4 + water} and {betaine + K2HPO4 + water} systems were measured at 298.15 K to evaluate the possible interactions between these components. There is no report on the water activity or vapor pressure for these systems; and only binodal curves for the two latter systems containing potassium salts and extraction of proteins in the corresponding aqueous two-phase systems have been reported6. In this work, similar vapor–liquid measurements were also carried out on the aqueous binary betaine solutions for which no data is available in open literature. There are several methods for obtaining osmotic coefficient or activity such as electromotive force method, headspace chromatography and vapor pressure osmometry7. In the present study, we have used the isopiestic technique which is an accurate method to determine solvent activity of solutions9. Since, quality of isopiestic measurements depends mainly on the temperature equality between the solutions, keeping the whole system at a constant temperature is required. In addition to reporting some thermodynamic properties of the mentioned mixtures, we attempted to provide solute–solute and solute–solvent interactions by calculating the solute activity coefficients and the transfer Gibbs energy , K2HPO4 (> 99.5%w/w) and NaCl (> 99.5%w/w) were supplied by Merck. All materials were dried in vacuum and kept over P2O5 before used. The water contents of materials were measured by a Karl-Fischer titrator . In preparing the sample solutions, the measured water contents was accounted and double distilled water was applied. The specifications for the used chemicals are listed in Table The betaine was obtained from Jinan Grace Industry (> 98%w/w).The salts K9. In measuring the water activity for binary aqueous betaine solutions, five leg manifold was used in which for each of pure betaine and standard pure NaCl, two flasks are employed and the central flask was considered for water supply. Also, this procedure with a seven leg manifold was employed in the case of water activity measurements for (betaine + PEGDME250 + water), (betaine + K3PO4 + water) and (betaine + K2HPO4 + water). In this case, for each of pure betaine, pure salt (K3PO4/K2HPO4) or polymer one flask was used. Two flasks were poured with standard sodium chloride. The remaining two flasks of the manifold are considered for {betaine + K3PO4/ K2HPO4} or {betaine + PEGDME250}. Before doing anything, it was necessary to degas and remove the air of the solutions which was made by evacuating the apparatus slowly and frequently. To reach the equilibrium between solutions, the apparatus was slowly immersed in a bath for about 120 h. The temperature controller with uncertainty of 0.01K was used. Then, the apparatus was removed from the bath and the mass of each flask and therefore solute mass fractions were determined with an analytical balance with a precision of ± 1 × 10–7 kg. We used the differences between mass fractions of two NaCl solutions as an equilibrium criteria; so that the equilibrium is assumed when this difference is less than 0.1%. In isopiestic equilibrium vapor pressure and water activity in the sample solutions and reference are the same. This enables us to calculate the osmotic coefficient for the reference solution, from which the osmotic coefficient of the each sample in isopiestic equilibrium with the standard NaCl is readily determined. In this work, for the required osmotic coefficient values of the reference solutions the correlation relation suggested by Colin et al.12 was considered.In this study, for obtaining water activity and vapor pressure at 298.15 K the improved isopiestic apparatus was applied250 + water}, {betaine + K3PO4 + water} and {betaine + K2HPO4 + water} solutions at different molalities of betaine as a solute and different molality of {polymer or salt + water} as a solvent  mol kg−1 were prepared in glass vessels and the corresponding density T = 298.15 K and atmospheric pressure For volumetric and ultrasonic studies, {betaine + PEGDME−3 and 0.5 m s−1 were estimated for density and speed of sound, respectively. Before doing anything, it was necessary to calibrate densitometer device; for this purpose dried air and double distilled water were used. The accurate temperature controller built in the apparatus enable us to carry out these measurements within ± 0.01 K which is a required condition for such measurements13.The uncertainty values of 0.15 kg m3PO4/K2HPO4) or PEGDME250, water activity measurements were