Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique  Rif1, originally reported as a telomere-binding factor in yeasts, is evolutionally conserved and regulates chromosome transactions including DNA replication, DNA repair, transcriptional silencing, and telomere homeostasis. We and other groups previously reported that Rif1 plays a crucial role in organization of DNA replication timing during S phase in fission yeast  and in mZscan4 (zinc finger and SCAN domain containing 4), previously identified as a gene expressed in late 2-cell zygote in vivo, has been also reported to be activated in ES cells in transient and sporadic manner in a rare population  and noncoding RNA of major satellite, all of which is specifically transcribed in embryos at two-cell (2C) stage (Zscan4 and ERVs expression possibly through the maintenance of histone H3 lysine 9 trimethylation (H3K9me3), H3K27 trimethylation (H3K27me3), and DNA methylation . NotablC) stage . This ceC) stage , but we C) stage . FollowiC) stage , 16, 17.hylation , 19, althylation . Thus, tMERVL in ES cells, N-terminal HEAT-repeat of Rif1 is required . Rif1 is more highly expressed in ES cells (>100-fold) than in fibroblasts, as previously reported  or induced differentiation by embryoid body formation, concomitant with the loss of Oct4 expression  in ZHBTc4 cells  and its overexpression increased Rif1 protein by 1.7-fold in 3 days , showing that Rif1 expression is correlated with the Oct4 protein level and undifferentiated states.The expression levels of DNA replication-regulatory proteins vary widely among cell types. In pluripotent ES cells, prereplicative complex components, Cdc6 and Orc2, are abundantly present compared with embryonic fibroblast cells, while DNA replication sliding clamp PCNA is at similar levels in both cells A. Rif1 iFig. 1A; , 18). RiFig. 1C; ). Since Fig. 1C; , 27, in c4 cells . As expeF). As a result, most population of cells retained alkali phosphatase activities with minor changes in ES cell-like morphology, showing that Rif1 knockdown (KD) has marginal effects on the maintenance of undifferentiated states (G and H). The mRNA and protein levels of Oct4 and Nanog along with other pluripotent markers were not markedly reduced by Rif1 KD (F and S1A). To achieve complete depletion of Rif1, we generated Rif1 conditional knockout (KO) ES cell lines (mRif1flox/floxCreERT2) and confirmed that the levels of pluripotent marker transcripts were not reduced by Rif1 KO, except for moderate upregulation of Nanog (approximately 1.8-fold) (B). In addition, the major differentiation markers were unchanged by Rif1 KO, although some genes were dysregulated (B). On the basis of these data, we have concluded that Rif1 is dispensable for maintenance of undifferentiated state of ES cells.Meanwhile, the effects of Rif1 depletion on undifferentiated states of ES cells have been controversial , 26, 29,Fig. 1 Rif1 KD F and S1A.8-fold) B. In addegulated B. On the22-fold, 1511 entities; Fig. 2A), and 43 out of the top 100 transcripts belong to a group of family genes that are specifically expressed in embryos at 2C stage and are sporadically and periodically activated in a limited population in ES cells  along with moderate dysregulation of differentiation-related genes . In addition, the transcripts of endogenous retrovirus MERVL and noncoding RNA of major satellite repeats were also extensively induced in Rif1 KO ES cells, as seen in 2C embryos and naturally occurring Zscan4-positive state cells . We cloned full-length Rif1 cDNA and established stable transformant cell lines expressing ectopic Rif1 for complementation assays. Rif1 siRNA-mediated derepression of Zscan4, Usl17l, AA684185, and MERVL was rescued by exogenous Rif1 expression in two independent stable clones . Taken together, our results firmly establish that Rif1 inhibits the dysregulated transcription of 2C genes in ES cells.To evaluate the effects of mRif1 depletion, we conducted gene expression analysis using whole mouse genome microarray. While pluripotent markers were unchanged, a fraction of genes were upregulated in Rif1 KD cells  with the following three features; (1) All of the nine genes are transcribed in ES cells and possibly express proteins (C); (3) Various endogenous retrotransposable elements and their remnants are frequently present within this gene cluster.  by Rif1 knockdown (D). In contrast, the transcripts for the non-Zscan4 family gene (Gm7789) localized between Zscan4b and Zscan4c were moderately upregulated by Rif1 depletion (3.4-fold). This indicates that Rif1 depletion may suppress the transcription of the whole Zscan4 gene cluster region but that it affects the Zscan4 promoter more profoundly. Next, we asked whether the regulatory sequence of each gene is sufficient for repression of the Zscan4 gene by Rif1. To answer this, we used 2 cell lines; Zscan4-Emerald GFP (EmGFP) transgene MC1 cells carry EmGFP fused to the 2.6 kb upstream sequence of the Zscan4c gene integrated in its genome . EmGFP-positive population increased by 6.5-fold in Zsc4-EmGFP Tg cells, as was reported previously . Not only EmGFP-positive (+) population but also the fluorescent signal intensities increased by Rif1 depletion in both reporter cells (A), suggesting that both the probability and extent of promoter regulation are affected (B–F). The results indicate that the upstream regulatory region of Zscan4 contains information sufficient for repression by Rif1, but some other genetic elements present in the gene cluster may also contribute to the repression.Zscan4 is required for maintenance of telomere functions in ES cells and in embryos and is essential for development . Nine Zsmosome 7 D and S1Cproteins , 30; (2)proteins C; (3) Vaig. S1C; ). To invnockdown D. In conene Gm778 localizeI cells; ) carry Eene loci E. EmGFP-F, left; ), while er cells A, suggesaffected , B–F. ThMERVL, a 2C-specific endogenous retrovirus (ERV) transcript, was also extensively upregulated in Rif1 depleted cells  as in intrinsic Zscan4-positive cells. It has been suggested that DNA methylation regulates certain ERV expression (Zscan4e gene by HpaII assay (A). We found similar levels of methylation in both untreated and Rif1-depleted ES cells, although sensitivity to HpaII (incapable of cleaving CpG methylated CCGG) slightly increased in the Rif1 knockdown cells (A). We further conducted bisulfite sequencing of four targeting sites in the Zscan4 gene loci; two upstream regions (BS1 and BS2), transcription start site , and gene body (BS4) (A). The upstream regions and gene body are highly methylated  as previously reported (B and C). When depleted of Rif1, DNA methylation was moderately reduced at BS1 (4.5 kb upstream of Zscan4c) from 81% to 62% [×0.77] or at BS3 (TSS) from 63% to 35% [×0.57], but less affected at BS4  (B and C). Next, we examined BS2, targeting the 3.8 kb upstream of Zscan4b or Zscan4e, and the 3.5 kb upstream of the Zscan4d gene where MERVL is inserted in head-to-head direction, to confirm whether the adjacent MERVL influences the DNA methylation of the Zscan4d locus. To do this, analyzed sequences were identified either as Zscan4b/e-derived or Zscan4d-derived on the basis of single nucleotide polymorphism information in NCBI database. Although the MERVL near Zscan4d was actively transcribed in Rif1 depleted cells  (B and C). We concluded that genomic DNA methylation of the Zscan4 gene cluster region is largely maintained at a high level in mRif1-depleted ES cells, regardless of the presence or absence of adjacent MERVL.pression , thus weII assay A. We fouwn cells A. We furdy (BS4) A. The upreported , while Treported , B and C[×0.87]) , B and Ced cells , DNA met[×0.91]) , B and CZscan4 gene region (D). Active marks, H3K4me3 and H3K9/K14ac, were low in untreated ES cells and unexpectedly, did not change by Rif1 knockdown . In contrast, H3K27 acetylation (H3K27ac), generally known as an enhancer mark, significantly increased in the all tested sites including the ones at TSS, in the gene bodies and the surrounding regions . The broadly spreading H3K27ac mark is reminiscent of “super-enhancers,” which is characterized by colocalization of another enhancer mark H3K4me1 and binding of pluripotent transcription factors and mediators , unlike in super-enhancers. We then asked whether the prominent increase of H3K27ac was caused by removal of H3K27 silent marks, but the Zscan4 loci had no H3K27me3 mark in untreated cells, and Rif1 depletion had no effects . Furthermore, another silent mark H3K9me3 was moderately present in untreated cells and was reduced in Rif1 knockdown cells  but was clearly remained (at the levels higher than in Nanog locus and lower than in differentiation marker locus of Sox17 or T). Similar results were obtained also in Rif1 KO ES cells derived from the different mouse strain (B). Other marks of histone H4 including H4K5ac, H4K8ac, H4K5/K8/K12ac, and H4K20me1 were also unchanged at most of the locations examined, except for moderate increase of H4K16ac in Rif1 knockout cells (F and S3C). Importantly, we found no significant changes (>2-fold) of histone modification in biochemically fractionated samples , H3e region D. Activeediators . Howevere strain B. Other ut cells F and S3C samples , indicatA, b and f), consistent with the results of the immunoblot analysis . Normally, H3K27ac is localized at transcriptionally active euchromatin domains, but is temporarily and strongly localized in heterochromatin in spontaneously appearing Zscan4-positive and other 2C gene-positive cells . Dispersion or reorganization of DAPI-dense chromocenter is one of the characteristic features of Zscan4-positive state in ES cells , but chromocenters were mostly unaffected , suggesting that there might be some differences in heterochromatin structures in Zscan4-positive cells induced by depletion of Rif1 and Chaf1.To monitor the changes in chromatin structure in single nuclei, we detected H3K27ac by indirect immunofluorescent microscopy in control and Rif1 KO cells. Although the overall signal intensities were unchanged Fig. 5 events; ). We als events; ES cells , and ChaES cells  along wis  and GFP-negative (−) cells, and H3K27ac-high and H3K27ac-mid/low . In control, only 0.87 ± 0.07% of cells were GFP(+)/H3K27ac-high, and Rif1 depletion increased this fraction to 7.8 ± 0.26% . GFP(+) population in control cells of H3K27ac-high and H3K27ac-mid/low cells was 7.7% and 2.3%, respectively, and Rif1 knockdown upregulated GFP(+) ratio in both populations , resulting in the increase of GFP(+) cells by similar ratios in H3K27-high and H3K27ac-mid/low cells . Next, we gated H3K27ac-“hyper” population, constituting approximately 3% of the total cells, consistent with the estimation from the immunofluorescent images . The GFP(+) ratio in this population was 14% in control, but increased to 70% in Rif1-depleted