The brain, spinal cord, and retina are supplied by capillaries that do not permit free diffusion of molecules between serum and parenchyma, a property that defines the blood-brain and blood-retina barriers. Exceptions to this pattern are found in circumventricular organs (CVOs), small midline brain structures that are supplied by high permeability capillaries. In the eye and brain, high permeability capillaries are also present in the choriocapillaris, which supplies the retinal pigment epithelium and photoreceptors, and the ciliary body and choroid plexus, the sources of aqueous humor and cerebrospinal fluid, respectively. We show here that (1) endothelial cells in these high permeability vascular systems have very low beta-catenin signaling compared to barrier-competent endothelial cells, and (2) elevating beta-catenin signaling leads to a partial conversion of permeable endothelial cells to a barrier-type state. In one CVO, the area postrema, high permeability is maintained, in part, by local production of Wnt inhibitory factor-1. Throughout the body, capillaries exhibit tissue- and organ-specific specializations of structure and function . In the Ndp null mouse [Norrie disease protein (Ndp) is the gene coding for Norrin] restores BBB integrity in the cerebellum. Intriguingly, constitutive activation of beta-catenin signaling in ECs in the choroid plexus, the site of cerebrospinal fluid (CSF) production, leads to the cell autonomous expression of a BBB marker [the tight junction protein Claudin5 (CLDN5)] and repression of a marker of non-BBB vasculature  omplexes . Curiousomplexes .R26-8xTCF/LEF-loxP-stop-loxP(LSL)-H2B-GFP; Consistent with the accumulation of LEF1 in CVO ECs in response to beta-catenin stabilization, an EC-specific H2B-GFP reporter of beta-catenin signaling  Based on RiboTag RNA-seq, 184 transcripts were enriched in WT cortex ECs compared to WT choroid plexus ECs  and 311 transcripts showed the reverse enrichment  in WT cerebellum ECs, WT anterior pituitary ECs, and WT posterior pituitary ECs, respectively, in at least one pair-wise comparison . These pIn beta-catenin stabilized ECs, the appearance of newly accessible chromatin in the neighborhood of newly activated genes suggests a causal relationship . To explTo link the accessible chromatin analyses to the actions of individual TFs, we used Hypergeometric Optimization of Motif EnRichment (HOMER), a suite of tools for discovering enriched motifs in genomic sequences , to perfMotifs corresponding to the ETS family of transcription factors, which play a central role in EC development and function throughout the body , were hiIn sum, this analysis shows that TCF/LEF motifs are highly enriched in genomic regions that are constitutively accessible in BBB-competent ECs (e.g. cerebellum ECs) or that become accessible in pituitary ECs upon activation of beta-catenin signaling. This finding lends strong support to a model in which beta-catenin regulates the BBB gene expression program via direct engagement of cis-regulatory elements adjacent to genes encoding BBB proteins .The experiments described up to this point have involved artificially activating beta-catenin signaling in ECs. If, as these experiments suggest, the high permeability state of CVO ECs requires low or absent beta-catenin signaling, then we might expect that the local environment created by CVO neurons and glia would be characterized by a low concentration of those canonical Wnt ligands that activate receptors on ECs and/or a high concentration of secreted Wnt inhibitors. Importantly, whatever mechanisms maintain the low level of beta-catenin signaling in CVO ECs must have a spatial distribution that closely matches that of the CVOs, since the BBB vs. non-BBB character of the vasculature switches abruptly at the border between each CVO and the adjacent CNS tissue, as seen in Wnt Inhibitory Factor-1  is expressed in the area postrema and also in the hippocampus and cortex. Our previous in situ hybridization analyses showed that sFRP1 is specifically expressed in the choroid plexus and ciliary body . However, loss of Wif1 produced a ~2 fold increase in the fraction of vessels in the area postrema that express BBB markers  were given a single dose of 400 ug of 4HT intraperitoneally and sacrificed at ~P40. Following intra-cardiac perfusion in Karnovsky’s fixative , tissues of interest were dissected and immersion-fixed in Karnovsky’s fixative overnight at 4°C. Tissues were rinsed with 0.05 N cacodylate buffer supplemented with 1.5 mM CaCl2 and 1.5 mM MgCl2, and fixed in 1% OsO4 on ice. Tissues were then rinsed with water, dehydrated, and embedded in Epoxy resin. Sections were cut with a diamond knife, placed on copper grids, stained with uranyl acetate in methanol (filtered twice through a 0.22 um filter) and viewed with a Hitachi transmission electron microscope.For TEM of brain and eye tissues, P10 mice . Homogenates were centrifuged at 10,000xg for 10 min at 4°C to create a postmitochondrial supernatant. 