carried out at 298.15 K using isopiestic method9. Considering equal water activity of standard and sample solutions at isopiestic equilibrium, and relation 14:To study the vapor–liquid equilibrium behavior of binary aqueous solutions of betaine and ternary aqueous betaine solutions containing the salts (Km and −1) of the sample and reference (NaCl) solutions, respectively;3PO4/K2HPO4) or PEGDME250, solutions. The osmotic coefficient values calculated from Eq. , (betaine + K3PO4 + water) and (betaine + K2HPO4 + water), and the corresponding iso-activity lines are shown in Figs. The for binary (betaine + water) solutions, the values of water activity, osmotic coefficient and vapor pressure are collected in Table 250 + H2O)18], (K3PO4 + H2O)19 and (K2HPO4 + H2O)20 make it possible to check the quality of our water activity data for these binary solutions by comparisons of these data, as shown in Figs. The available water activity data for (PEGDME250 + water), (K3PO4 + water) and (K2HPO4 + water) solutions may be used for comparison of the extent of solute–solvent interactions in these binary solutions. For this purpose, the vapor pressure depression values P values reported in Tables The vapor pressure data for binary (betaine + water), (PEGDMEer), K3PO + water 3PO4 + water) are higher than (K2HPO4 + water) at the same solute concentrations. In other words solute–solvent interactions for salts are stronger than that of betaine or polymer. The differences in interactions between these salt systems are related to their anions; so that, a more vapor pressure depression is observed for the salt which has a higher anion charge (K3PO4 ˃ K2HPO4) leading to stronger salt-water interactions. In other words, since vapor pressure depression is a colligative property, its value is increased by increasing the number of moles of ions. The higher 250 may be due to the fact that betaine is a hydrogen-bond accepter and PEGDME250 is more hydrophobic than the betaine. The more negative logarithm of octanol/water partition coefficient value for betaine (log Kow = − 4.6)21 than that the polyethylene glycol (log Kow = − 1.36)22, which has similar structure to PEGDME250, indicates lower affinity of PEGDME250 to water molecules compared to betaine.The plot of calculated vapor pressure depression values for these binary solutions versus molalities of betaine, polymer and salts have been given in Fig. 250 + water), (betaine + K3PO4 + water) and (betaine + K2HPO4 + water) systems may be investigated. The Zdanovskii rule was used for this purpose using (Zdanovskii-Stokes-Robinson) relation at constant water activity24:Based on Figs. 250 or salts and betaine  in the ternary systems, respectively. Similarly, 250 or salts and betaine in the binary solution which has an equal aw. The results of applying Zdanovskii rule23 are also presented in Figs. 3PO4 and K2HPO4) have been observed by Zeng et al.6. Also, it was found that betaine can form an ATPS with polyethylene glycol (PEG)6. These ATPSs have found important applications in protein extraction6. As expected, we observed that betaine can also form an ATPS with PEGDME250 which has similar structure with PEG.In Eq. , \documementclass2pt{minim10 has been frequently used for obtaining relation between activity coefficients of solutes and their molalities in ternary systems26. According to this method, for each component i of the ternary system, the activity coefficient in molality basis,m1 and salts (K3PO4/K2HPO4) or PEGDME250 molality, m2 as follows:The polynomial methodEquation i in aqueous binary solutions which is expressed in terms of its molality, mi, as follows:In Eq.  the term27:Within this thermodynamic framework a quantity with Eq. 8b\docume3PO4/K2HPO4) or PEGDME250.In above relations, 27:Then, using the Gibbs–Duhem relation the Eq.  can be d3PO4/K2HPO4) or PEGDME250. Then, the parameters 3PO4/K2HPO4) or PEGDME250.Using the Eq. 8, the values for 3PO4/K2HPO4) or PEGDME250 at each molality m1 listed respectively in Tables 3PO4/K2HPO4) or PEGDME250, the obtained corresponding osmotic coefficients were correlated with the Eq.  or PEGDME250Then, the following relation is used for calculation of the activity coefficients of betaine, salts (KE denotes the parameters of Eqs. (The symbol  of Eqs.  and 11)E denotes3PO4/K2HPO4) or PEGDME250 solutions, the Gibbs energy of transfer,28. The sigh of the Using the calculated activity coefficients of betaine in binary and ternary salts (K the Eq.  using th3PO4/K2HPO4) and