In this study, Kang et al. sought to understand the underlying principles that mediate transcriptional memory and reactivation in the daughter cells after mitosis. They used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications, and combined with EU-RNA-seq and Hi-C analyses, they found that during prometaphase promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac, thus providing new insights into the histone modification landscape during cell division. During mitosis, transcription of genomic DNA is dramatically reduced, before it is reactivated during nuclear reformation in anaphase/telophase. Many aspects of the underlying principles that mediate transcriptional memory and reactivation in the daughter cells remain unclear. Here, we used ChIP-seq on synchronized cells at different stages after mitosis to generate genome-wide maps of histone modifications. Combined with EU-RNA-seq and Hi-C analyses, we found that during prometaphase, promoters, enhancers, and insulators retain H3K4me3 and H3K4me1, while losing H3K27ac. Enhancers globally retaining mitotic H3K4me1 or locally retaining mitotic H3K27ac are associated with cell type-specific genes and their transcription factors for rapid transcriptional activation. As cells exit mitosis, promoters regain H3K27ac, which correlates with transcriptional reactivation. Insulators also gain H3K27ac and CCCTC-binding factor (CTCF) in anaphase/telophase. This increase of H3K27ac in anaphase/telophase is required for posttranscriptional activation and may play a role in the establishment of topologically associating domains (TADs). Together, our results suggest that the genome is reorganized in a sequential order, in which histone methylations occur first in prometaphase, histone acetylation, and CTCF in anaphase/telophase, transcription in cytokinesis, and long-range chromatin interactions in early G1. We thus provide insights into the histone modification landscape that allows faithful reestablishment of the transcriptional program and TADs during cell division. Mitosis marks a dramatic transition during which cells move from a transcriptionally active to a largely repressed state in which most genes are transcriptionally inactive . This drcis-regulatory elements (cis-REs), such as promoters and enhancers, and regulate gene expression via interaction with transcription factors. H3K4me3 and H3K27ac are highly enriched at active promoters near the transcription start site (TSS) and positively correlated with transcription. Enhancers play a crucial role in the activation and fine-tuning of their target promoters. Enhancer elements can exist in two major chromatin states, primed or active. Primed enhancers (PEs) are marked by H3K4me1/2 and their target genes are weakly or not expressed, whereas active enhancers (AEs) are additionally marked by the histone acetyltransferases CBP/p300-mediated H3K27ac and are associated with actively transcribed genes , and DNA loops are lost during prometaphase and reestablished in G1 phase . TADs arIn this study, we combined histone modification ChIP-seq, EU-RNA-seq, and Hi-C from prometaphase to G1 phase to comprehensively examine on a genome-wide level the temporal relationship between histone modification binding, nascent transcription, and long-range chromatin interactions during the mitosis–G1 transition. We found that H3K4me3 remains associated with almost all promoters and that H3K4me1 is highly retained at enhancers of cell type-specific genes during prometaphase. In contrast, H3K27ac is largely reduced during prometaphase, although a subset of H3K27ac remains at enhancers of cell type-specific genes and promotes early expression of these cell type-specific genes. During the anaphase/telophase transition, most promoters gain H3K27ac, and this is essential for the accurate transmission of gene expression programs during cell division. Furthermore, TAD boundaries during prometaphase remain associated with H3K4me3 and H3K4me1 in the absence of CTCF, but CTCF is recruited and enriched at the boundaries during anaphase/telophase together with H3K27ac before the appearance of loops, TADs, and compartments. Altogether, the multilevel data of epigenetic landscape, nascent transcription, and TAD interaction shed light on not only the temporal order of genome organization after mitosis but also indicate a potential bookmarking role for histone modifications in accurate re-establishment of transcriptional activation pattern and long-range chromatin interactions.Supplemental Fig. S1A). Thus, our synchronization approach enables us to collect enriched populations of cells at prometaphase, anaphase/telophase, and interphase, respectively.Transcription is reactivated at the time of mitotic exit, as the NE starts reassembling and RNA Pol II globally rebinds to the chromatin . It is tSupplemental Fig. S1B,C). To comprehensively examine the extent and genomic localization of mitotic occupancy of histone modifications, we performed ChIP-seq for H3K4me3, H3K4me1, and H3K27ac in U2OS and RPE1 cell lines during interphase, prometaphase, and anaphase/telophase . When there is a global signal change under different experimental conditions , typical normalization by the total number of mapped reads can be misleading since similar distributions of positions are sampled during sequencing. It is thus often challenging to quantitatively compare histone modification ChIP-seq data sets from different phases of the cell cycle. To overcome this, we used a spike-in based normalization strategy, whereby adding an equal amount of exogenous chromatin from a different species into each sample can be used as a more accurate normalization control  or active enhancers . We performed immunofluorescence in asynchronous cells and Western blotting in histone extracts from interphase, prometaphase, and anaphase/telophase enriched cells. We observed that all three histone modifications  were detected in all phases of mitosis from both U2OS and RPE1 cells , PE , and AE  elements based on our ChIP-seq data  and H3K4me1 peaks were depleted at TSS . Only a small fraction of interphase AEs retained H3K27ac in prometaphase (18.0%). Of AEs, 69.2% lost H3K27ac, but retained H3K4me1 in prometaphase cells . We found that the majority of PEs remained stable in prometaphase and anaphase/telophase  similar to H3K4me3+ promoters . These data suggest that promoters and enhancers continue to retain H3K4me3 and H3K4me1 respectively, as potential bookmarks, although they lose H3K27ac during prometaphase.We next wanted to determine how gene regulatory elements “remember” their histone modification dynamics across mitosis. To answer this question, we selected all promoters and enhancers from interphase and compared the dynamics of each histone modification from interphase to prometaphase or to anaphase/telophase cells. Promoters can be marked by either H3K4me3 only or by both H3K27ac and H3K4me3. We found that the majority of H3K4me3+ promoters remained in prometaphase (98.3%). In contrast, only 1.1% of promoters remained associated with H3K27ac in prometaphase and 87% of H3K4me3+ promoters were able to regain H3K27ac in anaphase/telophase . Notably, promoters retaining H3K4me3 during prometaphase, were strongly enriched for genes involved in fundamental cellular processes, such as RNA and protein metabolism, whereas AEs either retaining or losing H3K27ac during prometaphase were predominantly linked to differentiation or development-related genes in both cell lines. Specifically, we observed that AEs losing H3K27ac, but retaining H3K4me1 were most strongly associated with genes involved in bone mineralization in U2OS . Together, these results indicate that enhancers use H3K4me1 to maintain their cellular identity upon loss of H3K27ac throughout mitosis.Next, we asked whether continuous retention of H3K4me1 during prometaphase associates with a bookmark of cell type-specific genes. For this, we used U2OS cells, which are derived from cells of mesenchymal origin that differentiate to osteoblasts. There are several genes identified as essential for bone formation and differentiation to osteoblasts. These so-called osteoblast-specific genes include and SOST . Strikincis-REs, we performed de novo motif analysis of the bookmarked elements in U2OS cells. Our analysis showed that both AEs and PEs were preferentially enriched with motifs of the activator protein-1 (AP-1) family of TFs including FOSL2, JUN-AP1, FRA2, FRA1, and JUNB . Then, we captured nascent RNA by adding biotin-azide and generated cDNA libraries for sequencing. As there was a large difference in the amount of nascent RNA between interphase and mitotic cells, we used external controls that appropriately normalize the RNA-seq data . Consistent with the previous study, we found a hierarchy of gene reactivation at the mitosis to G1 transition (Histone modifications are found on mitotic chromatin with either a stable (H3K4me1/3) or reduced (H3K27ac) abundance when compared with interphase chromatin . Togetheansition . We alsoansition B. Gene Oansition C. HoweveR = 0.60) . AEs are associated with active transcription by bringing DNA loops physically close to gene promoters. However, for AEs, either H3K27ac or H3K4me1 level in mitosis was not positively correlated with gene expression  D. FurtheSupplemental Fig. S3). We thus asked whether H3K4me1 occupancy throughout mitosis associates with rapid transcriptional activation of cell type-specific genes. We obtained the list of 113 genes specifically expressed in bone or bone marrow, which are also expressed in U2OS, using the Tissue-specific Gene Expression and Regulation (TiGER) database . We then washed out both nocodazole and A-485 and released cells for 1 h or 24 h. Reduced H3K27ac binding from prometaphase chromatin was able to regain as cells exit mitosis to enter 1-h cytokinesis or 24-h next cell cycle . Second, to inhibit H3K27ac regaining from anaphase/telophase, we released cells from mitotic arrest while treating with A-485 for 1 or 3 h. The H3K27ac levels successfully decreased in both 1-h and 3-h treatment with A-485 . Next, nascent transcripts were pulse-labeled with the EU followed by sequencing and normalized by spike-in controls . We confirmed by EU-RNA-seq analysis that the biological replicates for each treatment were highly correlated  and that A-485 treatment resulted in extensive transcriptional changes for all treatments .Mitotic occupancy of histone modifications is associated with postmitotic gene activation suggestive of mitotic bookmarking. However, a recent study showed that inhibition of mitotic BRD4 binding did not affect transcriptional activation, suggesting that mitotic occupancy can be passively bound to mitotic chromatin and dispensable for transcription . To test2 fold change  upon A-485 treatment. When inhibiting mitotic H3K27ac, 56% (48% down and 8% up) of the genes in prometaphase and 45% (29% down and 16% up) of the genes in cytokinesis were dysregulated. Since the length of the cell cycle is ∼24 h for dividing mammalian cells, we let cells enter the next cell cycle upon mitotic inhibition of H3K27ac. Surprisingly, 45% (22% down and 23% up) of the genes in 24-h release cells were dysregulated, suggesting