25 μl and 40 μl aliquots of choroid plexus homogenate and cortex homogenate, respectively, were saved as input samples. Four μl of mouse monoclonal anti-HA antibody  were added to the supernatants and rotated for 4 hr at 4°C. 100 μl protein G magnetic beads  were washed twice with polysome buffer and added directly to the antibody-coupled polysomes and rotated overnight at 4°C. The following day, samples were placed in a magnet on ice and supernatants recovered before washing the pellets 4 × 5 min in high salt buffer . To prepare total RNA, 350 μl of Qiagen RLT buffer was added to the beads or to the input samples. Total RNA was prepared according to manufacturer’s instructions using the RNeasy Plus Micro kit (Qiagen), and the quantity and quality of the RNA were assessed using a Bioanalyzer (Agilent).6–8 week old WT control and beta-catenin stabilized mice were used for the RiboTag experiments. Immunoprecipitation of the RiboTag-labelled polyribosomes were carried out as previously described . Cortex Tie2-GFP) and EC-specific beta-catenin stabilized . Pituitaries were dissected from the base of the skull and place in Dulbecco’s PBS (DPBS). Posterior pituitaries  was gently separated from the anterior pituitary and the 20–30 anterior and posterior pituitaries were collected in separate tubes for subsequent papain dissociation. RNA was extracted from GFP-positive and GFP-negative cells that were FACS sorted directly into QIAGEN Buffer RLT Plus and then processed using the RNeasy Micro Plus kit . For ATAC-seq,~50,000 GFP-positive FACS-sorted cells were gently centrifuged and then resuspended in ice-cold lysis buffer  and immediately centrifuged at 500 x g for 10 min at 4°C to prepare nuclei. The resulting nuclear pellet was resuspended in a 50 ul reaction volume in Tn5 transposase and transposase reaction buffer , and the tagmentation reaction was incubated at 37°C for 30 min.To control for the possibility of sex-dependent differences, male mice were used for RNA-seq and ATAC-seq. Viable ECs were isolated using the Worthington Papain Dissociation System  and a MoFlo XDP Sorter  as previously described , with prEach RNA-seq and ATAC-seq analysis was conducted on two biological replicates. Libraries for RNA-seq and ATAC-seq were prepared as previously described . For RNAMost data analyses were performed as previously described . For basSalmon version 0.10.2  was usedrlog and plotPCA function . Peaks were called using MACS2  .ATAC-seq data were aligned using Bowtie2 (Version 2.3.2 t -X 2000 --no-mixed --no-discordant) and then duplicate reads were removed (summits) . Peaks wsummits)  with Edgsummits) . For eaciscovery , in partBedtools intersect (-u) was used to determine features that overlapped with other features by greater than or equal to 1 bp. Bedtools window (-w 100000) was used to identify ATAC-seq peaks within 100 kb of various sets of genes. For binom.test. For fisher.test, nrow = 2), alternative=”greater’).GSE122117. Processed RNA-seq data can be accessed at the VECTRDB website (https://markfsabbagh.shinyapps.io/vectrdb/).Data files are available at GEO accession  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Elisabetta Dejana as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Dritan Agalliu (Reviewer #2); Christer Betsholtz (Reviewer #3).Thank you for submitting your article "Beta-catenin signaling affects barrier vs non-barrier gene expression in circumventricular organ and ocular vasculatures" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary and essential revisions:eLife. The two major aspects to revise are:Reviewers agreed that the study is interesting and the major conclusions of the paper, once substantiated by additional data as outlined in the reviews, would in principle be sufficient to warrant publication in 1) Better characterization of the permeability properties of the CVO endothelial cells;2) A more precise characterization of the composition of endothelial cell junctions in the different conditions.Reviewer #1:In the present paper Wang et al. study the role of canonical Wnt /beta-catenin signaling on the permeability properties of the capillaries of the circumventricular organs and choriocapillaris. These vessels present different anatomical and functional characteristics that strongly influence their low control of