PEGDME250. It is interesting to note that in Sect. 3.1.1 we arrived at the same result regarding these unfavorable interactions by observing the negative deviation of iso-activity lines from semi-ideal solution for the investigated solutions. Table The calculated transfer Gibbs energy values are presented in Table 30. According to this theory, at constant temperature and pressure the Gibbs energies of transfer for betaine from water to aqueous salt or polymer solutions are given by Eq.  are triplet interaction parameters. These interaction parameters were determined by fitting m1 and m2. From the obtained g12 values and the following relation the salting coefficient In Eq. , g12 is g12, found for betaine indicates that pairwise interactions between betaine and salts (K3PO4/K2HPO4) or PEGDME250 are energetically unfavorable. Also the calculated positive salting constant, 3PO4/K2HPO4) or PEGDME250 have the salting-out effect on betaine in these ternary systems. Consequently, the McMillan–Mayer theory also confirms the unfavorable interaction between salts (K3PO4/K2HPO4) or PEGDME250 and betaine that we concluded in Sect. 3.1.1 from the negative deviation of constant water activity lines with respect to semi-ideal solutions, and from the positive sign of Gibbs energies of transfer for betaine from water to aqueous salts (K3PO4/K2HPO4) or PEGDME250 solutions discussed above.The pair and triple interaction parameters obtained from Eq.  were tab250 and salts (K3PO4/K2HPO4) are collected respectively in Tables 250, K3PO4, and K2HPO4 solutions were compared with the corresponding literature values33, which show fairly good agreement. The density data presented in Tables 250 or salt + water} solutions at T = 298.15 K the following equation34 was used:M is the molar mass of betaine and m is its molality; ρ0 and ρ stand for density of the (betaine + H2O) and ternary mixtures (betaine + PEGDME250 + H2O) or [ + H2O], respectively. The obtained 35 were used to evaluate the values for apparent molar volume at infinite dilution The experimental density and speed of sound values for binary aqueous betaine and ternary aqueous betaine solutions containing PEGDME35.The obtained apparent molar volume at infinite dilution The values of 3PO4 is negative the corresponding one for the K2HPO4 is positive. This difference may be due to the more pronounced chemical reaction between betaine and K3PO4 than K2HPO4 which leads to decrease of solute–solute interactions in ternary solutions containing betaine and K3PO4 as indicated with the negative Sv values reported in Tables 3PO4. The betaine is an amphoteric molecule and therefore it can react with the alkaline solution. However, this reaction can be occurred readily with K3PO4 than K2HPO4.The solute- solute interactions in the studied ternary solutions can be deduced from the behavior of apparent molar volumes as plotted against betaine molality in Figures trVφ0) is another important thermodynamic property that can be used for investigating the solute–solvent interactions. Here, the ΔtrVφ0 is the difference between 3PO4, K2HPO4 or PEGDME250 and binary aqueous betaine solutions as follows:The standard partial molar volumes of transfer  polymer or salts were calculated and tabulated in Table 37.The apparent molar isentropic compressibility 38 was used for calculation of the required isentropic compressibility κs of mixture:The Laplace – Newton's equationu and density ρ, the measured values presented in Tables For speed of sound κs0 is the isentropic compressibility of pure solvent. The measured speed of sound data obtained for (PEGDME250 + H2O), (K3PO4 + H2O) and (K2HPO4 + H2O) may be compared with literature data as Figures 33. The calculated κφ values reported in Tables κφ0 was also determined from fitting of κφ values to the following relation38:κφ0 and  σ (κφ0) obtained from least square fitting of κφ values to betaine molality m are reported in Table κφ values against betaine molality shown in Figures κφ values were satisfactorily correlated with Eq. 21.In Fig. κφ0 values were plotted against concentration of polymer or salts for different studied systems. It is seen from Table κφ0 values for the studied systems decrease with increasing concentration of betaine. Also these values decrease with increase of polymer or salts concentration. The trend observed in variation of κφ values again implies that while there is a strong interaction between polymer or salts and water, the betaine-polymer and betaine-salts