that mitotic H3K27ac is required for maintaining transcriptional memory. H3K27ac begins to get regained from anaphase/telophase . We assessed the reproducibility of Hi-C data between two biological replicates and observed a strong Pearson correlation coefficient at all time points . In agreement with previous observations, Hi-C contact maps showed that chromosomal interactions were no longer observed in mitotic cells . In concordance with our ChIP-seq analysis, immunofluorescence and Western blot analyses showed that CTCF is largely dispersed to the cytoplasm in prometaphase, then recruited on chromatin in anaphase/telophase . We then analyzed the distribution of CTCF binding peaks at the TAD boundaries in each cell cycle phase. As previously suggested, interphase CTCF binding was enriched at the TAD boundaries. We did not observe any enrichment at the boundaries in prometaphase, possibly due to the limited numbers of binding sites during this stage. Strikingly, anaphase/telophase CTCF binding was strongly enriched at the boundaries even higher than that seen in interphase  and 79% of H3K27ac   (1 peaks) B interph1 peaks) E,F. Thus1 peaks) I, indica1 peaks) J, indica1 peaks) K,L. TogeHere, we provide a temporal order of genome organization during and after mitosis. During prometaphase, histone methylation binding is largely retained, however only low levels of transcription occur and no long-range chromatin interactions are detected. Because histone modifications are established first, we investigated whether mitotic histone modification binding contributes to gene reactivation or to the formation of long-range chromatin interactions. We observed that levels of H3K4me3 and H3K4me1 are stable throughout mitosis at promoters and enhancers, respectively, and both at insulators. H3K4me1 remains at enhancers of cell type-specific genes and TFs binding motifs, which regulate cell type-specific gene expression during prometaphase. This indicates that histone methylations act as a widespread epigenetic memory of regulatory information previously active in the mother cells. Limited H3K27ac remains associated with mitotic chromatin for the accurate transmission of transcription programs and early expression of cell type-specific genes. Loss of H3K27ac in prometaphase is recovered in anaphase/telophase preferentially at promoters and insulators. This recovery of H3K27ac by anaphase/telophase is required for gene reactivation and is particularly relocalized at the TAD boundaries. When cells enter cytokinesis, most of the genes are transcribed. Long-range chromatin interactions gradually increase from cytokinesis, and are complete between 90 and 120 min G1. In addition to H3K27ac, CTCF lost in prometaphase is recruited in anaphase/telophase at the TAD boundaries, which may be involved in the reformation of TADs in the daughter cells. Overall, the genome is orderly reformed in which histone methylations are retained throughout mitosis, followed by histone acetylation and CTCF in anaphase/telophase, transcription in cytokinesis, then long-range interactions in 90 min G1 .cis-REs. First, H3K4me3 bookmarks promoters of genes during prometaphase. Ninety-eight percent of promoters retain H3K4me3 during prometaphase . Second, H3K4me1 bookmarks enhancers of cell type-specific genes during prometaphase. Cell type-specific genes tend to contain H3K4me1 during prometaphase and to be expressed early in the mitosis–G1 transition  that acetylates histones H3 and H4 such as H3K14ac, H3K27ac, H3K122ac, and H4K16ac . It bindRecently, a genome-wide RNA pulse-labeling study showed that some genes are actively transcribed in prometaphase arrested cells, and described waves of gene reactivation during mitotic exit . Using tMultiple studies show that CTCF remains associated with mitotic chromosomes using imaging and Western blot analyses , which p2. Cells were synchronized using thymidine and nocodazole treatments. The mitotic cells were collected by mitotic shakeoff. A-485  was used to inhibit H3K27ac levels. See the Supplemental Material for detailed description.U2OS cells were cultured in DMEM (Gibco) and 10% FBS. RPE1 cells were cultured in DMEM-F12 (Gibco) and 10% FBS. Cells were incubated at 37°C and 5% COCells were grown on glass coverslips and fixed with 4% paraformaldehyde in PBS for 5 min at room temperature. Fixed cells were permeabilized with cold PBS containing 0.5% Triton X-100 for 5 min. Cells were blocked with 1% BSA in PBS for 1 h at room temperature and incubated in primary antibody in blocking buffer overnight at 4°C, followed by secondary antibody for 1 h at room temperature. Cells were then briefly stained with 1 μg/mL Hoechst 33342 (Molecular Probes H-1399) in PBS and mounted in VectaShield (Vector Laboratories). Images were acquired in a Zeiss LSM710 confocal microscope. Images were analyzed and prepared for presentation in Photoshop.g for 10 min at 4°C. The supernatant containing the cytoplasmic fraction was transferred and saved. The pellet was resuspended in lysis buffer  for 30 min on ice, followed by centrifugation at 12,000g for 20 min at 4°C. The supernatant was collected as the nuclear fraction.Cells were resuspended in hypotonic buffer  for 10 min on ice , followed by centrifugation at 500For histone extraction, the protocol is adapted from previous work . BrieflyThe following antibodies were purchased from the indicated commercial sources: anti-H3K4me3 (Abcam ab8580), anti-H3K4me1 (Abcam ab8895), anti-H3K27ac (Abcam ab4729), anti-CTCF (Active Motif 61311), anti-spike-in (Active Motif 61686), anti-α-Tubulin (Sigma T5168), anti-Histone H3 , and anti-H3S10p .6) were cross-linked with 1% formaldehyde for 10 min, and ChIP-seq was performed as described previously  were cross-linked with formaldehyde. Chromatin was digested with a restriction enzyme MboI (NEB), biotinylated with biotin-ATP (Life Technologies), and then ligated with T4 DNA ligase (NEB). DNA was purified and sheared with Covaris LE220 instrument (Covaris). Biotinylated DNA interactions were pulled down with Dynabeads MyOne Streptavin T1 Beads (Life Technologies) and sequenced in a NovaSeq 6000 sequencing system (Illumina). See the Supplemental Material for detailed information about data analysis.Hi-C was performed using the in situ method as previously described . BrieflyThe Gene Expression Omnibus accession number for the ChIP-seq, EU-RNA-seq, and Hi-C data reported in this study is GSE141139.


P. betulaefolia and classified into 12 groups. Results were supported by the gene structural characteristics and phylogenetic analysis. These genes were unevenly distributed on 17 chromosomes. The gene structures of the C3H genes were relatively complex but conserved in each group. The C3H genes experienced a WGD event that occurred in the ancestor genome of P. betulaefolia and apple before their divergence based on the synonymous substitutions (Ks) values. There were 35 and 37 pairs of paralogous genes in the P. betulaefolia and apple genome, respectively, and 87 pairs of orthologous genes between P. betulaefolia and apple were identified. Except for one orthologous pairs PbeC3H66 and MD05G1311700 which had undergone positive selection, the other C3H genes had undergone purifying selection. Expression profiles showed that high salinity stress could influence the expression level of C3H genes in P. betulaefolia. Four members were responsive to salt stress in roots, nine were responsive to salt stress in leaves and eight showed inhibited expression in leaves. Results suggested important roles of PbeC3H genes in response to salt stress and will be useful for better understanding the complex functions of the C3H genes, and will provide excellent candidates for salt-tolerance improvement.Transcription factors regulate gene expression in response to various external and internal cues by activating or suppressing downstream genes. Significant progress has been made in identifying and characterizing the Cysteine3Histidine (C3H) gene family in several dicots and monocots. They are characterized by their signature motif of three cysteine and one histidine residues, and reportedly play important roles in regulation of plant growth, developmental processes and environmental responses. In this study, we performed genome-wide and deep analysis of putative C3H genes, and a total of 117 PbeC3H members, were identified in  The zinc finger protein (ZFP) is one of the largest and specific transcription factor families in plants. Members are characterized by common zinc finger (Znf) motifs in which cysteines and/or histidines coordinate with a few zinc ions to form the local peptide structures . The ZFP4−17-C-X4−6-C-X3-H (X represents any amino acid)   ZFPs are characterized by a Znf motif consisting of three cysteines and one histidine coordinated by a zinc cation , which ano acid) . A whole-C-X3-H) .Arabidopsis, HUA1, the first reported C3H-type ZFP with six tandem C3H motifs, was identified as an RNA-binding protein and likely participates in a new regulatory mechanism for flower development  and AtTZF11 (AtSZF1/AtC3H47) are involved in salt stress responses  are one of the most important fruit crops in the Rosaceae family. They are the third most important fruit crop in temperate zones, after grape and apple  of NCBI (https://www.ncbi.nlm.nih.gov/bioproject/) (P. bretschneideri were got from genome (version v1.1) project (http://peargenome.njau.edu.cn/). The Malus domestica C3H gene files were downloaded from the Genome Database for Rosaceae (https://www.rosaceae.org/). The Hidden Markov Model (HMM) profiles of C3H domains (PF00642) were obtained from the Pfam database (http://pfam.xfam.org/), and it was employed as a query to identify all possible C3H proteins using HMMER (V3.0) software (http://smart.emblheidelberg.de/) and MOTIF Search (https://www.genome.jp/tools/motif/).The latest version of the roject/) . The C3Hsoftware . Then thhttp://www.expasy.org/tools/). The motif analyses of the PbeC3Hs were detected using MEME online software (http://meme-suite.org/tools/meme/) with the default parameter settings, the width of motifs was set from 6 to 50, and the number of motifs was 2 to 15. The gene structures of the PbeC3Hs were parsed from the general feature format (GFF) files of the P. betulaefolia genome database, and diagrams of the exon-intron structures were drawn using the online program Gene Structure Display Server .Protein properties, including three fields  of each PbeC3H protein were calculated by online ExPasy program  (http://mapinspect.software.informer.com/).Positional information of all the C3Hs was parsed from the All obtained proteins were aligned using ClustalX2.0 . PhylogeKa)/synonymous substitutions (Ks) was evaluated to determine homologous relationships and divergence time of C3H genes. Ka and Ks values, and the ratio of Ka/Ks of C3H homologous gene pairs in P. Betulaefolia or in apple, and orthologous gene pairs between P. betulaefolia and apple were calculated using DnaSP v5  of 9.26 synonymous substitutions per 109 years . About 45 days later, at the eight-leaf stage, the roots of the P. betulaefolia plants were immersed into solution with 200 mM NaCl and the deionized water as controls as the treatment