cells . This indicates that Rif1 depletion increases the probability of the Zscan4-positive state in cells with H3K27ac-hyper mark, although Rif1 depletion-mediated enhancement of GFP(+) does not require high H3K27ac state since GFP(+) ratio is also increased in H3K27ac-mid/low populations . Conversely, GFP(+) cells are more associated with H3K27ac-high mark than in GFP(−) cells , although Rif1 KD did not significantly affect these ratios . The geometric mean of H3K27ac of GFP(+) population was not significantly affected by Rif1 knockdown , in accordance with the fact that the whole-cell H3K27ac level is not affected by Rif1 depletion.To examine whether elevated H3K27 acetylation is a determinant for derepression of Zscan4c-EmGFP signal intensities of GFP(+) population were much higher in Rif1-depleted Zsc4c-EmGFP KI cells than in control cells  due to the increased number of cells with higher GFP levels (F). Zscan4c-GFP signals of H3K27-high populations in Rif1 knockdown cells were also higher (p < 0.01) than those in control cells . Interestingly, activation of Zscan4c-GFP(+) by Rif1 depletion occurred only in the limited fractions of H3K27-high population and resulted in two-phase distribution of extremely high GFP(+) and GFP(−) . Consistent with this, fluorescent microscopic images have also indicated that only a fraction of GFP(+) cells are accompanied with high H3K27 mark and not all high H3K27ac cells are GFP-positive . In summary, global high H3K27 acetylation is strongly associated with derepression of Zscan4 but is not a sole factor for Zscan4 deregulation in Rif1-depleted cells.We next analyzed the GFP signal levels of selected populations. The P levels F. Zscan4A). To elucidate the segments required for suppression of 2C gene transcripts, the stable ES cell lines expressing N-terminal 1051 aa, ΔN984 or C-terminal 552 aa of Rif1, all of which localized in nuclei (B), were established. When endogenous Rif1 was depleted by siRNAs, none of the three mutants could fully rescue the deregulation of 2C transcript-positive state (C and S4B). Surprisingly, transient expression of Rif1 C552 or ΔN984 in Zsc4-EmGFP Tg cells resulted in significant increase of GFP(+) cells, while full-length and Rif1 N1051 did not affect the GFP(+) population (D). Single-cell imaging analysis revealed that signal intensities between ectopic expression of C552 or ΔN984 (FLAG) and GFP were significantly correlated , suggesting that expression of the C-terminal polypeptides is responsible for the induction of the Zscan4 transcripts (C and D). Furthermore, 2C-specific transcripts including Zscan4, Usp17l, AA684185, MERVL, and major satellite, the marks of Zscan4-positive state, were significantly elevated in cells expressing exogenous C552 at the approximately same levels of endogenous Rif1 (E and F). Interestingly, not all the populations expressing C552 or ΔN984 became Zscan4-positve (G); Only 19.5% or 17.9% of FLAG-positive cells were GFP(+) in C552 or ΔN984-expressing cells, respectively (H).Mammalian Rif1 protein consists of N-terminal HEAT repeat structure, C-terminal conserved domain, and the central long intrinsically disordered polypeptide region (IDR) A. To elun nuclei B, were eve state C and S4Bpulation D. Singlenscripts , C and Dous Rif1 , E and F-positve G; Only 1ectively H.Figure A, lanes 7, 8, 11 and 12). We previously reported that both N-terminal HEAT repeat and C-terminal segments can form homo-oligomers on their own , is highly soluble in cellular fractionation analysis , whereas both endogenous and ectopic full-length Rif1 proteins were distinctively fractionated into nuclear scaffold/matrix fraction like LaminB2 . A small amount of endogenous Rif1 was relocated to soluble fraction when C552 was overexpressed . On the other hand, ΔN984 was mostly insoluble with small portions detected in the soluble fraction , and its overexpression did not affect fractionation of endogenous Rif1. We also analyzed the localization using confocal immunofluorescent microscopy. As reported for a human ortholog, the endogenous mouse Rif1 was localized at nuclear membrane and to perinucleolar heterochromatin structures . In C552-overexpressing cells, the signal intensity of the endogenous Rif1 detected by anti-Rif1 N206 (Rif1N) antibody appear to be compromised, as well as by anti-Rif1 UCRIII (Rif1U) antibody to a lesser extent . Similarly, overexpression of ΔN984 reduced the endogenous Rif1 signals . Since the protein level of endogenous Rif1 was not affected by the ectopic expression of mutant proteins , the decreased Rif1 signals may reflect structural changes of chromatin and/or nuclear scaffold that block the accessibility of the antibodies to Rif1, or Rif1 protein conformation itself.Next, to investigate whether overexpression of C552 or ΔN984 would affect the localization of endogenous Rif1, we examined whether it affects the biochemical fractionation and cellular localization of endogenous Rif1. C552, which is exclusively localized to nuclei B, is higin vivo by indirect immunostaining microscopy using two anti-Rif1 antibodies. In fertilized oocytes of ICR mice, Rif1 accumulated in nuclei and was excluded from nucleolus with diffused week signals in cytoplasm (A) as previously reported (B–F). The signal intensities of Rif1U were mostly unchanged throughout early embryogenesis, whereas Oct4 were markedly decreased at two to four-cell and emerged again after eight-cell stage when zygotic gene became active (G) and regained its nuclear localization after the four-cell stage (H–K). This 2C stage-specific dislocalization of Rif1N signals was also demonstrated in C57BL/6J mouse embryos (A), both in early (possibly G1 or S phase) and in late stage (G2 phase) , while Rif1U signals were constantly present in nuclei . Inhibition of nuclear export by leptomycin B did not affect the Rif1N signals, suggesting the possibility that Rif1-derived polypeptide containing the N-terminal segment was intrinsically expressed in cytoplasm at 2C stage .The possible effects of truncated forms of Rif1 on induction of 2C genes prompted us to examine the expression and localization of Rif1 during early developmental stages ytoplasm A as prevreported . At the reported , B–F. The active . Unexpece active G and regll stage , H–K. Th embryos A, both iA). Next, we retrieved the public data, which were processed from a series of mass spectrometry datasets of mouse oocytes and preimplantation embryos . This pattern was similar to Pcna and Ctcf, which were expressed at constant levels . Interestingly, the proteins levels of other 2C gene suppressors, including Kdm1a, Chaf1, and Trim28, were also almost constant during these stages , whereas Igf2bp2 and Oog4 showed stage-specific changes as previously reported . Thus, these transcriptome and proteome data did not provide evidence for the presence of 2C stage-specific short forms of Rif1.To search for the presence of the alternative forms of Rif1, we first reanalyzed public RNA-sequencing data of mouse oocytes and embryos . We haveFig. 9 embryos . Data wareported   and detect the ectopically expressed Rif1 fragments with high specificity (B–D). These antibodies could detect Rif1 in the extract derived from as few as 100 ES cells (E). With these antibodies, Rif1 was detected as multiple short forms and only a small amount of the full-length form was present in unfertilized eggs . In 2C embryos as well, the majority of Rif1 protein was detected as truncated forms. Among them, a 25 kD polypeptide  and other forms ranging from 60 to 185 kD constituted 38% and 57%, respectively, of the total polypeptides detected by Rif1N that reacts to N-terminus-derived polypeptides. The antibody Rif1U, which was developed against the central IDR segment upstream of C-terminal conserved domain, detects the 67 kD fragment and other forms  constituting 53% and 17%, respectively, of total Rif1U-reacting proteins. At the morula stage, the full-length Rif1 accounted for 87% of the total polypeptides detected by Rif1U antibody . The ratio of the full-length Rif1 to truncated forms appears to continue increasing during the preimplantation stages . The truncation of Rif1 is probably not caused by nonspecific protein degradation during sample preparation, since other proteins, including Pcna and LaminB1, were detected as single full-length forms in the same samples .To examine more precisely the expression of Rif1 protein at the 2C stage, we collected mouse oocytes and embryos and investigated the size of Rif1 protein by immunoblot analysis. Two newly developed Rif1 antibodies, Rif1-N206 (Rif1N) and Rif1-UCRIII (Rif1U), react to the full-length Rif1 as a single band in the ES cell extracts A and detcificity , B–D. ThES cells E. With tD, a, lanes 1). In ES cells, the N-terminal Rif1 polypeptide coding N382 aa (43 kD) or N877 aa (95 kD) was localized in cytoplasm (I), which coincides with the Rif1N-reacting cytosolic-signals in 2C embryos , whereas small amounts of full-length and a minor ∼100 kD forms were bound to insoluble materials . At the morula stage, the full-length form was clearly detected in the insoluble chromatin/nuclear scaffold fraction . In conclusion, in 2C embryos, most of the truncated forms derived from the N-terminus (less that 95 kD) or from the central IDR segment are not localized in at nuclear scaffolds and thus, are likely to be nonfunctional.To characterize Rif1 short forms, the cell extracts were fractionated into the soluble (including cytoplasm and nucleoplasm) and the insoluble (including chromatin and nuclear scaffold) fractions. Most of the short forms in 2C embryo were detected in the soluble fraction, including the major N-terminal 25 kD fragments . Interestingly, Nanog, one of Rif1-interacting protein  Kdm1a , Chaf1 , Rif1 , 19; thi; thi19; Tmem92 and Tdpoz4 loci .The chromatin structure that underpins the transcriptional bursting of 2C-specific genes in ES cells has not been fully understood. Previous epigenetic analyses of intrinsic Zscan4-positive state of ES cells have indicated modest decrease of DNA methylation in oz4 loci  and globoz4 loci . Howevere states , 48. In antibody  versus bHistone modification is another important epigenetic regulatory mechanism for activating and silencing gene expression. Based on immunoblot analysis, we have found that Rif1 depletion does not globally change 15 histone modifications, in agreement with previous work reporting no effects on total levels and genome-wide distribution of H3K9me3, H3K27me3, H4K20me3, and H3K4me3 , or globZscan4 gene and neighboring regions, in both Rif1 knockdown and knockout cells with different genetic backgrounds. H3K9me3 of the Zscan4 gene loci has decreased to some extent , but is still maintained at higher levels than that of Nanog (as a control active gene) or the LaminB-associating domain (as a control silent site) in