interactions are unfavorable. These results are consistent with the ones we deduced from volumetric studies regarding solute–solvent and solute–solute interactions.The apparent molar isentropic compressibility at infinite dilution trkφ0) of betaine from water to aqueous PEGDME250 or phosphate salts solutions at infinite dilution were calculated by the help of the following relation:The transfer molar isotropic compressibility (Δ250, K3PO4, and K2HPO4 solutions, the 32. The corresponding trkφ0 values reported in Table For binary aqueous PEGDME250, K3PO4 or K2HPO4 in aqueous media, water activity measurements were made on the aqueous systems composed of {betaine + PEGDME250}, {betaine + K3PO4} and {betaine + K2HPO4} at T = 298.15 K and atmospheric pressure by the isopiestic method. The experimental water iso-activity lines showed that these systems have negative deviation from the semi-ideal solutions implying unfavorable interactions between betaine and polymer or salts. This means that these ternary solutions have tendency to form two-phase systems, which was confirmed experimentally. From thermodynamic treatment of water activity data, activity coefficients values for betaine, PEGDME250, K3PO4 and K2HPO4 in binary and ternary solutions were determined and these values were used to calculate transfer Gibbs energies from binary to ternary solutions. The positive transfer Gibbs energy values obtained for all the studied systems confirm the unfavorable interaction between betaine and PEGDME250, K3PO4 or K2HPO4. We found further evidence for these interactions by studying volumetric and acoustic properties of the systems by measuring density and speed of sound values. It was found that both of the transfer molar volume (In this work, to investigate a possible interactions between betaine and PEGDMESupplementary Information.


It is demonstrated that structural and magnetic transitions are strongly coupled by the presence or absence of the magnetic quasi-atomic IAEs and non-magnetic hydrogen anions in the interlayer space, which dominate exchange interactions between out-of-plane Gd−Gd atoms. Furthermore, the magnetic quasi-atomic IAEs are inherently conserved by the hydrogen desorption from the P1m structured Gd2CHy, restoring the original ferromagnetic state of the Rm structured [Gd2C]2+·2e− electride. This variable density of magnetic quasi-atomic IAEs enables the quantum manipulation of floating electron phases on the electride surface.In electrides, interstitial anionic electrons (IAEs) in the quantized energy levels at cavities of positively charged lattice framework possess their own magnetic moment and interact with each or surrounding cations, behaving as quasi-atoms and inducing diverse magnetism. Here, we report the reversible structural and magnetic transitions by the substitution of the quasi-atomic IAEs in the ferromagnetic two-dimensional [Gd 2C]2+ 2e− electride to canted antiferromagnet by reducing and restoring IQEs.Interstitial quasi-atomic electrons (IQE) have shown promising magnetic elements. Here authors present the reversible hydrogenation and dehydrogenation of ferromagnetic [Gd Benefited from the intriguing nature of IAEs, exotic physical and chemical properties of electrides, such as low work function4, high electronic mobility5, excellent electron reservoir7, efficient catalytic activity9, and quantum properties of magnetism11, superconductivity12, and topology13, have attracted considerable interests in both fundamental science and practical applications. This triggers the exploratory research for the discovery of a uncharted class of electrides over the past decades, leading to the success in finding several two-dimensional (2D) electrides; [Ca2N]+·e− and [Re2C]2+·2e−  with IAEs at interlayers14 and van der Waals [ReCl]2+·2e− (Re = Y and La) with IAEs at intralayers17. Emergent quantum properties of such 2D electrides are governed by the localization degree of IAEs and their hybridization with neighboring cations. In particular, topological Weyl and ferromagnetic states in [Gd2C]2+·2e− electride with strong hybridization of IAEs and Gd cations are of great interest in the field of quantum materials18.Interstitial anionic electrons (IAEs) construct an ionic crystal as an essential ingredient together with positively charged lattice framework, forming an electride, which is distinguished from conventional ionic compounds with defect color centers trapping electrons17. For example, the IAEs in the [Y2C]2+·2e− play as