before  of P. betulaefolia was used as internal controls for normalization  from leaf and root tissues according to the manufacturer’s instructions. The expression of PbeC3H genes was analyzed using a BIO-RAD CFX Connect Real-Time system  with the SYBR Green Master Mix . Gene-specific primers were designed based on the gene sequences using Primer Premier 5.0 . lization . The effP. betulaefolia  (The released whole-genome sequence of pear  and P. blaefolia  was usedbeC3H117 . Based o-C-X3-H) . CharactP. betulaefolia (117) than that in pear (99) and reported for Arabidopsis (68) and rice (67) (Arabidopsis (152) and rice (150)  and C-X8-C-X5-C-X3-H (31.0%)  . The numice (67) . the on ice (67) , Arabidoce (150) . We founce (150) , consistce (150) . We foune domain . As simi (31.0%) . The motbeC3H117 .PbeC3H65, PbeC3H95 and PbeC3H117 had one or two introns while PbeC3H81 showed much more complex gene structures  remained on unattributed scaffold or contig fragments  followed by chromosome 11 (ten) and chromosome 2, 9 and 11 (eight each), and sevsn each on chromosome 7 and 17. Notably, some C3Hs located on chromosomes 3, 6, 9, 11, 14 and 15 were arranged in clusters , prior to divergence of the two groups ∼22.4–29.4 MYA, but after their divergence from strawberry   event shared by rawberry . Analysionducted . Resultsectively . All theectively  (Krzywinectively . The Ks 9.4 MYA) . This sueciation . Moreoveancestor . This inGD event .Ks) and non-synonymous substitutions (Ka) per site between duplicated pairs; Ka/Ks = 1 indicates neutral selection, Ka/Ks <1 indicates purifying selection, and Ka/Ks >1 indicates accelerated evolution with positive selection  undergone strong positive selection  analysis was performed to illustrate the function classification of the 117 C3H genes   were highly induced by salt stress treatment in roots  were significantly induced by salt at least at two time points , followed by pear (218), Arabidopsis (152) and rice (150)  . The MEMce (150) . Similar5-C-X3-H . In addi5-C-X3-H . A previ5-C-X3-H .We also noted that the pI ranged from 4.72 (PbeC3H101) to 9.69 (PbeC3H101). It is because of the length of protein sequences were quite different between members and contained various motifs besides the basic C3H motif. The various values of pI may indicate different physicochemical property or three-dimensional structure to affect gene functions.AtHUA1, AtPEI and OsLIC1)  . The domK motifs . The othK motifs . These vP. betulaefolia and apple, prior to divergence of these two taxa  showed strong positive selection  than that in P. bretschneideri (99)  , grape (eri (99) , poplar eri (99) . The expPbeC3H30 was significantly induced after 24 h of salt stress, and PbeC3H59 responded to salt stress at 24 and 48 h (PbeC3H2, PbeC3H30, PbeC3H47, PbeC3H67, PbeC3H96, PbeC3H77, PbeC3H88, PbeC3H92 and PbeC3H113 were induced under salt stress in leaves, especially for PbeC3H77, PbeC3H88 and PbeC3H113 (OsC3H33, OsC3H37 and OsC3H50 were induced by salt stress (OsC3H12 positively and quantitatively regulates rice resistance to bacterial leaf blight caused by Xanthomonas oryzae pv oryzae, which is likely associated with the jasmonic acid dependent pathway (P. betulaefolia.In this paper, we identified some important candidate genes that were highly or specifically expressed after salt stress treatment; for example, in roots, and 48 h . Genes PbeC3H113 . A numbebeC3H113 . In ricet stress . Another pathway . These gPbeC3H genes were repressed under salt stress or sensitive to salt stress; for example, PbeC3H27, PbeC3H34, PbeC3H42, PbeC3H82, PbeC3H81, PbeC3H32, PbeC3H101 and PbeC3H110 (PbeC3H42 PbeC3H81, PbeC3H32 and PbeC3H110 were significantly reduced after salt stress treatment. Especially for PbeC3H81, this was about 10% of the expression of controls (P. betulaefolia.We also found that some beC3H110 . These gcontrols . These gcontrols  are alsoP. betulaefolia and conducted a detailed investigation of their classification, structure, gene evolution and expression profiles under salt stress. All the 117 PbeC3H genes were classified into 12 groups based on the organization of various characteristic domains and mapped onto 17 chromosomes. The identification and classifications were supported by structural characteristics of the genes and proteins, as well as by phylogenetic analysis. There were 35 and 37 pairs of paralogous genes in the P. betulaefolia and apple genome, respectively, and 87 pairs of orthologous genes between them. Except for one orthologous pairs PbeC3H66 & MD05G1311700 which had undergone positive selections, the other C3H genes had undergone purifying selection. And the C3H genes expanded in P. betulaefolia than that in P. bretschneideri. Expression profiles showed that high salinity stress could influence the expression level of C3H genes in P. betulaefolia and we found genes response to stress contained relative complex gene structure. Genes induced or inhibited by salt could be used as excellent candidates for further stress response research. The present study provides a foundation for understanding the complex functions of the PbeC3H gene family and will facilitate studies of them to salt stress response in P. betulaefolia salt tolerance improvement.The C3H-type Znf family transcription factors play vital roles in plant development and response to biotic and abiotic stresses. We performed the first genome-wide analysis of the C3H family genes in 10.7717/peerj.9328/supp-1Table S1Click here for additional data file.10.7717/peerj.9328/supp-2Table S2Click here for additional data file.10.7717/peerj.9328/supp-3Table S3Click here for additional data file.10.7717/peerj.9328/supp-4Table S4Click here for additional data file.10.7717/peerj.9328/supp-5Supplemental Information 5Click here for additional data file.


Arsenic trioxide (ATO) is widely studied for its antitumor efficacy and several recent studies suggested the immune modulatory effects of ATO in animal models. In this study we found ATO treatment induced increased ROS production and DNA damage in esophageal squamous cell carcinoma (ESCC) cells, led to DNA damage mediated degradation of Cyclin D1 and upregulation of PD-L1 in these cancer cells. Mechanistically, we found ATO induced a transient upregulation and nuclear translocation of Cyclin D1 by sumoylation. Followed with increased ubiquitination and degradation of Cyclin D1 through T286 phosphorylation, and at least partly mediated by Stat1 Y701 phosphorylation. We observed inversed correlations between Cyclin D1 and PD-L1 expression levels in human ESCC tissues. With 4NQO induced PD-L1 humanized mouse oral and esophageal squamous carcinoma model, we found combinatory administration of ATO and check point inhibitor resulted in a significant reduction of tumor volumes. Inversed correlation between Cyclin D1 with PD-L1 was also observed in the 4NQO induced mouse ESCC and OSCC model. Together, these data suggested ATO induced degradation of Cyclin D1 and functional suppression of CDK4/6 pathway sensitized OSCC and ESCC to checkpoint inhibitor treatment. T cell immunity is an important adaptive cellular immune mechanism that guards the host system against the abnormal cells, including cancer cells. Programmed death ligand-1 (PD-L1) expressed by the cancer cells can bind to programmed death 1 (PD-1) protein on the pro-activated T cells to suppress its activation Although checkpoint inhibitors are effective in the treatment of a number of cancers, there are still tremendous needs to increase the efficacies of PD-1/PD-L1 checkpoint inhibitors in cancer treatment  et al. reported enhances the NK cell cytotoxicity against acute promyelocytic leukemia. Combination of ATO treatment with NK cell therapy significantly increased the survival time in APL mouse model et al. reported the application of ATO as the immune adjuvant in the treatment of mouse hepatocellular carcinoma in vitro by decreasing CD4+ T lymphocytes and Tregs, and increasing CD8+ T lymphocytes. In another study, Wang et al. reported combination use of ATO with adoptive T cell therapy significantly enhanced cytotoxic T lymphocytes activity, which preferentially decreased the population of Foxp3+ Tregs in the tumor centers in the colon cancer lung metastasis mouse model Arsenic trioxide (ATO) is known as the first line chemotherapy drug for acute promyelocytic leukemia (APL). Combination use of ATO with all-trans retinoic acid (ATRA) can archive better complete remission rate in the treatment of APL CDK4/6 signaling pathway has been reported to modulate the function of PD-1/PD-L1 immune checkpoint ESCC cell lines KYSE150, KYSE450 were preserved in our laboratory. The ESCC cells were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) at 37°C under 5% CO2. OSCC cell line (SSC1), kindly provided by Professor Demeng Chen of Cancer Hospital of Sun Yat-sen University, was cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS at 37°C under 5% CO2.ESCC cancer tissues were collected from the cancer tissue bank of Zhejiang Cancer Hospital, of which were derived from 97 patients  diagnosed with ESCC who had undergone curative surgery at Zhejiang Cancer Hospital  in 2015. After being checked by the pathologist, the tumor tissues and paired normal esophageal tissue were immediately frozen in liquid nitrogen and stored at -80°C for further research. All tissue specimens used in our study were obtained from the tissue bank of Zhejiang Cancer Hospital and all patients were informed and consent with the study before surgery. This study was approved by the Institutional Ethical Review Board of Zhejiang Cancer Hospital.ESCC KYSE-150 cells were seeded on Millicell EZ slides, and stained with 25µM of DCFDA purchased from Sigma (D6883) for 40 mins. Then the cells were washed with PBS for 3 times, the cells were cultured back in 1640 full growth media and treated with ATO for 12 hours before fluorescence microscope observation with excitation/emission at 495 nm/529 nm.The comet assay was performed with DNA damage Assay kit (Nanjing Jiancheng bioengineering institute G010-1-1) following the manufacture protocol. Briefly, KYSE-150 cells were treated with ATO for 24 hours. After ATO treatment, the medium was removed and the cells were washed with PBS, the cells were harvested, and resuspended in PBS at a cell density of 1×106. The cells were mixed with molten LM Agarose (42°C) at a ratio of 1:8 (v/v), and 85μl of the mixed solution was immediately pipetted onto a glass slide. The slides were placed flat at 4°C for 30 min and then immersed in lysis solution on ice for 1 hr. The lysis buffer was removed, and the slides were immersed in alkaline solution  for 1 hr. Slides were washed three times for 10 min with pre-chilled Neutralization buffer (0.4 mmol/L Tris-HCl) after electrophoresed at 1 volt/cm (25 v) for 20 min. Slides were stained with propidim iodide for 10 min. The slides were viewed with fluorescence microscope  and analyzed using the Comet Assay Software Project (CASP).pGME2F-Luciferase reporter plasmid was purchased from Yeasen Biotechnology Company, the construct was transfected into KYSE-150 cells with Lipofectamine 3000®. 