Rif1-depleted cells. ChIP-qPCR analyses of specific genomic loci have advantages in quantitatively mapping histone marks especially for multicopy genes or repeats, when compared with ChIP-seq analysis in which results can be influenced by background signals and/or by multiple parameters in data processing. Importantly, H3K27ac, upregulated in Zscan4-positive ES cells  and silent marks (H3K27me3 and H4K20me1) of the ES cells , 33, is Zscan4 loci. First, H3K27 acetylation, the only notable histone H3 modification mark in the Zscan4 region that increases after Rif1 depletion, is not accompanied by another enhancer mark H3K4me1. Generally, H3K4me1 is a primary mark, generating “primed enhancers” decorated only with H3K4me1, which will be converted to “active enhancers” by additional H3K27ac , mediator complexes, and active enhancer marks H3K4me1 and H3K27ac, whereas the Zscan4 enhancer is associated only with the prominent H3K27ac mark in much longer range of sequences. As an atypical activated domain, pathogenic enhancer bound by BRD4-NUT with hyper H3K27 acetylation signature in NUT midline carcinoma was reported to form “megadomains” raging from 100 kb to 2 Mb , suggesting that factors other than H3K27ac mark also may affect the Zscan4-positive state. We note two features of Rif1 depletion-induced Zscan4-positive state, not observed in naturally occurring one. (1) The presence of hyper Zscan4-positive populations, which may be caused by strong derepression or long-term persistence of this state. (2) Intact chromocenters (heterochromatin at pericentromeric regions). They colocalize with hyper H3K27ac signals and DAPI-detectable, whereas they are clustered into larger and fewer regions  cannot provoke 100% transition from the normal to Zscan4/MERVL-positive state , ins study; , 60, or s study; .Zscan4 expression. We have recently reported that mouse Rif1 C-terminal 299 aa forms multimers  Rif1C exerts dominant-negative effect on endogenous Rif1 by forming nonfunctional hetero-oligomers. (2) The hetero-oligomers may dissociate from nuclear periphery/insoluble nuclear scaffold. The association of Rif1 with nuclear periphery may be crucial for its functions. Notably, 2C transcript-positive state is induced in only a fraction of Rif1-C-overexpressing cell (H), as in Rif1-depleted cells.We have also analyzed functional domains of Rif1 using transcription suppression assays in ES cells. Previous study has shown that Rif1 truncation mutant lacking C-terminal domain (ΔC231 aa) largely rescues the Rif1 KO phenotype, but that lacking N-terminal HEAT repeat domain (ΔN1299 aa) does not, leading to the conclusion that N-terminal domain is required for its function . In thisdecamer; ), and sidecamer; . We specing cell H, as in Using two highly specific Rif1 antibodies, we have found that the N-terminal polypeptide-derived signal is detected only in cytoplasm of the 2C embryos in two different mouse strains and in nuclei after the four-cell stage, whereas the IDR-derived signal is detected in nuclei throughout the cleavage stages of embryos. Unexpectedly, the majority of Rif1 protein appears as truncated short forms in oocytes and 2C embryos, which are likely to be nonfunctional and are dissociated from chromatin. Short Rif1 forms, which are detected by the Rif1-U antibody, could contain the C-terminal segment of Rif1 and exert a dominant negative effect on the remaining full-length Rif1. These Rif1U-reacting short forms, both soluble 67 kD and minor insoluble longer forms, could interact with the residual full-length form (such as Rif1 C552 and ΔN984 in ES cells), may promote dissociation of active Rif1 from nuclear insoluble structures, and/or generate nonfunctional hetero-oligomers, leading to derepression of 2C genes. It should be noted that the presence of the Rif1 short forms is evident in unfertilized oocytes, indicating that the accumulation of short forms alone is not a trigger for zygotic genome activation (ZGA) or 2C gene activation. Nevertheless, the temporal loss of functional nuclear Rif1 at the 2C stage may contribute to formation of permissive chromatin state for zygotic transcription events.Rif1 from public RNA-seq data, although further analysis by long read sequencing or cap analysis of gene expression may clarify the presence or absence of alternative Rif1 transcripts in 2C embryos. In oocytes, maternal proteins/transcripts are required for the events during fertilization, but a large part of them are removed prior to the onset of maternal-to-zygote transition. The two protein degradation systems, autophagy and proteasome-dependent degradation, play important roles in normal development of mouse embryos . Zscan4-EmGFP Tg cells  and the ARRIVE guidelines, and all studies using animal experiments were approved by the Animal Care and Use Committees of Tokyo Metropolitan Institute of Medical Science (permission Nos. 20-030 and 21-022).ICR (CD-1) and C57BL/6J mice were purchased from Charles River Laboratories Japan and CLEA Japan, respectively. C57BL/6J Rif1 cDNA were isolated from D3 cells and cloned into pBluescript KS II (−), and the sequence was validated from three independent clones (GenBank: JX255737). Resulting sequence is highly homologous to AY428571 derived from ES cells established from 129/Sv strain  with C552-F primer (containing substitution of G5598A to generate BamHI site as a silent mutation) and C552-R primer (containing XhoI and NotI site) using pBluescript-mRif1 C552aa as a template, was cloned to backbone vector at EcoRI site. Second, Rif1 cDNA 1 to 5595 bp was clone to this vector at EcoRI site using In-Fusion (Clontech). Rif1 C552 expression vector was derived from the products of the first step by digestion with BamHI, blunting by T4 DNA polymerase and self-ligation to make it in frame. Rif1 N1051 expression vector was generated from full-length construct by deletion of cDNA 3153 to 5497 bp (at EcoNI site 3152 and 5497 cut by EcoNI star activity) resulting in the coding N-terminal 1051 aa with additional two amino acids at the C-terminus. Rif1 ΔN984 was also derived from the full-length constructs by deletion of 1 to 2951 bp and self-ligation at EcoRI sites. For Rif1 N877 expression vector, Rif1 cDNA fragment 1 to 2631 bp was amplified by PCR with mRif1-N(RI)frame3 and mRif1_2626R_SpeI primers and cloned into pME18S. Then the insert DNA was isolated by digestion with EcoRI and NotI and cloned into pCAGIP-FLAG. Rif1 N382 expression vector was derived from pCAGIP-FLAG-N877 by digestion with XhoI and NotI, blunting by Mung Bean nuclease and self-ligation. Rif1 N1307 was generated from N1051 expression vector by digestion with EcoNI and NotI followed by insertion of Rif1 cDNA 3139 to 3921 bp amplified with mRif1_EcoNI_F3139 and mRif1_R3921stop primers by using In-Fusion. Rif1 ΔN782 was generated from ΔN984 vector, by insertion of Rif1 cDNA 2347 to 2967 bp amplified with mRif1_F2347_Flag_IF and mRif1_R2967_IF primers by In-Fusion. Since plasmid DNA containing Rif1 cDNA full-length and the N-terminal coding regions were highly unstable in bacterial cells for unknown reasons, transformation and culture have been carried out at 25 to 30 °C in XL10-Gold (Stratagene) and expanded at least three clones per constructs. The sequences were validated for every preparation before the use in the experiments.Three fragments of mouse v strain  as a temv strain , followeAY428571; a gift from I. Adams) as a template, and cloned into pGEX-4T1 (Pharmacia) at BamHI and XhoI sites, then 619 to 1137 bp of CDS was removed by digestion with XbaI and NotI, followed by blunting by Mung Bean Nuclease (Takara) and self-ligation. For GST-Rif1 1673 to 1851 aa protein expression vector, cDNA fragment was amplified with mRif1_F5017_BamHI and mRif1_R5553_XhoI primers using the same template, and cloned into pGEX-4T1 at BamHI and XhoI sites.For bacterial expression of GST-fused mouse Rif1 N206 aa protein, N-terminal Rif1 coding region (AY428571) was amplified by PCR with mRif1_N_BamHI and mRif1_R1137_XhoI primers shown in  using pBCreERT2-HSV thymidine kinase polyA digested from pMB80 vector (Addgene) was cloned into pBluescript II KS(−) at XbaI and EcoRI sites, followed by deletion of polyA at MluI and EcoRI sites and self-ligation. Third, IRES-pac-bGH polyA digested from pPyCAGIP vector at EcoRI and BamHI (blunted by T4 DNA polymerase) was ligated at EcoRI and blunted SalI sites of second construct, resulting pBluescript II KS(−)-SA-CreERT2-IRES-pac-bGH polyA. Finally, SA-CreERT2-IRES-pac-bGH polyA in third construct was cloned into first construct pZDonor-Rosa26-ZFNTSmut by In-Fusion. All primers used for constructions are listed in pZDonor-mRosa26-ZNFTSmut-SA-CreERT2-IRES-Puro vector was constructed as follows; First, three point mutations in zinc finger nuclease (ZFN) recognition sequences was introduced to original pZDonor-mRosa26 vector (Sigma-Aldrich) by PCR mutagenesis  to prevent cleavage of integrated donor alleles. The PCR fragment amplified by using F primer with intact sequence and R primer containing mutations was ligated to at NotI and XmaI sites, resulting pZDonor-mRosa26-ZFN-TSmut. Second, SA-https://www.expasy.org/). IDR was predicted by PredictProtein  and HEAT repeat regions of Rif1 protein were assigned from previously reported human Rif1 sequence , 24. NuctProtein .g for 20 min at 4 °C, GST-fused proteins were purified using glutathione Sepharose 4B . For generation of polyclonal antibodies, 0.3 mg of purified proteins was used to immunize a rabbit each time for total five times, and two animals for each antibody were used (Bio Regenerations). The antibodies were affinity-purified by AKTA Explorer 10S  using recombinant antigen protein-conjugated HiTrap NHS-activated HP columns following manufacturer’s instructions and validated the specificities. Other antibodies used in this study are listed in Recombinant proteins, mouse Rif1 N206 aa or Rif1 UCRIII  and used as a PCR template for a standard curve, and the amounts of total Rif1 (using the primers targeting both endogenous and transgene) or those of transgene (using the primer for 3′ UTR of transgene) were calculated for picograms of cDNAs derived from 1 μg total RNA (F).Total RNA extracted from ES cells with TRIZOL (Invitrogen) was purified by 2-propanol precipitation and quantified by NanoDrop (Thermo Fisher). After DNase treatment of 1 μg or 0.5 μg of total RNA, cDNA was synthesized from RNA with PrimerScript RT reagent Kit (Takara) or ReverTra Ace qPCR RT Master Mix (Toyobo). One to two microliters of tenfold diluted cDNA was subjected to real-time PCR using SYBR Premix Ex Taq (Takara) on LightCycler 480 (Roche). PCR cycle condition is; 95 °C for 1 min, followed by 45 cycles of 95 °C for 5 s and 68 °C for 30 s for most of primer sets, or 45 cycles of 95 °C for 10 s, 57 °C for 20 s and 72 °C for 16 s, for others. The relative