ferromagnetic particles in the lattice framework composed of only paramagnetic elements, exhibiting the superparamagnetism10. The [Gd2C]2+·2e− was found to be the room-temperature ferromagnetic electride with TC of 350 K due to the exchange interactions of interlayer Gd cations across IAEs11. Importantly, the IAEs in [Gd2C]2+·2e− are considered as magnetic quasi-atomic electrons with substantive magnetic moments that are responsible for the occurrence of ferromagnetism from the antiferromagnetic [Gd2C]2+ lattice framework. The concept of interstitial quasi-atomic electrons (IQEs), suggested by Miao and Hoffmann, has been esteemed to understand the nature of elemental and compound electrides on the basis of theoretical ground19. Indeed, potassium electride under high pressure is stabilized by ferromagnetic ordering of the IQEs20. Furthermore, the mixed-cation [YGdC]2+·2e− electride exhibited the ferrimagnetic state, which was attributed to the direct exchange interactions between magnetic IQEs at different crystallographic positions21. In addition to the magnetic ordering of IAEs in the electrides, the IAEs on the cleaved surface of 2D [Gd2C]2+·2e− electride are found to be spin-polarized Fermi liquid and crystallized into the hexatic phase by decreasing their density on the surface22.Meanwhile, the magnetism of 2D electrides originates from the existence of strongly localized IAEs at interlayer space, which have own magnetic moments and facilitate the magnetic interaction with neighboring cations23, indicating that the presence of the IQEs can provide a freedom to tune the magnetic properties and study their role in magnetic phase transitions of electrides. In the previous report11, the substitution of chlorine atoms for the IAEs in ferromagnetic [Gd2C]2+·2e− electride resulted in the transition to antiferromagnetic Gd2CCl and proved the presence of magnetic IQEs. On the other hand, hydrogen-substituted electrides, in which hydrogens absorb the IQEs and form the hydrogen anions, have been examined to find the crystallographic positions of IQEs and elucidate their contribution to the electronic density of state25. Recent computational studies suggested that the hydrogenation for the monolayers of 2D [Ca2N]+·e− and [Gd2C]2+·2e− electrides significantly altered their magnetic properties27. However, an experimental investigation of hydrogenation of the magnetic 2D electrides has been hardly ever reported in spite of a possibility to identify the critical role of magnetic IQEs for triggering the magnetism and tuning the magnetic properties. This might come from the experimental difficulty in handling the chemically unstable electrides as well as a common expectation for the hydrogen-induced embrittlement.The IQEs in the magnetic electrides can be regarded as analogous to the substituted or doped magnetic elements in typical magnetic alloys2C]2+·2e− electride, which simultaneously induced the magnetic and structural phase transitions. Depending on the relative concentration between IQEs and hydrogen anions, crystal structure, and magnetic phase exhibited the strongly coupled reversible transitions between the ferromagnetic layered structure of the Rm space group to the canted antiferromagnetic layered structure of the P1m space group, providing an experimental proof on the magnetic nature of IQEs.Here, we report the hydrogenation and dehydrogenation of the ferromagnetic [Gd2C]2+·2e− electride is crystalized in anti-CdCl2-type layered structure belonging to the rhombohedral Rm space group, where the IQEs are occupying the interlayer space  space group due to the penetration of excess hydrogens into the positively charged [Ca2N]+ layers28. On the contrary, hydrogens only occupied the interlayer space between positively charged [Y2C]2+ layers, leading to the structural phase transition to the different layered structures of Pm1 and P1m30 depending on the hydrogen concentration at octahedral and tetrahedral sites as shown in Fig. The pristine 2+·2e− electride .Pristine GdC2+·2e− eide Fig. . On the ure Fig. . The XRD2C]2+·2e− electride, yielding the P1m structure of Y2CH2.55, where 2 moles of hydrogen occupy all tetrahedral sites and additional 0.55 moles of hydrogen occupy the octahedral sites, as confirmed from neutron diffraction (ND) study30. Our total energy calculations on the Gd2CH3 compound, which showed the full occupancy at both octahedral and tetrahedral sites, is consistent to the P1m structure of Y2CH2.55  for the sample hydrogenated