24 h after transfection, the KYSE-150 cells were treated with ATO (10 µM) for 48 hours. Then the cell lysates were collected, and luciferase activity was measured using a Yeasen Luciferase reporter gene assay kit (11401ES60).6 cells from control (untreated) and treated cells were washed twice with cold PBS, and then fixed in 70% ethanol at 4°C overnight. The cells were then stained with propidim iodide (50 µg/ml) at 37°C for 30 min. The samples were analyzed with a FACSC antoTM Flow Cytometry System (BD Biosciences). And the data were analyzed using FlowJo VX software according to the manufacturer's instruction.Cellular DNA content was measured by flow cytometric analysis after 48 h incubation of KYSE150 cells with ATO (10 µM). Briefly, 1 × 106 of KYSE-150 Cells were harvested from the Petri dish after ATO (10 µM) treatment for 48 hours with a cell scrape stick, and suspended in 1ml PBS. The cell suspensions were briefly centrifuged, and the pellet were resuspended in 1 ml Trizol (Invitrogen). For the real-time PCR measurement of cyclin D1 mRNA levels in the human tissue samples, 97 cases of human ESCC tissue samples with paired normal esophageal tissues were randomly picked for real-time PCR assay. Then the Cell suspensions Tissue samples were immediately posited in 1 ml Trizol (Invitrogen) Reagent after taking out from -80°C refrigerator, and further processed with TissueLyser for RNA extraction. Total cellular RNA was extracted by the Trizol Reagent according to the supplier's instructions. cDNAs of the samples were synthesized with RevertAid First Strand cDNA synthesis Kit (Thermo Fisher). And real-time PCR amplification of the cDNAs were performed with SYBR® Premix Ex Taq™ (TAKARA) kit in ABI 7500 real-time PCR system, with the following specific primers in table For the real-time PCR measurement of cyclin D1 RNA levels in the ESCC cells, approximately 10The PCR conditions included a denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 58°C for 15 s, and extension at 72°C for 10 s. Detection of the fluorescent product was carried out at the end of the 72°C extension period. Each sample was tested at least in triplicate and repeated using three independent cell preparations.2, 10 mM Tris-HCl pH 8.5, 0.5% NP-40), incubated on ice for 5 min, and centrifuged at 5000 × g for another 5 min. The supernatant was then removed to a new tube, and 1 mL Trizol reagent was added (cytosol fraction). After washing with PBS, the nuclear pellet was also resuspended in 1 mL of Trizol reagent. Then the nuclear and cytosol fractions of proteins were further extracted from the Trizol solution according to the kit protocol.Nuclear/cytosol protein extraction was performed with Solarbio nuclear protein extraction kit (R0050). Briefly, the KYSE-150 cells were harvested and washed with phosphate-buffered saline (PBS), and then centrifuged at 500 × g for 5 min. After removal of the PBS, the cell pellet was resuspended with 100 µL of nuclear and cytoplasmic extraction reagent . Protein loading buffer for electrophoresis is composed with  dimethylammonium)-1- propanesulfonate (CHAPS), 50 mM dithiothreitol (DTT) and 24 mM Spermine). 2 µl anti-cyclin D1 mAb (Abcam), 20 µl of 50% slurry of protein G beads were added to each samples incubated at 4°C for 16hours at a rotator in the immunoprecipitation experiment. Beads were washed four times with 1 ml of 10-fold diluted urea buffer and 20 µl of 2 × SDS loading buffer was added. Immunoprecipitations were analyzed by western blot as indicated. Antibodie: anti-cyclin D1 antibody (Abcam), anti-γH2AX antibody (Santa cruz), anti-Cul3 (Proteintech), anti-PDL1 (Proteintech), anti-β-actin (Proteintech), anti-Lamin B1 (Proteintech), anti-pSTAT1 (CST), anti-pCyclin D1 (T286) (CST). Sumoylated cyclin D1 and ubiquitylated cyclin D1 was detected by co-immunoprecipitation using anti-SUMO-2/3 antibody or anti-ubiquitin antibody conjugated beads (Enzo Life Science), followed by immunoblotting with anti-cyclin D1 antibody or for cyclin D1 detection.Haematoxylin and eosin staining were performed to confirm the diagnosis, and analysis of pathological grades. All the tissues were fixed in 4% neutralized formaldehyde, embedded in paraffin. Blocks of paraffin-embedded donor tissue were sampled using a Manual Tissue Arrayer 1 instrument . Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples were hematoxylin and eosin (H&E) stained, and ESCC was confirmed by pathologist. The paraffin tissue blocks of 97 ESCC cases with paired normal esophageal tissues were used in the construction of tissue microarray. In brief, the H&E-stained standard slides were reviewed from each section of esophageal cancer tissues and one representative tumor area of each tumor (1.5 mm diameter) were removed from FFPE tissue blocks. A serial of 4-μm-thick sections were cut for the purpose of immunohistochemistry and transferred to adhesive slides according to manufacturer's instructions.Standard IHC analysis was performed with the primary antibodies against human Cyclin D1 (CST (92G2) Rabbit mAb #2978), Cul3 (Proteintech 11107-1-AP), Stat1-p (CST Phospho-Stat1 (Tyr701) (58D6)), PD-L1 (Proteintech 66248-1-Ig) at a dilution in 1:100. In brief, the tissue microarray slides were deparaffinized in xylene and gradient ethanol. Antigen retrieval was performed by placing the slides in a high-pressure cooker in a 0.01 mM citrate buffer, pH 6.0, for 2.5 min at 100°C; they were then cooled for 20 min. Endogenous peroxidase activity was blocked by incubating the section in 3% H2O2 for 10 min, followed by rinsing in PBS solution three times, tissue microarray slides were preincubated with blocking serum and then were incubated with the primary antibodies at 4°C overnight. After three washes in PBS, the slides were treated with the horseradish peroxidase (HRP)-labeled goat anti-mouse/ rabbit secondary antibody  for 50 minutes at room temperature. After washing with PBS, reaction products were visualized with 3, 3′-diaminobenzidine  and the slides were counterstained with hematoxylin. After being dehydrated, slides were mounted in resin. Immunohistochemistry results were evaluated by scanning each slide under low power magnification (×100) to identify regions containing positive immunoreactivity.KYSE-150 cells grown on Millicell EZ slides (EMD Millipore) were fixed in 4% PBS paraformaldeyde for 20 min, incubated in 0.3% Triton-X-100 for 15 min. After blocking with IF blocking buffer, the slides were stained with an anti-cyclin D1 antibody (Abcam), anti-γH2AX antibody (Santa cruz) overnight at 4°C. Antibodies were used at 1: 200 dilution in PBS. The cells were stained with the secondary antibody for 60 min and DAPI  for 10 min at room temperature away from light and EZ slides were visualized by a fluorescence microscope .6 cells into the flank of C57/B6 mice, purchased from Hangzhou Sike Biotech. Mice were housed in a standard experimental animal facility according to the national laws and policies. When the tumor volume reached approximately 100 mm3, the mice were randomized and intraperitoneally administrated with ATO (2 mg/kg) or PBS every other day for 2 weeks. The tumors were excavated and lysed for Western Blot analysis when reached approximated 2000 mm3.Murine Hepa1-6 cells were purchased from Sciencell. Hepa1-6 cells were cultured in DMEM medium. The Hepa1-6 tumor models were generated by subcutaneous injection of 2×10em2(hPD-L1)Smoc mice, purchased from Shanghai Model Organisms, were fed with 4NQO water (100 µg/ml) for 16 weeks starting at six weeks of age. 4NQO was withdrawn from the the drinking water of the mice after 16 weeks, and the mice were further kept for 4 weeks. Mice were grouped into nontreatment control group, ATO treatment group, checkpoint inhibitor Durvalumab treatment group, and ATO plus Durvalumab treatment group. Checkpoint inhibitor was delivered by intraperitoneal administration of Durvalumab (200 µg/mice in 200 µl PBS) every three days for 8 times. ATO treatment was delivered by intraperitoneal injection 2 mg/kg every other day for 3 weeks. By the end of the treatment period, mice were sacrificed, the anatomized and tumors of the oral cavity and esophagus were and measured. Afterward, the tumors were dissected and put into formaldehyde and prepped for IHC staining. ATO used in this study was purchased from Beijing Puxi Scientific. Checkpoint inhibitor Durvalumab was provided by AstraZeneca.Humanized C57BL/6J-Cd274Previous studies showed ATO treatment induced G1/S and G2/M cell cycle arrests in cancer cells ATO is a potent inducer of reactive oxygen species (ROS), which can cause DNA damages in cancer cells  et al. reported Sumo modified cyclin D1 primarily resided in the cell nucleus, and sumoylation of cyclin D1 is important for its nuclear translocation and oncogenic functions Cyclin D1 is involved in DNA damage repair by recruiting Rad51 to the DNA repair sites to promote homologous recombination mediated repair et al. reported ATO activated GSK3β by inhibiting ERK/AKT signaling in APL NB4 cells et al. reported phosphorylated Stat1 directly interacted with cyclin D1 to promote its proteasomal degradation DNA damage induced T286 phosphorylation of cyclin D1 by GSK3β has been reported to mediate the ubiquitination and degradation of cyclin D1 Cyclin D1-CDK4/6 pathway is known to regulate PD-L1 stability through transcriptional induction of Cul3-Spop E3 ligase, which mediates the degradation of PD-L1  et al. reported 49% of cases of human ESCC tissue samples showed with a strong positivity of Stat1 in immunohistochemistry analysis, and ESCC patients with strong Stat1 positive scores in the IHC analysis survived significantly longer than those with STAT1-weak/negative tumors Overexpression of cyclin D1 is found in a large number of cancers, including esophageal cancers, head and neck cancers, breast cancers, lung cancers etc. em2(hPD-L1)Smoc). By adding 4NQO into the drinking water for 16 weeks, PD-L1 humanized mice developed with oral and esophageal cancers. At week 20, PD-L1 humanized mice were treated with ATO plus PD-1/PD-L1 checkpoint inhibitor , compared with ATO or Durvalumab treatment alone, the mice were sacrificed at week 24. With anatomical and immunohistochemistry analysis we found significantly smaller sizes of ESCC and OSCC in the ATO and checkpoint inhibitor combinatory treatment group of mice  is a potent inducer of oral and esophageal squamous cell carcinomas (SCC) e Figure A-C.Immunohistochemistry analysis of the mouse OSCC and ESCC tissues of the 4NOQ induced OSCC/ESCC model showed downregulations of cyclin D1, Cul3 protein levels in the ATO treated groups. We also observed inversed correlations between the protein levels of cyclin D1/Cul3 and PD-L1 with IHC analysis in the 4NQO induced mouse SCC model Figure C. CollecOral and esophageal squamous cell carcinoma  are among the most common malignant cancers in China and worldwide Cyclin D1-CDK4/6 signaling pathway is frequently altered in SCC cancer cells to promote drug resistance and to maintain the malignant phenotypes of the cancer cells et al. reported cyclin D1-CDK4/6 signaling is an important upstream regulator of PD-L1 degradation, and upregulation of PD-L1 by CDK4/6 inhibition sensitized cancer cells to checkpoint inhibitor therapy et al. reported DNA damage induced upregulation of PD-L1 through activation of the JAK1/2-STAT1/2/3-IRF1 signalling pathway et al. reported IFN-γ activated Stat1 directly interacts with Cyclin D1 and promotes its proteasome degradation through its ser-701 phosphorylation site in fibrosarcoma cell line U3A cell line Zhang et al. reported ATO has the immunosuppressive effect of arsenic trioxide on islet xenotransplantation and they found ATO inhibited proliferation of T lymphocyte and increased the proportion of Foxp3+ regulatory T cells in recipient mice Zhao  in vivo. Additionally, the present study does not preclude some other molecular targets of ATO may also take part in the synthetic effects of co- administration of ATO with checkpoint inhibitor Importantly, ATO induced PD-L1 upregulation is just one of the many downstream effectors of CDK4/6 signaling. More systematic analysis indicates CDK4/6 regulate additional downstream signaling programs that confer cellular resistances to immune checkpoint inhibitors