quantification values were normalized by otal RNA F.For complementation assay, E14tg2a stable transformant cells expressing full-length Rif1, N1051, C552, or ΔN984 were generated by transfection of plasmid vectors and selected by 2 μg/ml puromycin. The clones with levels of ectopic expression of Rif1 transgenes similar to those of the endogenous gene were used for analysis. The effects on derepression of 2C transcripts between cells depleted of endogenous Rif1 alone  and those of both endogenous and transgene (using siRNA #2 or #3) were examined. For all qRT-PCR analysis, a minimum of three independent experiments were done, in technical triplicate. All primers used for qRT-PCR are listed in Rif1f/fCre-ERT2 mouse ES cells treated with or without 4-OHT were cultured for 60 h. Fixed chromatin was prepared as previously reported  containing protease inhibitors , 0.4 mM Pefabloc SC (Roche)) at 4 °C for 15 min, and centrifuged at 440g for 10 min. The pellets were lysed with Nuclear lysis buffer (FA-lysis buffer containing 1% SDS instead of 0.1% supplemented with protease inhibitors) at 4 °C for 15 min, and nuclei was centrifuged at 1750g  or 440g (for E14tg2a cells) for 10 min. Chromatin pellets were washed and resuspended with FA-lysis buffer (at the density of nuclei derived from 1 to 2 × 107 cells/ml) in 15 ml polypropylene tubes (Corning), and sequentially sonicated by Sonifier 250D (Branson) on ice at 20% with 50% frequency (1 s on and 1 s off) for 1 min, 2 to 5 times with 2 min intervals, followed by Covaris S-220 (Covaris) in 1 ml glass tubes at power 150W to 175W, Duty Factor 20, for 12 to 15 min at 4 °C. The size of sheared chromatin was assessed by agarose gel electrophoresis, ranging from 100 to 400 bp with average size at 200 bp. After centrifugation at 39,000g for 10 min and a preclear by beads for 6 h at 4 °C, the sonicates corresponding to 1 × 106 cells were diluted to 0.5 ml with FA-lysis buffer containing 0.5 mM DTT and protease inhibitors, and incubated with 1 μg of histone antibodies bound to 20 μl of Dynabeads Protein G (Life Technologies) for 12 to 16 h. The beads after ChIP were washed at 4 °C for 5 min with 1 ml of buffers as follows: with FA-lysis buffer without protease inhibitors (three times), FA-lysis buffer containing 350 mM NaCl, ChIP wash buffer , and TE (once for each step). DNA was eluted with 0.3 ml of ChIP elution buffer  at 37 °C for 30 min on rotary mixer (TAITEC) with constant tapping. The eluates were incubated at 65 °C for 6 h for de-cross-linking, treated with RNaseA (50 μg/ml) at 37 °C for 1 h and treated with proteinase K (0.5 mg/ml) at 50 °C for 2 h. DNA was extracted with phenol/chloroform/isoamyl alcohol, and the organic phase was re-extracted with TE containing 0.2 M NaCl. The combined aqueous phase was added with 1× vol of 2-propanol, 0.3 M sodium acetate and glycogen, and centrifuged at 17,000g for 30 min. DNA was repurified by MinElute (QIAGEN) and eluted with 50 to 80 μl of EB (Qiagen). One microliter of ChIP DNA (approximate 5 ng) was subjected to ChIP-qPCR using SYBR Premix Ex Taq (Takara) on LightCycler 480 (Roche). PCR condition for most of primer sets was: 95 °C, 30 s, followed by 45 cycles of 95 °C, 7 s and 62 °C, 30 s; for Zscan4-ChIP4, 45 cycles of 95 °C, 7 s and 62 °C, 40 s; for T-ChIP, 45 cycles of 95 °C, 7 s, 57 °C, 15 s and 72 °C, 10 s. Input samples were de-cross-linked and purified as above, and the ChIP efficiency (ratio of ChIP DNA to input DNA) was calculated. All experiments were done at least two times, with technical triplicates. All primers used for ChIP-qPCR are listed in E14gt2a cells were transfected with control or Rif1#2 siRNA twice and cultured for 72 h, or Fgf5; 30 cycles for Zscan4e, Bmp4, and T). The products were analyzed by gel electrophoresis using 2% (Bmp4) or 4%  agarose in TBE buffer. As a control, methylation-insensitive isoschizomer MspI was used for digestion. Two independent experiments were performed.DNA methylation analyses by HpaII-digestion/PCR were previously reported . BrieflyZscan4 gene family members and compared with corresponding genomic sequences, in order to distinguish between converted and intact TpGs, which were derived from polymorphism among gene family members. For BS2 region, 29 clones were sequenced, and only those derived from the Zscan4b, e, and d loci, which contain CpG sites, were shown. Since BS4 region in the Zscan4 exon 4 is well conserved, clones were not classified but compared with the Zscan4c reference sequence. Two independent experiments were performed. All primers used for DNA methylation analysis are listed in DNA methylation was analyzed also by targeted amplicon bisulfite sequencing by previous protocol . DenaturRif1f/f mice (ES cells were derived from blastocysts of C57BL/6J f/f mice  as reporf/f mice . Three c3), and the EmGFP signals were analyzed by FACSCantoII or LSRFortessa X-20 (Beckton Dickinson) at FITC channel. For immunostaining analysis, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with FACS buffer containing 0.5% Tween 20 were incubated with anti-H3K27ac or anti-FLAG M2, and with anti-GFP antibodies at 4 °C for overnight. The cells were washed with FACS buffer containing 0.1% Tween 20 three times followed by incubation with secondary antibodies, PE-conjugated anti-mouse IgG (BD Pharmingen), and Alexa Fluor 488-conjugated anti-rat IgG (Invitrogen), for 30 min. Washed samples were analyzed by LSRFortessa X-20 (Beckton Dickinson). Data was analyzed by using BD FACSDiva software (Becton Dickinson). Each analysis was done in two to five independent experiments with biological triplicates for each set of experiment.For live cell analysis, dispersed ES cells were washed with PBS and suspended with FACS buffer  containing 0.1% Triton X-100, lysed on ice for 5 min, and centrifuged at 800g for 3 min at 4 °C. The pellets were washed again with 100 μl of CSK buffer without detergent, and the first and second supernatants were combined and stored as cytosol and nuclear soluble materials. The precipitates were suspended with hypotonic buffer  supplemented with protease inhibitors and 100 U/ml of Benzonase (Novagen), and incubated on ice for 1 h. After addition of 10 mM EDTA, the samples were separated into the supernatants (chromatin-enriched fractions) and the precipitates (nuclear scaffold-enriched fractions). Five percent of each fraction was analyzed. Two independent experiments were performed for ES cell fractionation.ES cells were fractionated as previously described  with min2, 1 mM EGTA, 20 mM NaF, and 0.1% Triton X-100 with cOmplete). The samples were transferred to siliconized polypropylene 0.2 ml tubes, suspended with low retention pipet tips, and incubated on ice for 5 min. The samples were centrifuged at 800g for 5 min, at 4 °C in a swing rotor, and collected the 25 μl of supernatants. To the pellets and remaining supernatants, 15 μl of 1× CSK+ buffer was added and suspended again by pipetting. After centrifugation, the supernatants were combined and added with 0.25× volume of 5× sample buffer. To the precipitation fraction, ×4 volume of 1.25× sample buffer was added. The samples were boiled, and each half of them was loaded onto an acrylamide gel, and two sample sets were subjected to immunoblot analysis.For cellular fractionation of embryos, 220 2C embryos or 193 morula embryos were collected in 6 to 8 μl of KSOM without BSA and freshly frozen. The samples were thawed by addition of 1× volume of 2× CSK+ buffer ), and the total volume was adjusted to 30 μl with 1× CSK+ buffer , 0.4 mM Pefabloc SC (Roche), and 10 mM sodium butyrate), centrifuged at 700g, and homogenized with NIBi plus 0.2% NP-40. Cells were incubated on ice for 10 min and centrifuged at 1000g. After two-time wash of nuclei with NIBi without detergent, the pellets were suspended with 0.2 ml of chilled 0.2 M H2SO4 and rotated at 4 °C for 4 h. The supernatants were collected by centrifugation at 3400g twice and 100 μl of chilled 100% TCA was added and incubated on ice for overnight. The TCA precipitates were washed with cold acetone +0.1% HCl and then with acetone. The dried pellets were suspended in water, and cleared supernatants were quantified by Bradford assay. In total, 3 to 0.1 μg of histone-enriched extracts, for modified histones or pan H3/H4, respectively, were boiled in SDS-PAGE sample buffer and loaded on 15% acrylamide gels, and resolved by SDS-PAGE. The blots were incubated with blocking solution (5% BSA in TBS-T) for 1 h, then incubated with primary antibodies diluted in blocking solution for 2 h at RT. Chemiluminescent detection was done as described elsewhere. For control experiments, E14tg2a cells were treated with trichostatin A , 5-aza-deoxycytidine . At the harvest, cells were washed using PBS with or without 10 mM sodium butylate. The histone-enriched fractions were prepared by acid extraction methods and analyzed by immunoblot as above. The experiments were done 2 to 4 times, depending on target marks.Histone-enriched fractions were prepared from  methods  with mod2, 150 mM NaCl, 1 mM Na3VO4, 20 mM NaF, 5% glycerol, 1% Nonidet P-40, 1 μg/ml pepstatin A, 1.5 μg/ml aprotinin, 1 μg/ml leupeptin, and 0.4 mM Pefabloc SC) containing 0.3 M NaCl (buffer A/0.3 M)  by rotation for 30 min. The lysates containing 0.5 mg proteins were incubated with 30 μl of anti-FLAG M2 agarose (Sigma-Aldrich) or 1 μg of anti-Rif1N antibodies followed by precipitation with 30 μl of protein A/G (2:1) Sepharose . The beads were washed with buffer A containing 150 mM NaCl for 4 times and the boiled samples were divided into 2 to load on 7% polyacrylamide gels. For immunoblot analysis, samples were transferred to Immobilon-P (Merck) membranes, and blots were incubated with blocking solution (5% nonfat milk in TBS-T) for 30 min, then incubated with primary antibodies for 2 h at RT. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch) for 45 min, chemiluminescent signals were developed by using Lumi-Light PLUS (Roche) and detected by LAS 3000 (Fuji film) or FUSION FX7.EDGE (Vilber Lourmat). The signal intensity of bands was quantified by Multi Gauge software version 3.0 (Fuji Film). All experiments were done at least two times.For preparation of whole cell extracts, the cells were lysed with lysis buffer A   and quanF–H, 150 cells of fertilized eggs (6 hpf), 2C or eight-cell embryos were used and processed as above. Two independent experiments were done for oocyte/embryo immunoblot analysis. The size of Rif1 truncated forms was calculated by migration distances to be fit to logarithmic curve models.One hundred and twenty mouse unfertilized eggs, 2C or morula embryos were collected by counting under microscopy and snap-frozen in liquid nitrogen. The samples were directly suspended in SDS-PAGE sample buffer supplemented with protease inhibitors described above and heat-denatured at 95 °C, 5 min. After agitation by microtube mixer for 5 min to shear chromatin DNA, the whole extracts were loaded onto 5 to 20% acrylamide gradient gel e-PAGEL (ATTO) and resolved. The proteins were transferred to membranes by tank blotting system (BIO CRAFT). The membranes were incubated with 5% skim milk in TBS-T for 1 h and incubated with anti-Rif1N antibody for 4 °C overnight, then incubated with secondary antibody , Jackson ImmunoResearch) in 1% Blocking reagent (Roche) in TBS-T for 45 min. The signals were detected as above and exposed to X-ray films. After detection, antibodies were removed by shaking in stripping buffer  at 50 °C for 6 min, and the membranes were used for reblotting with different antibodies. In experiments in Preparation of cells for immunofluorescence staining was performed as previously described with modifications . BrieflyFluorescent images were acquired with LSM780 confocal laser scanning microscope (Carl Zeiss) and objective lens . Alexa Fluor 488 was detected using the 488 nm argon laser and a 495 to 551 nm filter; Cy3 was detected using the 561 nm DPSS laser and a 569 to 630 nm filter; DAPI was detected using a 405 nm blue diode laser and a 410 to 483 nm filter. Conventional fluorescent images were acquired with microscope BX-61, UPlanSApo 40×/0.95 lens (Olympus), and ORCA R2 CCD camera (Hamamatsu Photonics). For phase-contrast images, data were captured by microscope CKX41 (Olympus) equipped with COOLPIX P6000 (Nikon). For alkali phosphatase staining, samples were processed by alkali phosphatase kit (Sigma-Aldrich) according to provider’s instruction except for using 4% paraformaldehyde instead of citrate buffered acetone for cell fixation. Stained cell images were recorded using IX-71 inverted microscope equipped with digital camera DP72 (Olympus). The levels of images were adjusted by Photoshop in some data series. Brightness and contrast were not adjusted. Two independent experiments were performed for alkali phosphatase staining.A. Two independent experiments were performed for transcriptome analysis. Data not presented in this study are available upon requests.E14tg2a cells were transfected with control siRNA or mRif1#2 plus NC#1 and harvested at 48 or 72 h. Total RNA was purified from transfected or untreated cells by using RNeasy Micro (Qiagen) following manufacturer’s instructions. The quality of RNA was assessed with a 2100 Bioanalyzer (Agilent Technologies). Cy3-labeled cRNA was prepared using a Low Input Quick Amp Labeling Kit according to the manufacturer’s protocol (Agilent Technologies). The labeled samples were hybridized to the Mouse Gene Expression v2 Microarray Slides  for 17 h at 65 °C at 10 rpm in a rotating hybridization oven. The Slides were washed and scanned with SureScan Microarray Scanner (Agilent Technologies) using a scan resolution of 5 μm and dye channel of Green with PMT set to 100%. Data was obtained using the Agilent Feature Extraction software (Agilent Technologies) with set to default for all parameters. The normalized expression data files were imported in GeneSpring GX12.6 software (Agilent Technologies) and 27,732 entities with values filtered by percentile  and by flags  were selected and shown in a scatter plot in Figure 2Zsc4-EmGFP Tg cells transiently transfected with the full-length Rif1 or its truncated mutants were fixed at 48 h and were subjected to immunostaining using anti-FLAG and GFP antibodies and costained with DAPI. Images were acquired by LSM780 (Zeiss) and over 400 cells in three representative images per each transformant were used for correlation analysis. For segmentation and quantification of three channels of signals of each cell, LSM format files were processed by MetaXpress version 5.1 (Molecular Devices) using the custom macro. Briefly, DAPI-stained nuclear areas were firstly defined, followed by segmentation of FLAG-stained nuclei and GFP-stained cytosol areas, and integrated signal intensities of binarized areas were assigned to DAPI total signals. Then FLAG and GFP signals in individual double-positive (FLAG(+) and GFP(+)) cells were analyzed for Spearman’s rank-correlation coefficients (rho), and corresponding two-tailed probability  was shown for each transformant: full-length, C552, or ΔN984 Rif1. Scattered plots were depicted by Excel.2. Fertilized oocytes  and embryos at 2C , four-cell (48 hpf), eight-cell , morula (3 dpf) and blastocyst (3.5 dpf) were collected and fixed with 4% paraformaldehyde in PBS for 15 min, washed with PBS three times, permeabilized with 0.2% Triton X-100/PBS for 15 min, and blocked with 5% normal goat serum/PBS for 1 h at RT. Embryos were incubated with primary antibodies overnight at 4 °C, washed, and incubated with anti-mouse Cy3  and anti-rabbit Alexa Fluor 488 (Molecular Probes) for 1 h at RT. DNA was stained with 1 μg/ml DAPI for the following 10 min. The samples were once adapted to 40% glycerol and mounted with 90% glycerol/2.5% DABCO. For immunostaining of unfertilized eggs, collected samples were soaked with 0.15% hyaluronidase (Sigma-Aldrich) in KSOM for 15 min at 37 °C to remove cumulus cells.IVF of ICR mice was carried out as previously described elsewhere . BrieflyA) and LSM780  with PlanApo63×/1.4 lens.IVF and immunostaining of C57BL/6J embryos were conducted as above, except for several modifications. In this case, embryos were incubated primarily with anti-Nanog antibody, followed by labeling with secondary Cy-3-conjugated anti-rabbit IgG, and tertiary Alexa Fluor 488-conjugated anti-Rif1N antibody. For analysis at 2C stage, B6 embryos at 24 hpf for G1 or S phase and at 40 hpf at G2 phase  were colFig. S5For immunoblot analysis of C57BL/6J mouse oocytes and embryos, IVF was conducted as above with minor modifications; 0.2 ml CARD HyperOva (Kyudo Co Ltd) per body in place of PMSG was injected into 4-week-old animals, and homemade HTF and KSOM were used. The unfertilized eggs were treated with 0.1% hyaluronidase for 1 min at 37 °C. Before collection, eggs and embryos were washed in custom-made KSOM BSA(−) medium (depleted of BSA and supplemented with protease inhibitors) and were transferred with a small volume of the same medium to the stock tubes.Public total RNA-seq fastq data of MII eggs and embryos  was collected from ArrayExpress (#E-MTAB2950) , and mRNt test and shown by asterisk marks explained in the figure legends or shown with p-values. For qRT-PCR and ChIP-qPCR, the means of technical triplicates with ±SD were shown. For quantification and correlation, analyses of image data were described elsewhere.In most of this study, statistical significance of two sets of data was analyzed by two-tailed Student’s JX255737. Microarray data are available in GEO with accession number GSE158220.The nucleotide sequence of mouse Rif1 cDNA originated from 129/Sv strain is available at GenBank with accession number This article contains The authors declare no conflicts of interest in regard to this manuscript.


Here in this study, a type of composite nanoparticles, consisting CaO2 core and Co-ferrocene shell, is designed and synthesized for combinational tumor treatment. The findings indicate that CaO2 core can be hydrolyzed to produce large amounts of H2O2 and calcium ions at the acidic tumor sites. Meanwhile, Co-ferrocene shell acts as an excellent Fenton catalyst, inducing considerable ROS generation following its reaction with H2O2. Excessive cellular oxidative stress triggers agitated calcium accumulation in addition to the calcium ions released from the particles. The combined effect of intracellular ROS and calcium overload causes significant tumor inhibition both in vitro and in vivo.Nanoparticles, presenting catalytic activity to induce intracellular oxidative species, have been extensively explored for tumor treatment, but suffer daunting challenges in the limited intracellular HThe online version contains supplementary material available at 10.1186/s12951-021-01055-4. The excessive content of intracellular Ca2+ ions causes cell death via the induction of calcium-overload stress. The recent studies indicate that the uncontrollable accumulation of Ca2+ ions in tumor cells may interfere with cell signaling, cause cytotoxicity and trigger apoptosis [2+-ATPase (PMCA) [2 as a potential therapeutic platform for tumor treatment, the strong alkalinity of Ca(OH)2, the byproduct of CaO2 hydrolyzation, results in severe toxicity to normal cells and tissue. An effective strategy, to lessen its intrinsic toxicity while maintaining its expected antitumoral properties, is therefore highly demanded.Metal peroxides, including CuO2 , ZnO2 [22 [2 [2 , CaO2 [4[2 [2 [2 , and BaO[2 [2 [2 , have bepoptosis , 8. In apoptosis , 10. Thepoptosis  and plase (PMCA) . However2O2 is overexpressed in tumor cells [2O2 can be converted into highly toxic hydroxyl radicals (·OH) via Fenton reaction triggered by catalytic metal ions, leading to cell apoptosis [2O2 (~ 50 × 10–6 to ~ 100 × 10–6 M) remains inadequate to maintain the sustained production of ROS for effective tumor inhibition. A comment strategy is to induce exogenous H2O2, for example utilizing the glycolysis reactions of glucose oxidase (GOx) [2O2. However, the catalytic reaction is considerably restrained by tumor hypoxia. Thus, CaO2 may potentially serve as an alternative H2O2 sources, due to its strong capability in hydrolysis, to incorporate with CDT agents for promoted tumor inhibition [In general, Hor cells , inducinpoptosis . This phpoptosis , 15. Evese (GOx) –18. As ahibition .2O2 to ·OH [At present, ferric ion remains as the most comment and efficient catalyst in triggering Fenton reactions, and it is extensively used as an ingredient for the investigation of CDT agents . Ferroce2 to ·OH .2O2, calcium release and Fenton activity, is designed and synthesized for combinational tumor treatment with both tumor specificity and therapeutic efficacy . The findings indicate that CaO2@Co-Fc may remain stable in the neutral aqueous condition, and hydrolyze to produce H2O2 and release Ca ions due to the degradation of Co-Fc shell in the acidic TME. The H2O2 reacts with Co-Fc molecules to generate considerable content of cytotoxic ·OH. More interestingly, the induction of intracellular ROS further agitates the intracellular aggregation of Ca2+ ions, leading to calcium overload. The combined effects of strong CDT phenomenon mediated by self-supplied H2O2 and calcium overload in cancer cells enable significant anticancer properties both in vitro and in vivo.Herein, a type of composite nanoparticles, presenting unique self-supplied Hacy Fig. . In this2, AR), hydrogen peroxide , ammonia solution , N, N-dimethylformamide , and ethanol anhydrous were purchased from Sinopharm Chemical Reagent Co. Ltd. Cobalt (II) acetate tetrahydrate ((CH3COO)2Co·4H2O, 99.9%) and 1,1’-Ferrocenedicarboxylic acid (Fc(COOH)2, > 98%) were purchased from Aladdin-Reagent