at 600 K  decreased with the decrease in temperature, showing the same metallic behavior. The increased R/R400K and enhanced electron-phonon scattering in the hydrogenated Gd2CHx (x ≤ 1.0) imply the reduced concentration of the IQEs  showed the typical behavior of semiconductors, indicating that most IQEs are substituted by the hydrogens and metal-insulating transition occurs according to their concentration.The IAEs have played a key role in governing the electronic and magnetic behavior of the [Gdχ) for the pristine [Gd2C]2+·2e− electride and its hydrogenated samples. Compared to the ferromagnetic transition temperature (TC) of 350 K of the [Gd2C]2+·2e− electride, the hydrogenated Gd2CHx (x ≤ 1.0) sample exhibits the decreased TC of 220 K, which was determined from the temperature dependence of dM/dT curve  sample. This hydrogenated Gd2CHx (x ≤ 1.0) is clearly distinguished from the antiferromagnetic Gd2CCl, where the Cl anions occupy the only octahedral sites and lead to the substantial increase of the interlayer distance (0.414 nm) that is even larger than that (0.386 nm) of the [Ca2N]+·e− electride with fully delocalized IAEs  of the pristine [Gd2C]2+·2e− electride11, probably resulting in much stronger localization of the remained IAEs at both octahedral and tetrahedral sites and reinforcing the ferromagnetic Gd-IQEs-Gd exchange interaction in the hydrogenated Gd2CHx (x ≤ 1.0) sample. This ascribes to the enhanced χ and the random distribution of the IAEs at the octahedral sites may induce the bifurcation between ZFC and FC curves. Meanwhile, the role of hydrogen anions at the octahedral sites in the Gd2CH compound is worthy to be further investigated whether the hydrogen anions between 2D Gd arrays can impart a ferromagnetic exchange interaction.A profound effect of hydrogenation was found in the magnetic properties. Figure M−T curve in the P1m structured Gd2CHy (y > 2.0) exhibits a typical feature of antiferromagnetic materials with ZFC downturn behavior. The isothermal magnetization (M−H curve) was also measured at 2 K. No hysteresis loop was observed in every sample, discarding a hard ferromagnetic ground state. Although the M−H curves exhibited the saturation of M at small H in the Rm structured [Gd2C]2+·2e− electride and hydrogenated Gd2CHx (x ≤ 1.0) sample, the P1m structured Gd2CHy (y > 2.0) showed no saturation even at 5 T, suggesting an antiferromagnetic ground state. In the Curie-Weiss (CW) analysis on the inverse susceptibility curve (χ−1 vs. T) for all the samples  sample followed the CW fit. However, a slight deviation from the linear fit was observed in the hydrogenated Gd2CHy (y > 2.0) sample, suggesting the antiferromagnetic nature.Importantly, it should be noted that the magnetic transition occurs by the substitution of most IQEs with hydrogens. The 2CHy (y > 2.0), we further examined AC susceptibility. Figure HAC with different frequencies. Both the real part (χ’) and imaginary part (χ”) extracted from the AC susceptibility of ferromagnetic [Gd2C]2+·2e− and hydrogenated Gd2CHx (x ≤ 1.0) showed the constant temperature of peak position  regardless of frequency  showed a systematic shift towards higher temperatures with the increase in frequency .To clearly understand the antiferromagnetic properties of the hydrogenated Gdncy Fig. . On the ncy Fig. . This in2CCl, where the IQEs are substituted by Cl atoms and the 2D array of gadolinium cations was responsible for the magnetism without splitting between ZFC and FC curves11, the Gd2CHy (y > 2.0) demonstrates non-zero net magnetization and weak hysteresis in M−H curve , where the XY model is not available below the TN . Dehydrogenation was conducted by heating the hydrogenated Gd2CHy (y ≥ 2.0) under a vacuum of 10−5 Torr. Figure M−T curves of the dehydrogenated samples of Gd2CHy (y > 2.0) at different temperatures together with the [Gd2C]2+·2e− (gray) and its hydrogenated samples (blue and red). The decreased magnetic transition temperature upon the hydrogenation, from TC ~ 350 K of the [Gd2C]2+·2e− electride to TN ~ 20 K of the hydrogenated Gd2CHy (y > 2.0), is perfectly reversed by the dehydrogenation, which increases the magnetic transition temperatures with the increase in dehydrogenation temperature. Notably, the magnetic transition temperature increases up to TC ~ 350 K, which is exactly same as that of the pristine [Gd2C]2+·2e− electride. It is also noted that the dehydrogenated samples show the magnetic transition from antiferromagnetism (dehydrogenated at 