Hepatitis B surface antigen (HBsAg) promotes persistent hepatitis B virus (HBV) infection. It primarily corresponds to small (S) envelope protein secreted as subviral particles. We previously found that genotype D clones expressed less S protein than genotype A clones but showed higher extracellular/intracellular ratio of HBsAg suggesting more efficient secretion. The current study aimed to characterize the underlying mechanism(s) by comparing a subgenotype A2 clone (geno5.4) with a subgenotype D2 clone (geno1.2). Five types of full-length or subgenomic constructs were transfected to Huh7 cells at different dosage. HBsAg was quantified by enzyme linked immunosorbent assay while envelope proteins were detected by Western blot. We found that ratio of extracellular/intracellular HBsAg decreased at increasing amounts of DNA transfected. Conflicting findings from two types of subgenomic construct confirmed stronger secretion inhibitory effect of the genotype D-derived large envelope protein. Chimeric constructs followed by site-directed mutagenesis revealed geno1.2 specific V118/T127 and F161/A168 in the S protein as promoting and inhibitory of HBsAg secretion, respectively. In conclusion, more efficient HBsAg secretion by subgenotype D2 than subgenotype A2 is attributed to lower level of S protein expression in addition to V118 and T127 in S protein, although its F161 and A168 sequences rather reduce HBsAg secretion. Chronic infection with hepatitis B virus (HBV) is a leading cause of liver cirrhosis and hepatocellular carcinoma. According to divergence of their genomic sequences, HBV isolates worldwide can be classified into genotypes A–J. Genotypes B and C predominate in East Asia and are often transmitted vertically from infected mothers. Genotypes A and D co-circulate in many other parts of the world and are transmitted mostly in adulthood through sex, blood transfusion, or shared needles. Serological markers of ongoing HBV infection include hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg). HBeAg is a secreted soluble version of core antigen, the building block of core particle (capsid) that shields the viral genome. It was initially identified as the “Australia antigen” . HBsAg iHBsAg is routinely detected from patient sera by its antibody through enzyme linked immunosorbent assay (ELISA). Molecular cloning of the 3.2 kb HBV genome not only identified the S gene as the coding sequence, but also revealed extended coding capacity further upstream. Subsequent experiments in cell culture established that besides major surface protein, also called small (S) envelope protein, HBV expresses large (L) and middle (M) envelope proteins through translation initiation from two upstream in-frame ATG codons . That seExpression of S protein alone is sufficient to drive SVP secretion in cell culture, although during natural HBV infection the M protein is also incorporated into SVPs. L protein expressed alone is retained inside cells, and its co-expression with S protein can inhibit SVP secretion to variable extents depending on the L/S protein ratio ,6,7. VirReplication competent SphI dimer of HBV clone geno5.4 (GenBank accession number: KX827293) of subgenotype A2 and geno1.2 (KX827290) of subgenotype D2 have been described . ReplicaThe human hepatoma cell line Huh7 was grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum . Transient transfection was performed on cells seeded overnight in 6-well plates using Lipofectamine 3000 reagent , with pBluescript SKII(−) DNA to bring the total amount of DNA to 2 μg/well. The medium was replaced 12 h later. Both cells and culture supernatant were harvested at day 4 post-transfection. Cells were scraped off and lysed in 100 μL of lysis buffer (10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 1% NP-40).g and 4 °C for 1 h and the pellet was resuspended in 10 µL of TN buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl), followed by separation of proteins in 0.1% SDS–12.5% polyacrylamide gel (SDS-PAGE). In some experiments, cell lysate or culture supernatant corresponding to same OD450 values of HBsAg was used for Western blot analysis (with prior PEG precipitation for culture supernatant).A 1/10th volume of the cell lysate was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes. The blots were blocked at room temperature for 1 h with 5% non-fat milk dissolved in TBS–0.1% Tween 20 (TBST). Next, they were incubated at 4 °C overnight with a 1:3000 dilution of polyclonal rabbit anti-HBs antibody  or a 1:5000 dilution of mouse monoclonal anti-preS1 antibody (7H11 ) in 5% m450) values of the samples were between 1 and 2, which made the HBsAg concentration in the sample proportional to the optical density value in a linear fashion. Subsequently, the total OD450 values of culture supernatant and cell lysate were calculated, based on the dilution and sample volume.HBsAg in culture supernatant and cell lysate was detected by a commercial ELISA kit . The samples were properly diluted so that the measured optical density  or geno1.2 . Cells and culture supernatant were harvested four days later. ELISA confirmed dose-dependent increase of HBsAg titers in both cell lysate and culture supernatant A,B. Howe protein F,G. The  protein G, but le protein F. Thus, To compare HBsAg secretion between the two genotypes without complication of genome replication and other viral proteins, we used a 0.7 mer L/M/S construct for transcription of 2.4 kb and 2.1 kb RNAs under endogenous SPI and SPII promoters. Relative to the SphI dimer, the 0.7 mer L/M/S construct of geno1.2 released much less HBsAg to culture supernatant than geno5.4 compare B. On the450 values of S protein from Huh7 cells transfected with different doses of geno5.4 were compared in Western blot. Little difference in S protein (gp27/p24) intensity was found for 300 OD450 value of HBsAg from culture supernatant of cells transfected with 0.1–1.6 μg 0.7 mer S construct  but weaker signals of gp27/p24 . Also included were CMV-S construct with the S gene alone cloned downstream of the strong CMV promoter, and 1.1 mer construct with transcription of the 3.5 kb pregenomic (pg) RNA is driven by the CMV promoter. The 1.1 mer construct of geno5.4 produced less L protein and even less S protein than corresponding SphI dimer, in both cell lysate and culture supernatant A–C. For For the S protein of 226 residues, geno1.2 differed from geno5.4 at 22 positions A. ChimerChi4 covered six divergent residues at the C-terminus of S protein, which were part of 15 divergent residues covered by chi2 B. The opTo identify residues responsible for more efficient HBsAg secretion by geno1.2 than geno5.4, divergent residues 8–13 were individually mutated. For residues covered by chi3.1, the T10V substitution of DpreS/AS was more effective at increasing the ratio of extracellular/intracellular HBsAg than L8I, while T9S had little effect  is the therapeutic end point ,14. HoweTo further characterize more efficient HBsAg secretion by genotype D than genotype A, the present study focused on geno5.4 of the A2 subtype and geno1.2 of the D2 subtype. The two clones were compared in the context of different types of replication  or envelope protein construct , with different DNA dosage. Consistent with a role of the L protein in inhibiting S protein secretion ,7, the 0We suspect that the 0.7 mer L/M/S construct produces more L protein than corresponding SphI dimer through increased transcription of the 2.4 kb RNA. All the HBV RNAs are unidirectional and co-terminal, thus raising the possibility of transcriptional interference. Our previous work revealed ability of the 3.5 kb pgRNA to diminish transcription of the 2.4 kb, 2.1 kb, and 0.7 kb RNAs from dimer or 1.1 mer construct . With 0.450 value of 1–2 for all samples by different fold of dilution according to the type of construct, DNA dosage, viral genotype, and cell lysate vs. culture supernatant. Moreover, control experiments where same OD450 values of HBsAg from cell lysate or culture supernatant were analyzed by Western blot revealed no bias against the S protein of SphI dimer of genotype A or D  and HBsAg secretion efficiency could at least partly explain why the 1.1 mer construct of geno5.4 manifested higher secretion efficiency than corresponding SphI dimer A–C, and Surprisingly, the CMV-S construct produced much less S protein than the 0.7 mer S construct, as indicated by lower HBsAg titers in both cell lysate and culture supernatant A,B. ThatThe S region harbored 39 divergent nucleotide positions between geno5.4 and geno1.2, leading to 22 amino acid substitutions scattered on the S protein of 226 residues A. StartiResidues 118, 127, 161, and 168 are all located inside the major hydrophilic region (residues 99–169) of the S protein (exposed on particle surface), although only P127 lies inside the “a” determinant (residues 124–147), the major target of neutralizing antibodies. Interestingly, both T118K and P127S are immune escape mutations that we previously found to severely impair virion secretion when introduced to a genotype A clone (the impact on HBsAg secretion was not determined) . The P12In conclusion, the present work confirmed weaker SPII promoter of a genotype D clone leading to reduced HBsAg production. It also confirmed more efficient HBsAg secretion from the S protein of a genotype D clone but a stronger inhibitory effect of its L protein. The current study revealed that the efficiency of HBsAg secretion correlates inversely with the dose of plasmid DNA transfected and consequently amount of S protein expressed, which can partly explain more efficient HBsAg secretion by genotype D than genotype A. Subtype D2 specific residue V118 and subtype D2, D3, and C4 specific T127 in the S protein could promote HBsAg secretion, whereas genotype D specific F161/A168 rather reduced HBsAg secretion. It will be of interest to study the combined effect of V118/T127 in genotype A, to investigate the impact of F161Y/A168V substitutions in genotype D, and to determine the impact of divergent residues 118, 127, 161, and 168 on HBsAg secretion in the context of intact HBV genome (SphI dimer). Finally, whether such substitutions alter the secretion of virions in addition to SVPs warrants further investigation.