Co. Ltd. Polyvinyl pyrrolidone , Fluo-4 AM and Cell Count Kit-8 (CCK-8) were purchased from Meilunbio Co. Ltd. 3, 3’, 5, 5’-Tetramethylbenzidine (TMB), 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO), 2’,7’-Dichlorofluorescin diacetate  were purchased from Sigma-Aldrich. Horseradish peroxidase (HRP) was purchased from Hefei Bomei Biotechnology. The ECL western blotting system was purchased from Beyotime Biotechnology. Primary and secondary antibodies were obtained from Abcam. Calcein AM and propidium iodide (PI) probes were obtained from Dojindo.Calcium chloride anhydrous (CaCln65 T). The zeta potential and dynamic light scattering were measured by zetasizer . The X-ray diffraction pattern was obtained by X-ray diffractometer with Cu Kα radiation . The chemical composition and valence states of different elements were measured by X-ray photoelectron spectroscopy . The UV–visible absorbance was measured by UV–vis spectrophotometer . Hydroxyl radical signal was monitored by electron spin resonance spectroscopy . CCK-8 and intracellular H2O2 content were recorded using a microplate reader. The fluorescence images of Live and Dead, intracellular reactive oxygen species (ROS) and Ca2+ ions were obtained by an inverted fluorescent microscope .Scanning electron microscopy (SEM) images was obtained by field-emission scanning electron microscopy . Transmission electron microscopy (TEM) images was obtained by transmission electron microscopy  and PVP (0.35 g) were dissolved in 15 mL ethanol. Subsequently, NH3·H2O  was added under continuously stirring. Then, H2O2  was added dropwise to the mixture using a syringe pump. Finally, CaO2 nanoparticles were collected by centrifugation at 12,000 rpm for 10 min and washed three times with ethanol. The product was stored in 5 mL ethanol for further use. CaO2@Co-ferrocene was prepared via in situ assembly. Typically, CaO2 (5.3 mg) and PVP (160 mg) were dissolved into 18 mL DMF under ultrasound treatment. Afterwards, (CH3COO)2Co·4H2O  and Fc(COOH)2  were added. After stirring at 80 °C for 4 h, the product was obtained by centrifugation and washed several times with DMF and ethanol, respectively.CaO2@Co-ferrocene  was dissolved in 0.5 mL acetate buffer solution (ABS) at pH 5 or 7. Then, 20 μL supernatant was collected at the different time interval. HRP  and phosphate buffer solution  were added. After 10 min’ reaction, TMB  was added and the characteristic absorbance of ox-TMB at 371 nm was recorded to quantify the H2O2 concentration according to the standard curve.In brief, CaO2@Co-ferrocene under acidic condition, which shows a characteristic UV–vis absorbance at 655 nm. In brief, different concentrations of CaO2@Co-ferrocene and TMB  were added in ABS at different pH (5 and 7). The UV–vis absorption of various samples was recorded by UV–vis spectrophotometer. Subsequently, the reaction kinetic curve was monitored under a UV–vis spectrophotometer. The generation of ·OH was further identified by electron spin resonance (ESR) spectroscopy with 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap. In addition, CaO2@Co-ferrocene  was dissolved in ABS . pH value was recorded with a real-time pH meter.3, 3’, 5, 5’-Tetramethylbenzidine (TMB) can be oxidized to the oxidation state of TMB by ·OH produced by CaO2 at 37 °C.Human liver cell (7702), human umbilical vein endothelial cell (HUVEC) and murine breast cancer (4T1) cells were used for the in vitro study. The culture medium RPMI 1640 was supplemented with 10% fetal bovine serum (FBS) and the cells were cultured in an incubator with 5% CO4 cells per well) and incubated for 12 h. Subsequently, CaO2@Co-ferrocene at desired concentrations were added and incubated for another 24 h. Finally, culture medium containing 10% CCK-8 was added to each well and the absorbance was detected by a microplate reader at 450 nm after incubation at 37 °C for another 1 h.7702 and HUVEC cells were seeded in 96-well plates (104 cells per well). After incubation for 12 h, the culture medium was replaced with fresh medium at different pH values. To simulate acidic tumor microenviroment, the pH of cell culture was regulated to 6.5. HCl solution  was added into the medium (980 μL). The acidic medium was added along with CaO2@Co-ferrocene. Then, CaO2@Co-ferrocene at desired concentrations were added and incubated for another 24 h. Finally, culture medium containing 10% CCK-8 was added to each well and the absorbance was detected by a microplate reader at 450 nm after incubation at 37 °C for another 1 h.4T1 cells were seeded in 96-well plates . After incubation for 12 h, the cells were cultured in fresh medium containing different concentrations of CaO2@Co-ferrocene for another 24 h. Then, the medium was discarded, and the cells were washed three times with PBS before the addition of DCFH-DA . After staining, cells were washed with PBS for three times and DCF fluorescence images were observed by an inverted fluorescent microscope at an excitation wavelength of 480 nm. To detect intracellular Ca2+ ions, Fluo-4 AM, was applied as a green fluorescent probe according to the above steps. After treatments with CaO2@Co-ferrocene (60 μg/mL), 4T1 cells were lysed by RIPA lysis buffer with 1% PMSF. Supernatant containing H2O2 was collected by centrifugation at 12,000 rpm. Then, hydrogen peroxide assay kit was added and the absorbance at 560 nm was recorded with microplate reader.ROS was detected by a fluorescent probe, 2, 7- dichlorofluorescein diacetate (DCFH-DA). 4T1 cells were seeded in 6-well plates. After incubation for 12 h, the cells were cultured in fresh medium containing different concentrations of CaO2@Co-ferrocene, 4T1 cells were lysed by RIPA lysis buffer with 1% PMSF. The collected cells were further crushed with an ultrasonic probe. Supernatant containing proteins was collected by centrifugation at 12,000 rpm. After quantification, protein (50 μg) was loaded onto SDS-PAGE, further transferred to the PVDF membrane and blocked by 5% skim milk for 2 h. Then, the PVDF membrane was incubated with primary antibodies and secondary antibodies successively. After washing three times in Tris-buffered saline Tween buffer (TBST), the band intensity was measured.After treatments with CaO6) into the right side back of each mouse. The mice were randomly distributed into four groups for in vivo experiments (5 mice per group) when the tumor volumes reached about 80 mm3 and intratumorally injected with different formulations: Group 1: PBS, Group 2: Co-Fc MOF, Group 3: 5 mg/kg CaO2@Co-Fc, Group 4: 15 mg/kg CaO2@Co-Fc. The tumor size (V) was calculated as follows: V = width2 × length/2. Body weight, tumor size and images were recorded every 2 days. After 2 weeks, tumors were collected for immunohistochemistry analysis after sacrifice.Four-week-old female Balb/c mice were purchased from Shanghai Slac Laboratory Animal Co. Ltd. and used in compliance with guidelines of the Biological Resource Centre of the Agency for Science, Technology and Research, Zhejiang University. The tumor-bearing mouse model was built via injecting 50 μL PBS with 4T1 cells . Slices were incubated with ki67 overnight at 4 °C and then overlaid with secondary antibody for 20 min at room temperature. Finally, a diaminobenzidine tetrahydrochloride (DAB) working solution was applied and the slices were counterstained with hematoxylin.Data were expressed as mean ± SD. Comparison analysis between groups was conducted by student’s test.2 was firstly obtained through a typical wet-chemistry approach following the approach reported previously [2+ ions and ferrocene molecules were absorbed at the surface of CaO2 nanoparticles. Under the heating at 80 ℃, the Co-Fc coating was formed. As shown in the scanning electron microscopy  pattern of CaO2@Co-Fc corresponds to the characteristic peaks belonged to CaO2 , when CaO2@Co-Fc is immersed in an acidic solution for a certain period, a typical four-fold peak is observed, confirming the ROS generated belongs to hydroxyl radicals (·OH), and this is attributed to the Fenton catalytic reaction by the H2O2 generated and Co-Fc molecules  chromogenic method was used to evaluate the Fenton activity of as-prepared CaOt 650 nm , 25. As 2@Co-Fc was investigated using two types of normal cell lines. As shown in Fig. 2@Co-Fc shows negligible cytotoxicity toward human liver cells (7702) and human umbilical vein endothelial cells (HUVEC) at the concentration range of 0–80 μg/mL. In contrast, pure CaO2 nanoparticles cause clear negative effect to the viability of 7702 normal cells , H2O2 content is ~ 26% higher in cells treated with CaO2@Co-Fc. In addition, the intracellular ROS was also be detected using 2,7-Dichlorodi-hydrofluorescein diacetate . The intracellular ROS fluorescence images show that the CaO2@Co-Fc-treated cells present bright fluorescence . As shown in Fig. 2@Co-Fc-treated cells compared with the control group, indicating that CaO2@Co-Fc could be effectively disintegrated in 4T1 cells and generate significant Ca2+ aggregation. Western blot assay was used to investigate the underlying interaction behind these phenomena. In general, the surges of ROS and Ca2+ ions are intrinsically avoided by tumor cells to prevent apoptosis [2+ channels or its regulators. For example, high levels of oxidative stress can inhibit cytoplasmic Ca2+ extrusion through the downregulation of plasma membrane Ca2+ ATPase [2+efflux pumps). Meanwhile, transient receptor potential (TRP) channels can enhance Ca2+ entry [2@Co-Fc, indicating that nanoparticles induce considerable influence on the activity of Ca2+ channel-related protein by inducing oxidative stress. Mitochondria and endoplasmic reticulum play a crucial role in shaping the Ca2+ signal. When intracellular calcium surges, as the outcomes of calcium overload, the ER stress and mitochondrial dysfunction arise  with different treatments , and treated via intratumoral injection  CaO2 and (b) CaO2@Co-Fc. Fig. S3. High-resolution XPS spectra of (a) Co 2p, (b) Fe 2p and (c) O 1 s. Fig. S4. Elemental mapping of CaO2@Co-Fc. Fig. S5. XRD pattern of CaO2@Co-Fc. Fig. S6. Standard curves of H2O2 at the peak of 372 nm by TMB method: (a) UV–vis absorbance spectra and (b) plotting curve of TMB solution with the addition of different concentrations of H2O2. Fig. S7. The viability of 7702 cells cultured with CaO2 with varied concentrations. Fig. S8. Bright field images of 4T1 cells incubated with different concentrations of CaO2@Co-Fc after calcein-AM and PI staining for live & dead. Fig. S9. Colony formation of 4T1 cells incubated with different concentrations of CaO2@Co-Fc. Fig. S10. Bright field images of 4T1 cells incubated with different treatments after DCFH-DA staining for intercellular ROS. Fig. S11. Bright field images of 4T1 cells incubated with or without CaO2@Co-Fc after Fluo-4 AM staining for intercellular Ca2+ accumulation.