500 K and 800 K) to ferromagnetism (dehydrogenated at 1000 K and 1500 K) with proceeding the dehydrogenation. This is the reverse of the hydrogenation of the [Gd2C]2+·2e− electride accompanied by the coupled structural and magnetic phase transitions. Indeed, it was confirmed from the XRD measurements of dehydrogenated samples  and its dehydrogenated sample at 500 K to Rm structure of dehydrogenated samples at 800 K, 1100 K, and 1300 K. Most of all, the TC as well as M−T curve of the dehydrogenated sample at 1300 K indicate that the [Gd2C]2+·2e− electride is re-formed by the re-generated magnetic IQEs at the original Wyckoff positions, re-inducing the ferromagnetic Gd−IQEs−Gd exchange interactions  and dehydrogenated samples at 500 K, 800 K, and 1100 K are also maintained by the coexistence of IQEs and hydrogen anions in the positively charged [Gd2C]2+ layered lattice framework. These results indicate that the continuous substitution between IQEs with hydrogens occurs during the both processes. Although the reaction of IQEs with hydrogens, which produces the hydrogen anions in the hydrogenated samples, can be reasonably expected, the conservation of IQEs by the desorption of hydrogens in the dehydrogenated samples is, to the best of our knowledge, an unreported phenomenon when considering that the formation of vacancy at the site of hydrogen ions is a general feature in the dehydrogenated materials33. Many theoretical studies have explored the nature of electride by the substitution of IQEs with hydrogen anions and experimental results have been reported to provide evidence on the existence of the IQEs in the electrides35. However, to our best knowledge, the dehydrogenation producing the electride has never been reported yet. Indeed, proof-of-demonstrations on the magnetic IQEs of the electrides have been rare in experiments. In contrary to the well-known intercalation and deintercalation processes of elements in 2D materials37, the reversible transitions depending on the stoichiometric balance between IQEs and hydrogen anions strongly indicates that the IAEs at a specific Wyckoff position behave as quasi-atoms to keep the charge neutrality and to ensure the thermodynamic and electronic stability of the electrides38. In particular, because the continuous change in magnetic transition temperatures is exclusively ascribed to the relative concentration of magnetic IQEs and hydrogens, the nature of IAEs can be regarded as magnetic IQEs, allowing the freedom to tune the magnetic properties of electrides.It is worthwhile to consider that the magnetic IQEs are inherently conserved from the dehydrogenation of the hydrogenated electride. When both processes of hydrogenation and dehydrogenation were completed, the positively charged 2+·2e− and hydrogenated Gd2CHx (x ≤ 1.0), the δ values of 3.3 and 4.9 are of ferromagnetic systems. Furthermore, the temperature dependence of the ΔSM from the following equation,SM showed a broad maximum around TC for the ferromagnetic [Gd2C]2+·2e− and Gd2CHx (x ≤ 1.0), whereas the canted antiferromagnetic Gd2CHy (y > 2.0) exhibited a sharp increase around TN. The maximum entropy change at TC was found to be as high as 17.3 J·kg−1K−1 for the Gd2CHy (y > 2.0), which is almost three times higher than those of [Gd2C]2+·2e− and Gd2CHx (x ≤ 1.0) samples, indicating that a weaker exchange interaction is present for the hydrogenated Gd2CHy (y > 2.0). In addition, the power-law fitting of −ΔSM ∝ Hn  with n greater than 1.0 follows the antiferromagnetic behavior, which is clearly distinguished from the ferromagnetic [Gd2C]2+·2e− and Gd2CHx (x ≤ 1.0). Besides of the identification of the change in the magnetism according to the concentration of magnetic IQEs, the magnetic properties such as relative cooling power (RCP) can be also controlled by the magnetic IQEs. We calculated the RCP with the equation of |ΔSMmax| × δTFWHM  sample is quite larger (~350 J·kg−1 at 5 T) than those of the [Gd2C]2+·2e− and Gd2CHx (x ≤ 1.0) samples, showing a comparable capability to that of magnetocaloric AlFe2B, Gd5Si4 and Gd2FeAlO6 materials45. These exotic properties based on the magnetic IQEs can provide a possibility to explore a refrigerant from electride material.A magnetocaloric effect (MCE) can also provide an insight into the nature of magnetic phase transition and the existence of magnetic IQEs, which are related to the difference in the degree of freedom of spin alignment. The MCE of a system with a weaker exchange interaction shows a