Excellent mechanical performances were achieved by tuning the incorporation and chain length of the higher α-olefins. When the comonomer content reached above 12 mol.%, polypropylene-based elastomers were obtained with high ductility. A combination of excellent elastic recovery and flexibility was achieved for the P/C16 copolymers with about 20 mol.% monomer incorporation. The monomer incorporation and side chain length played a crucial role in determining the mechanical property of the outstanding polypropylene-based elastomers.In this contribution, we explored the copolymerization of propylene with higher α-olefins, including 1-octene (C8) 1-dodecene (C12), 1-hexadecene (C16) and 1-eicosene (C20), by using a dimethyl pyridylamidohafnium catalyst. A series of copolymers with varied comonomer incorporation, high molecular weight and narrow molecular weight distribution were obtained at mild conditions. The effects of the insertion of the comonomers on the microstructure, thermal and final mechanical properties were systemically studied by  Polyolefin represented by polyethylene (PE) and polypropylene (PP) is one of the most important kinds of thermoplastics in the market. It enjoys tremendous production volume and wide applications in our daily life because of their unique combination of chemical and physical properties ,2,3,4,5.Tm) and a lower chain conformational mobility compared to PE, which can reduce the viscous flow and chain entanglement during stretching to some extent  [B(C6F5)4] and AliBu3were purchased from Albemarle Corporation and Aldrich, respectively, and used without further purification. Anhydrous solvents used in this work were purified by a Solvent Purification System purchased from Mbraun . All the higher α-olefins were dried over CaH2 before distillation. It should be noted that for 1-eicosene, a vacuum distillation equipped with air condenser was applied and the fraction was collected at the temperature around 190–210 °C under vacuum.All work involving air- and/or moisture-sensitive compounds was carried out under a nitrogen atmosphere by using standard Schlenk technique. The dimethyl (pyridylamido)hafnium precatalyst was prepared following the procedure reported previously ,34.  [B(C6F5)4]. The reaction mixture was stirred at 25 °C. After the desired time, the reactor was vented. The resulted copolymers were precipitated from acidic ethanol, filtered, washed three times with ethanol and acetone, and then dried in vacuo at 40 °C to a constant weight.The copolymerization was carried out in a 50 mL Schlenk flask under propene atmosphere. The flask was repeatedly evacuated and refilled with propene. The prescribed amount of Al3C] [B(C6F5)4] 20 μmol, AliBu3 0.20 mmol, propylene 1 atm, Vtotal = 40 mL, copolymerization for 0.5 min, 1-octene 6.0 mmol. After the precipitated copolymer was isolated by filtration, the filtrate was concentrated before analyzed by ESI-MS.Following the procedure described above, the copolymerization of propylene with 1-octene was conducted under the reaction conditions: Catalyst 10 μmol,  [B(C6F5)4] and AliBu3, which is well-known for the excellent ability to incorporate higher α-olefins  [B (C6F5)4], dimethylpyridylamidohafnium complex exhibited excellent performance  in the copolymerization of propene with higher α-olefin, including C8, C12, C16 and C20. PP copolymers with high molecular weight (up to 160 × 104 g/mol) and unimodal distributions (PDI < 1.6) were easily obtained under mild condition. The 13C NMR analysis showed the isolate distribution of α-olefin and the absence of continuously incorporated α-olefin sequence. Thus, the randomly distributed α-olefin units shortened the average length of iPP sequence and resulted in a decrease in Tc values of the copolymers. Two crystallize peak assigned to the PP backbone and side chain, respectively, was observed in the P/C20 copolymers. All of the copolymers exhibited higher ductility and flexibility than iPP because of the reduced crystallinity. The copolymers with about 20 mol.% monomer incorporation contains exhibited elastomeric behavior, in which a small amount of crystalline  was dispersed in the large fraction of amorphous matrix. The side chain length has important effects on the elastic recovery. The elastic recovery of P/C8 and P/C20 with about 20% monomer incorporation decreased significantly at higher strain, while the copolymer P/C16 and P/C12 exhibited high elastic recovery (higher than 80%) in the whole range of deformations. Subsequent hysteresis tests revealed a large amount of unrecovered strain after the first cycle and significant deformation on each subsequent cycle in the copolymer P/C12. Although nearly 35% unrecovered strain occurred after the first cycle, minimum deformation and good recovery were observed on each subsequent cycle. Thus, P/C16 copolymers with about 20 mol.% hexadecane units were proved to be an outstanding PP-based elastomeric material.Activated by [Ph


Plants using the C4 photosynthetic pathway have an efficiency advantage, particularly in hot and dry environments. They account for 23% of global primary production and include some of our most productive cereals. While previous genetic studies comparing phylogenetically related C3 and C4 species have elucidated the genetic diversity underpinning the C4 photosynthetic pathway, no previous studies have described the genetic diversity of the genes involved in this pathway within a C4 crop species. Enhanced understanding of the allelic diversity and selection signatures of genes in this pathway may present opportunities to improve photosynthetic efficiency, and ultimately yield, by exploiting natural variation. Here, we present the first genetic diversity survey of 8 known C4 gene families in an important C4 crop, Sorghum bicolor (L.) Moench, using sequence data of 48 genotypes covering wild and domesticated sorghum accessions. Average nucleotide diversity of C4 gene families varied more than 20-fold from the NADP-malate dehydrogenase (MDH) gene family (θπ = 0.2 × 10−3) to the pyruvate orthophosphate dikinase (PPDK) gene family (θπ = 5.21 × 10−3). Genetic diversity of C4 genes was reduced by 22.43% in cultivated sorghum compared to wild and weedy sorghum, indicating that the group of wild and weedy sorghum may constitute an untapped reservoir for alleles related to the C4 photosynthetic pathway. A SNP-level analysis identified purifying selection signals on C4 PPDK and carbonic anhydrase (CA) genes, and balancing selection signals on C4 PPDK-regulatory protein (RP) and phosphoenolpyruvate carboxylase (PEPC) genes. Allelic distribution of these C4 genes was consistent with selection signals detected. A better understanding of the genetic diversity of C4 pathway in sorghum paves the way for mining the natural allelic variation for the improvement of photosynthesis.C This reaction produces toxic phosphoglycolate which has to be converted to useful metabolites requiring substantial metabolic energy [2 is termed photorespiration. Photorespiration becomes a major constraint of photosynthesis in situations where CO2 to O2 ratios are low and temperatures are high. The evolution of C4 photosynthesis coincided with declining atmospheric CO2 concentrations [2 in the vicinity of ribulose-1,5-bisphosphate carboxylase (Rubisco).Cant taxa . The maic energy . This watrations ,4 as a m4 plants, this is achieved via spatial separation of the initial CO2 fixation and the Calvin–Benson–Bassham cycle in two different cell types, most often mesophyll cells and bundle sheath cells [2 concentration in C4 bundle sheath cells is up to 10-fold higher than that found in C3 mesophyll cells [4 photosynthesis is not only more efficient compared with C3 photosynthesis in terms of reducing energy losses from photorespiration, but due to the improved efficiency of this pathway, it renders plants more nitrogen- and water-use efficient [4 plants are more productive than C3 plants in areas with high light intensities, warm temperatures, and low rainfall, such as the sub-tropical and tropical areas around the globe.In the majority of Cth cells . CO2 conll cells . At highfficient ,8. C4 plmiscanthus, and switchgrass, use the C4 pathway [4 crops account for an estimated 23% of global primary production [4 photosynthesis pathway is a good starting point to improve photosynthetic capacity and resource efficiency in crop plants. Attempts are currently being undertaken to integrate characteristics of the C4 pathway into C3 crops [Many of the major crops that originated from warm and dry regions of the world, such as maize, sorghum, millet, sugarcane,  pathway . C4 cropoduction . Improveoduction . The C4 C3 crops ,12,13,144 photosynthesis in different plant taxa [4 species in terms of the biochemical pathway. It has long been known that three major biochemical subtypes—nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME), nicotinamide adenine dinucleotide-malic enzyme (NAD-ME) and phospho-enol-pyruvate carboxykinase (PCK)—exist among C4 species [4 cereals belong to, differences in pathway and performance are likely to exist, as C4 photosynthesis has evolved at least 25 times in this group of plants [4 photosynthetic efficiency [However, possibly due to multiple independent evolutions of Cant taxa , large v species . More re species  and that species . Especiaf plants . Explorificiency .4 crop well-known for its adaption to drought and high temperatures. It provides staple food for over 500 million people in the semi-arid tropics of Africa and Asia; in addition to being an important source of feed, fiber, and biofuel. Due to these characteristics, it is expected to play an increasingly important role in meeting the challenges of feeding the world’s growing population under the threat of global warming. Substantial variation in photosynthesis and related traits has been revealed in sorghum [Sorghum is an NADP-ME subtype C sorghum ,21,22,234 photosynthetic pathway. Several high-throughput comparative transcriptomics and evolutionary studies using C3 and C4 phylogenetically related species and cell-specific gene expression have elucidated the key genes and regulatory networks that underpin the C4 photosynthetic pathway [4 genes, mined their allelic diversity and investigated signatures of selection during domestication in sorghum.The recent assembly of whole-genome sequences for a wide range of wild and cultivated sorghum species ,25,26 pr pathway ,35,36,374 genes), and their non-C4 isoforms in sorghum were extracted from two previous studies [4 isoforms are homologous of C4 genes but there was no evidences supporting their involvement in the NADP-ME photosynthetic pathway. Homology between these sorghum C4 genes and their non-C4 isoforms was further verified via a local blast strategy. Protein sequences of these 9 core C4 genes were extracted from the sorghum reference genome V3.1 and were blasted against the reference genome. Blast hits of each gene were filtered using the criteria: E-value <−10, sequence identity >60%, and alignment length >80%. All hits of the same gene satisfying the criteria were plotted based on –log ; only hits of top –log  class were considered if clear differentiation among them was visualized, otherwise all hits were used.This study focuses on 8 key proteins in the NADP-ME photosynthetic pathway in sorghum . A total studies ,39  reported in previous studies [Sequence data of the identified C studies ,25,26. TST [Population genetic parameters including nucleotide diversity (θπ) , Tajima’ST , which mST . The BioST between the group of cultivated sorghum and the group of wild and weedy sorghum > 95% of the population pairwise distribution, (3) Tajima’s D < 0. Criteria for balancing selection included: (1) θπ and hW > 25% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum < 90% of the population pairwise distribution, (3) Tajima’s D > 5% of the empirical distribution.The criteria used in Mace et al. (2013) were employed to identify genes under purifying selection and balancing selection, respectively. Criteria for purifying selection included: (1) θπ and hW < 5% of the empirical distribution in the cultivated group, (2) FST between the group of cultivated sorghum and the group of wild and weedy sorghum were computed for these 27 genes using CDS sequence in PopGenome, a population genomics