Lens culinaris Medik.) is a widely consumed edible crop possessing a high level of Zn and Fe micronutrients. Thus, the present study was conducted to examine the influence of foliar application of Zn and Fe on productivity, concentration, uptake and the economics of lentil cultivation (LL 931). For this, different treatment combinations of ZnSO4·7H2O (0.5%) and FeSO4·7H2O (0.5%), along with the recommended dose of fertilizer (RDF), were applied to the lentil. The results of study reported that the combined foliar application of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at pre-flowering (S1) and pod formation (S2) stages was most effective in enhancing grain and straw yield, Zn and Fe concentration, and uptake. However, the outcome of this treatment was statistically on par with the results obtained under the treatment ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage. A single spray of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage enhanced the grain and straw yield up to 39.6% and 51.8%, respectively. Similarly, Zn and Fe concentrations showed enhancement in grain  and straw (27.5% and 27.6% respectively) of the lentil. The increase in Zn and Fe uptake by grain was 54.8% and 68.0%, respectively, whereas uptake by straw was 93.6% and 93.7%, respectively. Also the benefit:cost was the highest (1.96) with application of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage. Conclusively, the combined use of ZnSO4·7H2O (0.5%) + FeSO4·7H2O (0.5%) at S1 stage can contribute significantly towards yield, Zn and Fe concentration, as well as uptake and the economic returns of lentil to remediate the Zn and Fe deficiency.Biofortification of pulse crops with Zn and Fe is a viable approach to combat their widespread deficiencies in humans. Lentil ( Lens culinaris Medik.) is a vital grain legume crop grown worldwide, and is also considered as the cheapest source of protein and micronutrients such as Zn and Fe with the concentrations ranging from 44–54 mg·kg−1 and 73–90 mg·kg−1, respectively [Pulses and legumes belong to the nutritionally rich Fabaceae family and are an important source of plant proteins, having a low glycemic index. Apart from that, they are great reservoirs of vitamins, minerals and complex carbohydrates that are crucial for optimum growth as well as development . Among dectively ,3. Howevectively ,7. Howevectively ,9,10. Moectively .Intense agricultural activities due to the burgeoning human population have resulted into micronutrient deficiencies in soil. Zinc deficiency is prevalent worldwide, especially in calcareous soils. In India, 51.2 and 19.2% of soils were found to be deficient in Zn and Fe, respectively . MicronuVarious approaches such as biofortification, nutrient supplementation, fortification, and diet diversification have been implemented in order to overcome micronutrient malnutrition . HoweverSeveral research studies have reported the increase in bioavailability of micronutrients through foliar application. The foliar application of Se at full bloom stage of lentil has been demonstrated to increase the seed Se concentration significantly . AnotherRabbi season (November-April) at the experimental farm, Department of Soil Science, Punjab Agricultural University, Ludhiana, Punjab. The research trial was conducted under the bio-fortification mandate of AICRP-MSN project, fully funded by IISS (ICAR), Bhopal, India. The sandy loam soil of this experimental field exhibited a pH of 7.21, electrical conductivity (EC ) 0.34 dS·m−1 and organic carbon (OC) 0.31%. The initial level of diethylene triamime penta acetic acid (DTPA) -extractable micronutrients viz. Zn, Cu, Fe and Mn in soil were 1.16, 0.65, 4.86 and 3.91 mg·kg−1, respectively. The climate in the region is subtropical, with hot, wet summers and dry winters. The annual rainfall of 400–600 mm was observed with the months of July to September receiving the majority of the rainfall, which is approximately 70% of the total. The total rainfall during the crop season from October to April was 219 and 68.9 mm during 2019–2020 and 2020–2021, respectively. During the growth season of lentil, the average monthly maximum temperature in the study region varied between 15.9 °C and 32.8 °C in 2019–2020 and 16.4 °C and 34.2 °C in 2020–2021, but the minimum temperature varied between 6.7 °C and 18.4 °C in 2019–2020 and 7.1 °C and 17.0 °C in 2020–2021 (The two-year experiment (2019–2020 and 2020–2021) was conducted during 020–2021 .4.7H2O (0.5%) and FeSO4.7H2O (0.5%) i.e., 1.25 kg·ha−1 fertilizer applied at pre-flowering (S1) and pod formation (S2) stages i.e., 80 and 100 days after sowing, respectively. For fertilizer application, 250 L·ha−1 water was used, thus, 1.25 kg·ha−1 fertilizer was applied for each 0.5% fertilizer application. In total, seven treatments were involved along with three replications in a complete randomized block design. The treatment details are as follows . The sowing was performed by the pora method with row to row spacing of 22.5 cm and plot size 3 m × 3.2 m.The study involved different combinations of RDF with ZnSO follows . The fie3 and HClO4 acid in ratio 3:1 on an electric hot plate [Plants were harvested manually when they attained physiological maturity followed by the collection of grain and straw samples for analysis (500–550 plants per plot). The samples were air-dried before drying in an oven at 65 °C for 48 h in order to determine the dry weights of plant components. Oven-dried plant samples were further grounded to fine material using a mechanical grinder. A representative grounded straw sample of 1.0 g and grain sample of 0.5 g were digested using a di-acid mixture comprising of HNOot plate . The micMEI) was calculated by using the following equation:The mobilization efficiency index (Zn), (PEFe), the apparent recovery efficiency of Zn (ARE-Zn), Fe (ARE-Fe) and the mobilization efficiency index (MEI-Zn), (MEI-Fe) of foliar-applied Zn and Fe were determined with the following equations [tY and cY represent the grain yield (kg·ha−1) of lentil in fertilized plots and in control, respectively; tNU and cNU represent the total nutrient  uptake (kg·ha−1) of lentil in fertilized plots and in control, respectively.The physiological efficiency of Zn and Fe viz. (PEquations .(3)PE=Y−1 for various treatments in the experiment was calculated separately, considering the prevailing prices of fertilizers in USD at the time of their use. The price of ZnSO4.7H2O (0.5%) and FeSO4.7H2O (0.5%) were 6.67 USD kg−1 and 8.01 USD·kg−1, respectively. The price of seed and the selling price were taken as 1.60 USD·kg−1 and 0.80 USD·kg−1, respectively, whereas the standard labour cost was 4.94 USD. Gross return  was calculated based on the MSP (price for minimum support) of lentil by the Indian government during the years of study. Net return (USD·ha−1) was obtained by subtracting the fertilizer cost from the gross return as given below.The cost of fertilizer in US dollars (USD) haB:C ratio was calculated by using the following equation:The data was analysed using SPSS version 16.0  packages. Analysis of variance (ANOVA) followed by a Duncan Multiple Range test was performed to test the significant difference between the treatment results.The two-year mean data demonstrated that the foliar application of Zn and Fe at S1 + S2 stages posed a significant impact on grain and straw yield of lentil .−1), which was not statistically different with the results of treatments T4 (961 kg·ha−1) and T6 (933 kg·ha−1). The minimum value of grain yield was observed in treatment T1 (688 kg·ha−1), which was not statistically different with treatment T2 (725 kg·ha−1). In case of straw yield, the highest value of two years of mean data was achieved in treatment T7 (3539 kg·ha−1), which was not statistically different from treatments T4 (3290 kg·ha−1) and T6 (3288 kg·ha−1), whereas the minimum straw yield was observed in treatment T1 (2166 kg·ha−1), which was not statistically different with treatment T2 (2417 kg·ha−1). Thus, statistically, foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) was equally effective at enhancing the grain and straw yield of lentil as FeSO4.7H2O (0.5%) at S1 + S2 stages as well as ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) at S1. Also, the foliar application of ZnSO4.7H2O (0.5%) along with RDF at S1 did not significantly affect the grain and straw yield of lentil.The highest value of grain yield was recorded in treatment T7 (987 kg·ha−1) was most effective in comparison to treatment T1, in which the least value of grain Zn concentration (55.9 mg·kg-1) was observed. Thus, foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) along with RDF at S1 + S2 stages was most effective to enhance the grain Zn concentration in lentil. The results of grain Fe concentration in lentil demonstrated that the maximum Fe concentration was recorded in treatment T7 (82.9 mg·kg−1), which was not statistically different with treatment T4 (78.9 mg·kg−1) and T6 (77.6 mg·kg−1). The minimum Fe concentration in grain was recorded in treatment T1 (65.5 mg·kg−1), which was not statistically different from treatment T2 (68.6 mg·kg−1).The two-year data for grain Zn and Fe concentration in lentil in response to foliar application of Zn and Fe at various stages of growth is given in The results of two years of data concerning the Zn and Fe concentration in the straw of lentil with foliar application of Zn and Fe are given in −1), which was not statistically different with treatment T4 (47.2 mg·kg−1). Minimum straw Zn concentration was observed in treatment T1 (37.0 mg·kg−1), which was not statistically different with treatment T3 (40.0 mg·kg−1). Thus, foliar application of FeSO4.7H2O (0.5%) along with RDF at S1 stage did not significantly affect the straw Zn concentration of lentil. The recorded data for Fe concentration in straw showed that treatment T7 was most effective to enhance the Fe concentration in straw (159 mg·kg−1), which was not statistically different with treatment T6 (149 mg·kg−1) and T4 (148 mg·kg−1). The Fe concentration in straw was minimum in treatment T1 (116 mg·kg−1). Thus, statistically, foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) either applied at the S1 stage or S1 and S2 stages are equally effective at enhancing the Zn and Fe concentration of lentil in straw.Maximum straw Zn concentration in two years was observed in treatment T7 (51.8 mg·kgThe Zn and Fe uptake by grain in lentil significantly increased with sole and combined use of Zn and Fe at different stages of growth as shown by the results presented in −1), which was not statistically different with treatment T4 (59.6 g·ha−1). The least Zn uptake by lentil grain was observed in treatment T1 (38.5 g·ha−1), which was not statistically different with treatment T2 (43.1 g·ha−1). The scrutiny of two-year data for grain Fe uptake in lentil demonstrated that the maximum Fe uptake was observed in treatment T7 (81.8 g·ha−1), and the minimum was observed in control (45.1 g·ha−1), which was not statistically different with treatment T2 (49.7 g·ha−1). Thus, foliar application of ZnSO4.7H2O (0.5%) at S1 stage did not significantly affect the grain Zn and Fe uptake of lentil. Overall, foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) either applied at the S1 stage or the S1 and S2 stages are equally effective at enhancing the grain Zn uptake in lentil.Maximum Zn uptake was recorded in treatment T7  and the minimum was observed in control . Thus, foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) along with RDF at the S1 and S2 stages was most effective in enhancing the Zn and Fe uptake in the straw of lentil. Moreover, the micronutrient uptake was higher in the treatments in which FeSO4 (T3 and T6) was applied as compared to ZnSO4 (T2 and T5).The two-year data of Zn and Fe uptake by straw in lentil as affected by sole and combined foliar application of Zn and Fe at different growth stages has been given in The results of 4 and FeSO4 is shown in The economic analysis of lentil cultivation as affected by foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) either at S1 or at the S1 and S2 stages significantly increased the grain yield of lentils, which might be ascribed to the synergistic interactions between Zn and Fe. Zinc acts as an important structural component of various enzymes involved in the metabolism of plant growth and yield component. Zinc also plays a crucial role in sugar and protein synthesis as well as seed production. Zinc application to lentil at the reproductive phase might have induced the structural and functional alterations in pollen grains and stigma of the plants, thus increasing the seed setting of lentils in contrast to unfertilized plots [4.7H2O and 0.5% FeSO4.7H2O was found to be more effective at increasing the grain and straw yield of chickpea as compared to the sole application of Zn and Fe. Moreover, the treatment in which fertilizers were applied at the pre-flowering and pod-formation stages was more effective as compared to the treatment when fertilizers were applied only at the pre-flowering stage [Grain yield is a crucial parameter of the crop grown for commercial purposes. The results of ed plots . Anothered plots . Thus, fed plots . Apart fed plots . In gooded plots ,33. Habied plots . Likewisng stage .4.7H2O and 0.5% FeSO4.7H2O was found to be more effective to increase the Zn and Fe concentration as compared to the sole application of Zn and Fe. Moreover, the treatment involving the fertilizer application at pre-flowering and pod-formation stages was more effective as compared to the single spray of fertilizers at the pre-flowering stage of Indian mustard [Although the micronutrient (Zn and Fe) requirement of the crop is very small, yet it is essential for optimum plant growth. Foliar application of Zn (single or double spray) resulted in higher Zn concentrations in the grain and straw of lentil in comparison to untreated plants, which might be ascribed to the immediate absorption of Zn by plant leaves as the nutrient spray is directed toward the leaves. Higher Zn concentration in grain