larger magnetic entropy , which perfectly conserved the IQEs. The structural phase transition between higher symmetric Rm structure of the [Gd2C]2+·2e− electride and lower symmetric P1m structure of the hydrogenated Gd2CHy (y > 2.0) is accompanied with the magnetic phase transition in a wide temperature range between ferromagnetism at the TC of 350 K to canted antiferromagnetism at the TN of 20 K. The reversible magnetic transition is governed by the spin exchange interactions in the out-of-plane Gd–Gd atoms, which are mediated across the magnetic IQEs or non-magnetic hydrogen anions. Our results clearly identified the nature of magnetic IQEs and proved their critical role in tuning the magnetic properties, providing the IQEs as a unique ingredient in magnetic materials. These IQEs, which can have interactions with each other or surrounding cations, can thus trigger antiferromagnet, ferromagnet, or permanent magnet, all the magnetism. Finally, the reversible substitution and conservation between magnetic IQEs and hydrogen anions can provide a possible platform to study the exotic magnetic state of quantum electron phases such as Wigner crystal on the electrides46.In summary, we explored the non-magnetic hydrogen substitution for the magnetic IQEs in the 2D [Gd2C]2+·2e− electrides is performed by the arc-melting process with mixed Gd metal pieces and graphite pieces in a 2:1 molar ratio under high-purity Ar atmosphere. Before synthesizing the hydrogenated sample, we pulverized the polycrystalline [Gd2C]2+·2e− electrides to powder and pelletize for handling. As displayed in Supplementary Fig. 2 mixed gas. To synthesize the Gd2CHy (y > 2.0) composition, pelletized [Gd2C]2+·2e− electrides were heat treatment of around 600 K under a 1 atm environment. The Gd2CHx (x ≤ 1.0) was synthesized around 1000 K and under 10−1 Torr pressure, which is created by flowing the H2-mixed gas and vacuum pumping at the same time. The dehydrogenation process was performed by pelletized Gd2CHy (y > 2.0) under 10−5 Torr by vacuum pumping with different temperatures.All samples were handled in glove boxes filled with high-purity argon gas (Ar 99.999%) to prevent the oxidation of raw materials and synthesized samples. The synthesis method of a polycrystalline ingot of GdC2+·2e− e2C]2+·2e− electride, hydrogenated samples (Gd2CHx (x ≤ 1.0) and Gd2CHy (y > 2.0)), and dehydrogenated samples were investigated by XRD using a Rigaku SmartLab diffractometer with monochromatic Cu Kα radiation (8.04 keV) at room temperature. The well-ground powder samples were prepared in glove boxes and measured in a plastic dome-type stage filled with Ar gas to avoid oxidation during measurements. A high resolution neutron powder diffraction of hydrogenated Y2CHy (y > 2.0) sample was measured at the HANARO, a research reactor of the Korea Atomic Energy Research Institute. The wavelength of the neutron beam of HRPD is λ = 0.1834528 nm, and the measurement error of the lattice change rate using this beam is about ± 0.004%. General Structure Analysis System (GSAS) software package was applied to perform Rietveld refinement.The crystal structure of the [GdThe sampling for resistivity and magnetic properties measurements were performed in the high-purity Ar-filled glove boxes. The temperature-dependent resistivity measurements were performed by the physical property measurement system . The four-electrode is made by silver epoxy on the pelletized samples. After that, samples were covered with Apiezon N grease to block the oxidation during the sample loading to PPMS and measurements. The measurement of magnetic properties used a vibrating sample magnetometer  and Squid magnetometer  for AC magnetic susceptibility. A plastic capsule copula containing a weighted sample was coated with N grease to prevent the oxidation of samples.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


In Jinyou Li et al.The exosome identification result of A549 cell line in Figure 2a was misused which was the 80 000x result of A549/PTX cell line . The correct one should be the A549 cell line 100 000x exosome identification result .FIGURE A Correct A549 cell line exosome electron microscope result(100 000x).FIGURE B The article is now regarded as the electron microscopy results of the A549/PTX cell line misused by A549(80 000x). FIGURE C Electron microscopy results of the A549/PTX cell line correctly used in the current article(100 000x).The authors apologize for the errors and any inconvenience they may have caused.