package implemented in the R environment (http://cran.r-project.org/) [ST, and Tajima’s D for each single nucleotide polymorphism (SNP), respectively, with a slide window of 1-bp and 1-bp step size. Functional annotation of each SNP was conducted using get.codons command. Fold decrease of θπ in the cultivated sorghum group compared to the group of wild and weedy sorghum was calculated to represent reduction of diversity (RoD). The following criteria were adopted to identify sites with signature of purifying selection: (1) A RoD greater than the average of neutral genes; (2) FST > 0; (3) Tajima’s D < 0. The following criteria were adopted to identify sites with signature of balancing selection: (1) An increase in diversity (IoD) in the cultivated group and the group of wild and weedy comparison; (2) FST > 0; (3) Tajima’s D > 0.Population genetics parameters including θπ, Tajima’s D, and Fct.org/) . Specifi4 gene families using the neighbor-joining method with default settings  in Geneious 8.1.2 . Analysis of haplotype network was conducted using a combination of the R package ape [Guinea margaritiferum and S. propinquum  . Nucleotide diversity varied dramatically among the C4 gene families, with the NADP-MDH genes displaying the lowest levels of diversity across all genotypes (average θπ = 0.25 × 10−3), followed by NADP-ME genes (θπ = 0.93 × 10−3), PPCK genes (θπ = 1.20 × 10−3), PEPC genes (θπ = 2.11 × 10−3), CA (θπ = 2.26 × 10−3), and PPDK-RP (θπ = 2.96 × 10−3), while PPDK genes showed the highest level of diversity (θπ = 5.21 × 10−3)  A. The on4 genes with non-C4 isoforms in each gene family with the average overall genetic diversity of C4 genes being comparable to that of their non-C4 counterpart (4 PPDK-RP gene (Sobic.007G166300) and C4 NADP-MDH gene (Sobic.002G324400) had an overall θπ which was 161.76% and 79.85% higher than their non-C4 isoforms, respectively, whereas the θπ of the C4 PPDK gene (Sobic.009G132900) was 75.16% lower than that of the non-C4 PPDK isoform. Nucleotide diversity of C4 genes in the other gene families was within the range of variation of their non-C4 isoforms.Mixed trends were found when comparing Cnterpart . The C4 4 gene families was significantly reduced during sorghum domestication . Averaged across all C4 gene families genetic diversity was reduced by 22.44% in the domesticated compared with the wild and weedy group and when just the 9 core C4 genes were considered, the reduction was 22.98%. However, the reduction of genetic diversity during domestication in C4 genes was not significantly different from that in housekeeping genes . Among the 27 genes, Sobic.003G292400, a non-C4 NADP-ME isoform, exhibited the most severe reduction in genetic diversity, with a reduction of 98.23%. The C4 version of that gene, the NADP-ME gene (Sobic.003G036200), showed the greatest loss of genetic diversity (51.89%) among the C4 genes, with an FST between the cultivated and wild and weedy groups of 0.06 (4 isoform of NADP-ME (Sobic.009G069600), a non-C4 isoform of PPCK (Sobic.006G148300), and a non-C4 CA isoform (Sobic.003G234600) showed a more than 2-fold increase in genetic diversity in the cultivated group.Genetic diversity across Cng genes  , only one gene  was identified as being under balancing selection, which maintains diversity of selected genes, during sorghum domestication, while no gene was identified as being under purifying selection, which reduces diversity of selected genes (4 PPDK-RP isoform (Sobic.002G324700) to 0 in the C4 PPDK (Sobic.009G132900). The C4 PEPC gene (Sobic.010G160700) had the highest number of non-synonymous SNPs (9) among the 9 C4 genes  and CA (Sobic.003G234200), three of 4 non-C4 NADP-ME , both two non-C4 PPDK-RP , and a non-C4 PEPC gene (Sobic.007G106500). Among the 2 C4 genes with SNPs under selection, Sobic.009G132900 had 3 synonymous SNPs under purifying selection, while Sobic.003G234200 had a non-synonymous SNP under purifying selection.The selection signature of these Ced genes . SubsequC4 genes . In contC4 genes . Genes w4 PPDK (Sobic.001G326900) had 24 SNPs under balancing selection including 5 non-synonymous SNPs, and additionally had an overall gene-level signature of balancing selection based on the previous analysis. Two C4 isoforms, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with 3 and 2 SNPs under balancing selection, respectively, although none of them were non-synonymous SNPs. Two non-C4 PEPC  were identified with SNPs under balancing selection, with Sobic.003G100600 having 21 SNPs including 2 non-synonymous SNPs exhibiting signatures of balancing selection. The other 2 genes with SNPs under balancing selection were a non-C4 CA isoform, Sobic.002G230100, and a non-C4 PPCK isoform, Sobic.004G219900.A total of 60 SNPs across 8 genes were identified as being under balancing selection, 7 of which were non-synonymous SNPs distributed across 2 genes . The nonS. propinquum, e.g., 4 unique haplotypes were observed for the C4 isoform of PEPC, with the 2 S. propinquum accessions sharing a single private haplotype. To investigate allelic variation of 4 core C4 genes with SNPs under selection in sorghum, haplotype networks were constructed using CDS SNPs. Based on 16 SNPs within the CDS of the PPDK gene (Sobic.009G132900), 8 haplotypes were identified. Five haplotypes were identified in the wild and weedy genotypes, with 3 being private haplotypes and two of them being maintained in cultivated sorghum; two new haplotypes arose in cultivated sorghum after domestication (A phylogenetic tree was constructed using the CDS of these 27 genes to depict the genetic relationship of 48 accessions . The inttication A. Ten hatication B. The lo4 PEPC gene (Sobic.010G160700), 4 haplotypes were identified. Wild and weedy genotypes encompassed 3 haplotypes and all of them were maintained in cultivated sorghum  had 22 SNPs in the CDS, based on which 5 haplotypes were identified. Two haplotypes were characterized by the wild and weedy genotypes, with the main wild haplotype maintained and further diversifying into two new haplotypes in the cultivated group C. Based  sorghum D. S. pro4 photosynthesis has been studied extensively at the cross-species level with signals of adaptive evolution identified on key genes in the C4 pathway [4 photosynthesis is driven by environments characterized by low CO2 availability, such as hot and dry environments in which CO2 uptake is limited by stomatal closure, it is likely that within-species adaptive variation also exists. However, to our knowledge, studies of within-species allele diversity and signatures of selection on key genes in the C4 pathway have not previously been undertaken.The evolution of C pathway ,48,49,504 photosynthesis. In this study, we performed the first investigation of the genetic diversity of C4 gene families within a C4 species using a collection of 48 sorghum lines. We focused on 9 C4 genes due to their reported key roles in C4 photosynthesis. Our collection of sorghum represents all major cultivated sorghum races, landraces, and wild progenitors, and captures a good proportion of genetic diversity within sorghum. Substantial variation of nucleotide diversity was observed among these 8 C4 gene families in sorghum, with the NADP-MDH gene family showing the least diversity and the PPDK gene family showing the greatest diversity. Nine core C4 genes also exhibited varying degrees of genetic diversity, ranging from θπ values of 5.04 × 10−3 and 4.32 × 10−3 in PPDK-RP and rbcS to θπ values of 0.33 × 10−3 and 0.67 × 10−3 in NADP-MDH and NADP-ME. However, despite such low levels of diversity, non-synonymous SNPs were identified in both NADP-MDH and NADP-ME , indicating the function of this gene is highly conserved.Knowledge of existing natural variation and levels of genetic diversity is a pre-requisite for the optimization of C NADP-ME . C4 PPDK4 gene families, indicating that wild sorghum, as a repository for genetic diversity, might harbor alleles useful for improving C4 photosynthesis.Cultivated sorghum was domesticated more than five thousand years ago in Africa ,52,53. T4 gene families was not significantly different from the genome-wide average, indicating that this gene family has not been under particularly strong selection pressure. Similarly, none of the 9 core C4 genes showed a domestication signal at the gene level. The absence of large sequence variation at the gene level is also consistent with previous evolutionary studies suggesting that relatively minor changes to pre-existing regulatory networks and the use of pre-existing cis-elements were often sufficient to recruit genes into the C4 pathway [4 isoform of the NADP-ME gene found in maize and sorghum is one such gene that has been found to be activated for C4 photosynthesis via subtle changes to its promoter, while the rest of the gene is highly conserved [However, the overall reduction in diversity of C pathway ,57. The onserved . This is4 genes, PPDK (Sobic.009G132900) and CA (Sobic.003G234200), with SNPs under purifying selection, while the other 2 C4 genes, PPDK-RP (Sobic.002G324400) and PEPC (Sobic.010G160700), were identified with SNPs under balancing selection. Previous studies have demonstrated that SNP-level analysis using less stringent criteria is superior for capturing soft selection signals compared with genome-wide ranking [A further high-resolution investigation of domestication signature at the SNP level revealed 2 C ranking ,58. Howe ranking  and the 4 isoforms of PPDK and PEPC were also found to show signals of positive selection in a previous cross-species evolutionary study using orthologous groups from closely related C3 and C4 grass species including sorghum [2 assimilation rate [4 pathway and its residues and regions involved in catalyzes are highly conserved in C4 species [4 isoform of the PPDK gene in our study.The C sorghum . PPDK anion rate , especiaion rate ,63. Howe species , possibl2, and variation of its affinity for CO2/HCO3− amongst species has been documented [4 photosynthesis as it catalyzes the first step of the C4 pathway, converting CO2 to HCO3− [4 dicot Flaveria bidentis, where antisense plants with <10% of wild-type CA activity required high CO2 for growth and showed reduced CO2 assimilation rates [PEPC is also regarded as a potential limiting step in the assimilation of COcumented ,66,67. Cto HCO3− . It was on rates ,70. Receon rates .4 pathway was indirectly improved during sorghum domestication. Without photosynthetic rate being a direct selection target in breeding programs, a steady increase in leaf photosynthetic rate over time of cultivar release has been shown in other cereals, e.g., in Australian bread wheat [4 PPDK-RP and PEPC may reflect adaptation to diverse environments, as both PPDK-RP and PEPC are associated with abiotic stress [4 genes all showed selection signals contrasting with their C4 counterparts with both two non-C4 PPDK-RP  containing SNPs under purifying selection and the non-C4 PPDK (Sobic.001G326900) containing SNPs under balancing selection.The signal of soft purifying selection on PPDK and CA may suggest the Cad wheat . The balc stress ,74. Inte4 genes in cultivated sorghum does not mean these haplotypes are inferior in terms of photosynthetic efficiency, as photosynthesis was not specifically targeted during sorghum domestication [After domestication, sorghum was introduced from tropical to temperate areas, and adapted to divergent local environments. New mutations also arose during this diversification process, and played an important role in local adaptation. In the haplotype analysis, these haplotypes unique to cultivated sorghum are likely to be young alleles arising after domestication, while haplotypes unique to the wild progenitor indicate that some haplotypes were lost during domestication of sorghum. Nevertheless, the loss of wild haplotypes of Ctication . On the 4 photosynthesis has been well studied over the past 50 years and key components of this complex pathway have been identified following the advent of transgenic and sequencing technologies [4 pathway is an important step towards mining the natural allelic variation for the improvement of photosynthesis. Cnologies . UnderstFurther investigation of these allelic variation to link them with agronomical traits will provide new targets for sorghum improvement .