and straw with two Zn sprays over the single Zn spray was associated with higher Zn availability through two sprays. For instance, foliar application of micronutrients in oilseed crops at the S1 and S2 stages has resulted in a significant increase in micronutrient concentration in the edible part of the crop . Another mustard .4 + FeSO4 was most effective at increasing the uptake of Zn and Fe in grain and straw. Moreover, the Zn and Fe uptake was higher with foliar Fe application as compared to the foliar Zn application due to significantly higher yield with Fe supplementation.The results of the study showed that micronutrient (Zn and Fe) uptake has been found to increase significantly with external supplementation. The trend can be coupled with the combined effect of yield and concentration. Overall, the combined application of ZnSO4 at the S1 and S2 stages is most effective at increasing the lentil production as compared to the ZnSO4 (0.5%) and ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%). The results of MEI indicated that the MEI of Zn and Fe were higher in control and the values were higher for MEI-Zn as compared to MEI-Fe, which suggests higher mobility of Zn as compared to Fe. The ARE measured the extent of nutrient loss from the cropping system and effectiveness of management practices. The results suggested that foliar application of a particular nutrient is effective in overcoming the nutrient losses. The results of PE indicate the increase in grain production with the absorbed nutrient. The higher values PE-Zn and PE-Fe were observed in treatment where the FeSO4 was applied as compared to the treatment in which ZnSO4 was applied. A similar increase in efficiency indices has also been observed in Indian mustard with Zn and Fe application [The agronomic efficiency reflects the impact of fertilizer applied on economic returns. The trend suggested that the application of 0.5% FeSOlication .4.7H2O and FeSO4.7H2O improved the economic outcomes of lentil cultivation. The results are in alignment with previous studies, where micronutrient application has increased the B:C ratio of sesame cultivation [4.7H2O (0.5%) + FeSO4.7H2O (0.5%) is more effective over the single spray. Moreover, the combined application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) is more effective over the sole application of nutrients. In agreement with the present results, the combined application of 0.5% ZnSO4.7H2O and 0.5% FeSO4.7H2O was found to be more profitable as compared to the sole application of Zn and Fe for chickpea cultivation. Moreover, the double spray of 0.5% ZnSO4.7H2O + 0.5% FeSO4.7H2O at the pre-flowering and pod-formation stages was found to be more economical as compared to the single spray of 0.5% ZnSO4.7H2O and 0.5% FeSO4.7H2O at the pre-flowering stage of chickpea [The results of the economic analysis indicated that the application of ZnSOtivation . Also, tchickpea .Lens culinaris Medik) is a leguminous crop predominantly grown in Asia whose productivity can be influenced by the application of micronutrients in order to enhance their availability to consumers worldwide. The combined foliar application of Zn and Fe through ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) at the pre-flowering + pod formation stages was found to be efficient in improving the grain and straw yield, Zn and Fe concentration and uptake, apparent recovery efficiency and benefit:cost ratio. The results of foliar application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) at the pre-flowering + pod formation stages were statistically at par with the results obtained at the pre-flowering stage. Thus, the present study points towards the potential application of ZnSO4.7H2O (0.5%) + FeSO4.7H2O (0.5%) at the pre-flowering stages as the most effective treatment for enhancing yield, nutrient concentration and economic returns of lentil grown in sandy loam soils in northwestern India. In sum, dual-biofortified lentil can contribute significantly in increasing the bioavailability of Zn and Fe to the population at risk of Zn and Fe deficiency.Lentil (


Although X atoms possess very high ionization energies, these resulting X@36adz complexes possess low adiabatic ionization energies (AIEs) of 0.78–5.28 eV. In particular, the adiabatic ionization energies (AIEs) of X@36adz  are even lower than the ionization energy (3.89 eV) of Cs atoms, and thus, can be classified as novel nonmetallic superalkalis. Moreover, due to the existence of diffuse excess electrons in B@36adz, this complex not only possesses pretty low AIE of 2.16 eV but also exhibits a remarkably large first hyperpolarizability (β0) of 1.35 × 106 au, indicating that it can also be considered as a new kind of nonlinear optical molecule. As a result, this study provides an effective approach to achieve new metal-free species with an excellent reducing capability by utilizing the cage-like organic complexants as building blocks.In this study, to examine the possibility of using cage-like complexants to design nonmetallic superalkalis, a series of X@3 In MLk+1, one more alkali metal atom will bring an extra valence electron for the electronic shell of M according to the octet rule. Consequently, such an MLk+1 complex has a great tendency to lose the extra valence electron and thus possess strong reducibility (Reducing agents with low ionization energies (IEs) play a crucial role in chemical synthesis. As is well-known, alkali metal atoms possess the lowest ionization energies (5.39–3.89 eV)  among alcibility .2 and N2 molecules  superalkali cations by using hydrogen atoms as ligands. Following a similar rule, the other two series of nonmetallic superalkali cations, namely, FnHn+1+ (n = 1–10) and CxHx+14+ (x = 1–5), have been proposed by In view of the great importance of superalkalis in chemistry, various superalkalis have been theoretically  and expeinuclear  and polyinuclear  superalkinuclear , Zintl-iinuclear , hyperalinuclear , alkali-inuclear , and so inuclear , particuinuclear , have beinuclear  designedMore recently, 6adamanzane (36adz) has been chosen as a representative to design a series of X@36adz  by encapsulating nonmetallic atoms into the cavity of this cage-like complexant in this work  of 8.15–17.42 eV  molecule with a remarkably large first hyperppolarizability of 1.35 × 106 au because the valence electron of boron atom is pushed out of cage to form diffuse excess electrons. We hope that this work will not only provide new nonmetallic members for the superatom family, but will also open the door to design strong reducing matters by embedding nonmetallic atoms into the various cage-like complexants.To verify this hypothesis, the 3work see . The 36ac chains , which hadz]+Na− . In thisadz]+Na− . Under t17.42 eV . In part6adz  level. Based on the optimized structures, the single-point energies, nature population analysis (NPA) charges, and static electric properties were calculated at the CAM-B3LYP/6-311++G  level.In this work, all the calculations were carried out by using the coulomb-attenuated hybrid exchange-correlation functional (CAM-B3LYP) , which h6adz see  to verif6adz  were calculated as the energy difference between the optimized neutral complex and the cation in the geometry of the neutral complex, while their adiabatic ionization energies (AIEs) are defined as the energy difference between the neutral and cationic complex at their respective optimized structures. In addition, the TD-M06-2X calculations were performed to obtain the transition energies and oscillator strengths of the crucial excited states as well as the difference of the dipole moments between the ground state and crucial excited state of X@36adz by using the 6-311++G  basis set. Herein, the dipole moments (µ0), polarizabilities (α0), and first hyperpolarizabilities (β0) are defined as follows,i, j = {x, y, z}.In this work, the vertical ionization energies (VIEs) of X@3All the above calculations were performed by using the GAUSSIAN 16 program package . The dim6adz  compounds have been constructed by encapsulating one X atom into a 36adz cage. After optimization, the geometric structures of X@36adz are illustrated in 6adz compounds are summarized in Initially, seven X@36adz is a cage-like complexant with S4 symmetry. From 6adz cage is well-preserved in these X@36adz compounds. However, the geometric symmetries of these compounds are lowered to C1 and C2, except for H@36adz, which maintains the S4 symmetry of 36adz. To be specific, the encapsulated hydrogen atom located at the central position of 36adz in H@36adz, yields the newly formed N-H bonds of 2.11 Å and ∠N1-H-N2 of 113.5°. As for B@36adz, the boron atom tends to bind with 3 N atoms of the complexant, forming 3 N-B bonds of 1.63 Å ∼ 1.66 Å, while the distance between the uncombined N and B atoms is as long as 3.02 Å. The C@36adz complex possesses a C2-symmetric structure, where the introduced carbon atom prefers to bind with 2 N atoms of 36adz by forming two N-C bonds of 1.52 Å. Differently, the more electronegative N, O, and F atoms are linked to only 1 N atom of the cage complexant via N-N, N-O, and N-F bonds of 1.41, 1.34, and 1.87 Å, respectively, generating the very similar structures of X@36adz . Similar to B@36adz, the introduced silicon atom tends to bind with 3 N atoms of complexant via 3 N-Si bonds of 2.06–2.35 Å in Si@36adz.As shown in 6adz, it is found that only the optimized structure of [B@36adz]+ cation almost coincides with the geometry of the corresponding neutral one , quite different geometries of cationic and neutral complexes were found. For instance, the H+ is attached to 1 N atom of the complexant in the resulting [H@36adz]+, while the doped N atom turns to combine with 2 N atoms of 36adz in [N@36adz]+ and Si atom almost moves to the center of the cage in [Si@36adz]+. The geometry of C@36adz is distorted from C2 symmetry to C1 with the changes of 0.29 Å for the C-N2 bond and 7.3° for ∠N1-C-N2. As for [F@36adz]+, the N-F bond is shortened from 1.87 Å to 1.38 Å because the introduced F atom further loses 0.333e  species.By turning to the cations of X@3 one see . For ins333e see  and thus6adz  complexes, although X atoms possess very high ionization energies (IEs) of 8.15–17.42 eV , B@36adz (2.16 eV), C@36adz (2.72 eV), N@36adz (3.15 eV), and Si@36adz (1.79 eV) are even lower than the IE of 3.89 eV  than that (6.80 eV) of 36adz. In particular, B@36adz exhibits the highest HOMO level of −1.81 eV, and thus possesses the lowest VIE of 2.18 eV among these X@36adz complexes. This is because that the valence electron of embedded boron atom is pushed out of the cage by the lone pairs of N atoms of the complexant, forming a electride-like molecule [B+@36adz](e‒) with obvious diffuse electrons in the HOMO of B@36adz , which show obvious antibonding character with respect to the central atom-complexant interaction. Such antibonding HOMOs destabilize the neutral structures of X@36adz  and result in their low VIE values  are larger than that of 5.39 eV for Li atom, although their HOMOs also possess obvious antibonding character. This is attributed to the larger elertonegativities of N, O, and F atoms than H, C, and Si atoms, which hinders the ionization of the valence electrons on their np orbitals in the HOMOs of X@36adz .Differently, as shown in E values . Hence, 6adz. To be specific, the geometric structure of B@36adz is hardly changed after its diffuse excess electron of HOMO is lost, resulting in its nearly equal VIE (2.18 eV) and AIE (2.16 eV) values. However, the destabilization of antibonding HOMOs for the neutral X@36adz  complexes drives the embedded X atom to lose nearly one valence electron . Note that the AIE of H@36adz is as low as 0.78 eV because the formed [H@36adz]+ is very stable and has been identified in various synthesized ionic compounds, such as [H@36adz]+X‒   .6adz, it is highly expected that this superalkali also exhibits considerable nonlinear optical (NLO) response. Thus, the static electric properties of these studied X@36adz compounds and 36adz complexant were calculated and listed in 6adz has the largest dipole moment (3.326 au) and polarizability  among these X@36adz complexes because of the existence of diffuse electrons in the HOMO of this superalkali. In particular, the first hyperpolarizability (β0) of B@36adz is as large as 1.35 × 106 au, which is significantly larger than those of the reported superalkalis and superalkali-based NLO materials, such as the aromatic organometallic superalkali Au3(Py)3 (3.74 × 104 au) M‒  (1.18 × 104–3.33 × 104 au) (3O@Al12N12 (8.73 × 105 au)  , superal 104 au) , and sup 105 au) , indicatβ0 value of B@36adz, we focus our attention on the simple two-level model  compounds, which rationalizes its largest β0 value among these studied X@36adz species. In addition, the proposed C@36adz and Si@36adz superalkalis also show considerable β0 values of 4.05 × 103 au and 1.95 × 104 au, respectively, because of their relatively smaller ΔE values and larger Δµ values.To understand the eminently large el model ,β0∝Δμ⋅fzability ,c. Hence6adamanzane (36adz) as a complexant, a series of X@36adz  compounds were constructed and studied based on the density functional theory. It is interesting to find that the X@36adz  complexes possess lower AIE values than the IE of Cs atoms though the X atoms and 36adz possess very high IE values. Thereby, they can be regarded as a new kind of nonmetallic superalkalis. In particular, different from other complexes, the low IE of B@36adz is derived from the diffuse excess electron formed by the repulsion between the valence electron of the embedded boron atom and lone pairs of N atoms of the complexant. Due to the existence of diffuse electrons, this superalkali also possesses a remarkably large β0 of 1.35 × 106 au, which can serve as a new kind of NLO molecule. Hence, it is highly hoped that the theoretical design and characterization of these nonmetallic superalkali species could provide meaningful references to further design novel reducing matters or NLO materials by using such cage-like molecules as complexants.By using 3