In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion  Further, recombinant T cell receptor (TCR) engineered T cells, TCR-T that are directed against MHC-complexed peptides, are also being evaluated primarily in solid tumors [Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies . To dateIt is unclear why some patients respond to treatment with adoptive cell therapies such as CAR-T, and others do not, though the tumor immune microenvironment is a likely contributor to variable effect of cell therapy in both hematologic and solid cancers. To this end, preclinical and clinical studies have shown that regulatory T cells (Tregs) can have an impact on the effect of ACT in mice and in patients with melanoma , 5. In tAnother important area for improvement in CAR-T therapies involves the serious adverse events that have been reported, particularly with the drugs directed against CD19 such as tisagenlecleucel and axicabtagene ciloleucel. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) occur at very high rates following CD19 CAR-T administration, with CRS occurring in greater than 50% of patients and at least 10–30% patients experiencing high grade ICANS . Recent in vivo following infusion, and represents a potential point of intervention to decrease serious toxicities following CAR-T treatment. Most CAR-T programs exploit the use of the combination of fludarabine and cyclophosphamide (flu/cy) as a lymphodepletive conditioning regimen prior to CAR-T. These drugs are often administered 2–7 days (2–5 day course of therapy) prior to ACT infusion. However, the commonly used flu/cy regimen is a non-specific and cytotoxic treatment that some patients may not be able to tolerate and may not offer tumor control. Additionally, flu/cy has been correlated with toxicities such as prolonged cytopenias and cytokine release syndrome (CRS) following CAR-T administration [Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion stration .In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. The CD45 antigen is found on all nucleated immune cells, with increased expression on mature lymphoid and myeloid lineages, leading to preferential depletion of mature immune cells compared to progenitor hematopoietic cells . Importa131I)-apamistamab (Iomab-B), is in a Phase III clinical trial as a myeloablative targeted conditioning regimen prior to allogeneic stem cell transplant in patients with active relapsed/refractory acute myeloid leukemia (AML). Results from patients following dosimetry testing have shown that low non-myeloablative doses of 131I-apamistamab were able to safely induce transient lymphodepletion [131I and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Our results support CD45 targeted RIT lymphodepletion prior to adoptive cell therapy using a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody.Anti-CD45 RIT with 131Iodine (epletion . This da111In (111In radiolabel was used in these experiments as imaging surrogate for therapeutic radionuclides 131I and 177Lu) showed that the antibody homed to immune system organs such as lymph nodes, spleen, and bone marrow as well as liver  but preserved bone marrow cells, platelets, and red blood cells. Experiments in which variable amounts of 30F11 were radiolabeled with 3.7 MBq 131I revealed no effect of the antibody amount on the efficacy of lymphodepletion  (microSPECT/CT imaging of mice administered CD45-targeting antibody 30F11 radiolabeled with as liver . The imaas liver . Dose fiepletion . Based os (HSCs) .131I-30F11 and received 106 OT I CD8 CD45.2 OVA reactive T cells on day 4. 131I-30F11 antibody enabled engraftment and persistence of adoptively transferred CD45.2 OT I CD8+ T cells in the spleen 17 days post injection. Similar to conditioning in non-tumor bearing mice, 131I-30F11 lymphodepletion mediated decreases in multiple lymphoid cell subsets such as spleen CD4+, CD8+, B and NK cells as well as Tregs. , while in the treated group which received immunodepletion followed by OT I cells, 50% of mice (2/4) achieved CR (p = 0.02).Following E.G7 tumor engraftment, mice were conditioned with 3.7 MBq s Tregs. . Importar growth . In the 177Lu-30F11 antibody revealed that 1.48 MBq 177Lu-30F11 was effective in transiently depleting various immune populations in the spleen including Tregs  (Pilot treatment of non-tumor bearing C57B/6 mice with 0.74 or 1.48 MBq ng Tregs . Subsequlatelets . The exp = 0.03) .131I-radiolabeled anti-CD45 30F11 antibody to effectively lymphodeplete in a targeted manner in experimental models prior to administration of adoptive cell therapy. Significantly, targeted conditioning with pan-CD45 RIT, which selectively targets all nucleated immune cells, depletes not only lymphocytes, but also macrophages, as well as immune suppressive regulatory T cells and myeloid-derived suppressor cells in the immune microenvironment. It can potentially also exert a direct anti-tumor effect on CD45+ hematopoietic cancers.Prior to a patient receiving a dose of an adoptive cell transfer such as engineered autologous or allogeneic CAR-T cells, it is common to perform a lymphodepletion step often using high dose chemotherapy , 6. This177Lu, for mediating lymphodepletion in mouse models. Both 131I and 177Lu are beta emitters with physical half lives of 8 and 6.6 days, respectively, and maximum beta energies of 0.6 and 0.5 MeV, respectively. The principle difference lies in the chemistry of these radionuclides, with 131I being a halogen and 177Lu – a trivalent radiometal. 177Lu-labeled somatostatin receptor targeting peptide (Lutathera) has been approved for treatment of neuroendocrine tumors [177Lu-labeled small molecule binding to PSMA is currently in advanced stage clinical trials for treatment of metastatic prostate cancer [177Lu over another beta-emitting radiometal 90Y as it has been shown in long term follow up studies that patients treated with 177Lu-peptide therapy survive significantly longer than those treated with 90Y-peptide therapy due to more side effects from 90Y [177Lu is a low energy beta emitter. We determined that 1.48 MBq 177Lu-30F11 could effectively deplete various immune cell subsets in mice but spare bone marrow cells, red blood cells, and platelets. In a model of adoptive cell therapy using CD45.1 OT1 mice bearing E.G7-OVA tumors, mice that received either 131I-30F11 or 177Lu-30F11 RIT-mediated lymphodepletion demonstrated enhanced tumor control over mice that did not receive lymphodepletion. Interestingly, lymphodepletion with 131I-30F11 resulted in somewhat greater tumor control than with 177Lu-30F11  to perform targeted lymphodepletion in mice. Radiolabeling of 30F11 antibody with 131I was carried out by adding 111 MBq Na131I to 15 μL PBS followed by 100 μg 30F11 antibody  in a microcentrifuge tube. Chloramine-T  was added and the solution was shaken at RT for 5 minutes. Sodium thiosulfate  was added followed by 1 μL Sodium Iodide solution (10% w/v) and 7.5 μL of Ascorbic acid (500 mg/mL concentration). The antibody was then purified using spin filtration via an Amicon Ultra 0.5 mL centrifugal filter . Radiochemical purity was then checked via instant thin layer chromatography (iTLC) and was greater than 99%. For radiolabeling with 177Lu and 111In (imaging surrogate of 177Lu) 30F11 antibody was conjugated to bifunctional chelating agent DOTA  at a ratio 20:1 DOTA: Ab and then radiolabeled with 177Lu or 111In with the specific activity of 0.185 MBq /μg antibody as in [PE. The EC50 for naked and modified 30F11 was 394 ng/mL and 393 ng/mL, respectively .The anti-mouse pan-CD45 binding antibody 30F11  was used in all experiments as a surrogate for pan-human CD45 dy as in , 17. Imm111In-30F11 antibody with a specific activity of 0.185 MBq/μg and antibody distribution was monitored by microSPECT/CT at 1, 24, 48, 72, 96 hours timepoints and then again at 6 days after antibody administration. A MILabs VECTor4 (Netherlands) microSPECT/CT scanner was used to collect images. PMOD  was used for comprehensive image analysis. An Extra Ultra High Sensitivity Mouse (XUHS-M) collimator for 20–350 keV range was used to collect SPECT data using spiral trajectories. MILABS reconstruction software was used to reconstruct SPECT images using both 111In gamma emissions (245 keV and 171 keV) on a 0.4 mm voxel grid. For visual representation of accumulation, MIP images were utilized.Female 5 weeks old C57Bl/6 mice were injected intraperitoneally (i.p.) with 60 μg 111In-labeled 30F11. Immediately after labelling male C57bl/6 mice were injected i.v. with 1.11 MBq (30 μg) 111In-30F11. Five mice were euthanized at 1, 4, 24, 48, 72, 96 and 240 hours post injection, the organs were collected into pre-weighted tubes, and activity was assessed with a 2470 Wizard2 Gamma counter . A standard that contained 10% of the injected dose was also read to perform decay correction. Percent injected dose per gram (%ID/g) was determined using the equation:To determine the pharmacokinetics of 30F11 antibody in immunocompetent mice, a biodistribution was carried out with *(standard CPM*10)*radiolabeling yield)) × 100%, where CPM are counts per minute.%ID/g = ((sample CPM)/(organ weight131I-30F11 labeled with 1.85–7.4 MBq to determine the most effective and safe radioactivity dose required for transient lymphodepletion. Mice were euthanized at varying time points (2–21 days) and various immune cell subsets were quantified using flow cytometry as described below. The goal of the second series of experiments was to determine the optimal amount of antibody to be used for lymphodepletion. This was accomplished by varying the amount of 30F11 antibody administered to a mouse in 20–100 μg range, but with a constant specific activity of 3.7 MBq.In the first series of experiments, female 5 weeks old C57Bl/6 mice were injected i.p. with 20 μg of Single cell suspensions were made by passing spleens through a 70 μm cell strainer, washed three times with FACS buffer , incubated with blocking antibody for 10 min at 4°C, then labeled with surface marker-specific fluorochrome-labeled antibodies (Supplementary Table 1) for an additional 20 min at 4°C and unbound Ab was washed away with FACS buffer. Cells that were probed for intracellular markers were permeabilized  for 30 min, washed again with Perm buffer, incubated with blocking antibody in the same buffer for 10 min, and then labeled with marker-specific or isotype control fluorochrome-labeled antibodies for an additional 20 min. Stained cells were washed twice with Perm buffer and once with FACS buffer, and analyzed on a CytoFLEX flow cytometer . The data was processed using FlowJo software . Supplementary Figure 2 shows gating scheme for the identifications of various immune cells.3, two groups of mice were treated with 3.7 MBq 131I-30F11 (20 μg), while the third group was left untreated. Four days post-lymphodepletion, OVA-specific CD8+ T cells were purified using anti-CD8 magnetic beads from CD45.2 OT I mice according to the manufacturer’s protocol . One group of mice treated with 3.7 MBq 131I-30F11 was given a single i. v. injection of 106 CD45.2 OT I CD8+ cells. Tumor volume and body weight were monitored, and mice were sacrificed when tumor volume exceeded 4,000 mm3 or tumors became necrotic. Blood and spleen were assessed for immune cell subsets and presence of engrafted CD45.2 OT I cells by flow cytometry.Three groups of female 5 weeks old C57Bl/6 CD45.1 mice, five animals per group, were injected subcutaneously with OVA expressing CD45+ E. G7-OVA lymphoma tumor cells. Seven days post-tumor cell injection, when tumor volume reached ~100 mm177Lu or with 1.85 or 3.7 MBq of 131I to determine the ability to selectively deplete immune cell subsets. Immune cell subset quantitation was performed by flow cytometry as described above.Female adolescent C57Bl/6 mice were injected i.p. with 20 μg 30F11 labelled with 0.74 or 1.48 MBq of 3 tumor volume was reached. Seven days post-tumor cell injection, mice were treated with 177Lu-30F11 (1.48 MBq), 131I-30F11 (3.7 MBq), or received no lymphodepletion treatment. Four days post-lymphodepletion, OVA-reactive CD8+ T cells isolated from CD45.2 OT I mice were administered to tumor-bearing mice. The tumor volume, body weight, immune cell subsets and presence of engrafted cells were monitored as above.Female 5 weeks old C57Bl/6 CD45.1 mice were injected subcutaneously with OVA expressing CD45+ E. G7-OVA lymphoma tumor cells until 100 mmin vivo studies was estimated using PASS version 11  using simulations of different cells depletions/tumor volumes based on pilot or literature data and conservative assumptions regarding the groups treated with the radiolabeled antibodies. All simulations showed power of at least 83% with only five animals per group because of the large differences between treated and untreated animals. Thus, 5 mice per group were utilized in the in vivo studies. GraphPad Prism 7 was used to analyze all the data . Differences among the groups were assessed using Student t-tests and one-way ANOVAs for multiple comparisons. Differences were considered significant at p < 0.05.Power analysis for the