permeability. Applying excellent imaging analysis and a large set of murine strains of genetically modified mice the authors bring evidence that low beta-catenin signaling is responsible, at least to a significant extent, for low permeability control. This observation is intriguing since the same signaling pathway appears to be required for correct differentiation of the blood brain barrier but should be strongly reduced in the CVO vessels to favor low permeability and dynamic exchanges. This work also shows that brain endothelial cells maintain a certain degree of plasticity in the adult being capable to positively or negatively respond to Wnt signaling.The paper is well done and certainly of interest but some aspects should be better defined to make it more complete:1) Along the manuscript the way to define the blood brain barrier is mostly based on upregulation of claudin-5 expression, increase in GLUT-1 and decreased PLVAP. This may be an oversimplification also considering the extreme variety of the vasculature in the brain or CVO capillaries. I strongly recommend adding staining of other tight junction elements such as cingulin or ZOs and possibly of specific transporters, at least in some key figures.Sostdc1. These observations are novel and of interest. However, overall, the paper looks essentially descriptive. It would improve its impact by, at least, discussing the physiological and pathological meaning of these profound differences in barrier functions in the different regions of the brain.2) The analysis of chromatin accessibility and RNA confirm the fact that beta-catenin is a major player in the maintenance of permeability control in the different regions of the brain. However, beta-catenin concentration should be tightly controlled to avoid pathological reactions. In CVO, as reported in this paper, optimal level of beta-catenin activation is due to the fine balance between activators and inhibitors such as WIF-1 and Reviewer #2:In this manuscript, Wang et al., address a critical question regarding the mechanisms that prevent blood vessels in circumventricular organs (CVO) to acquire blood-brain barrier (BBB) properties compared to the rest of the central nervous system (CNS) blood vessels. The authors attribute the high permeability in CVO blood vessels to the low levels of beta-catenin signaling in these endothelial cells compared to the rest of the CNS. For example, endothelial cells in area postrema is permeable due to high expression of Wnt inhibitory factor-1 (WIF1) in this region. Moreover, overexpression of a stabilized form of beta-catenin in CVO endothelial cells led to a partial induction of BBB properties both structurally and based on the molecular signatures from the RNA seq and ATAC seq data.Overall, the study is well conceived and executed, and strengthens our understanding of the molecular mechanisms that regulate barrier properties in the CNS endothelial cells versus blood vessels in CVOs as well as the diversity among endothelial cells in distinct CNS regions. However, there are some concerns about the manuscript as follows:1) A major functional feature of the BBB is to exclude various circulating molecules from entering the CNS. However, a functional assessment of barrier properties in CVO blood vessels after beta-catenin stabilization is missing throughout the paper. The authors need to inject intravenously small or large MW tracers and assess their permeability into the CVOs under conditions of Wnt/beta-catenin activation in endothelial cells.Axin2, Apcdd1 etc.) . Several key transcriptional regulators of the BBB  are completely missing in CVO blood vessels after beta-catenin overexpression after examining carefully Supplementary file 1. There seems to be a complete absence of induction of these genes which likely reflects permanent changes in chromatin accessibility. This has not been discussed or addressed in detail in the paper. The data are there, but they are buried in supplementary files. This is a critical point that needs to be highlighted in greater detail.2) The transcriptional analysis shown in Figures 6, 7, Figure 7—figure supplement 1, Figure 7—figure supplement 2 and Supplementary files 1, 2 clearly demonstrates a partial induction of BBB-gene expression profile in CVO blood vessels. In my opinion, I do not think that this is only due to an incomplete recombinations which is obvious in the variability of the induction of Wnt downstream target genes (3) In Figure 5, it would be important to show TEM images of the structural changes in ciliary body blood vessels following beta-catenin stabilization in this organ.4) Do the blood vessels in CVO acquire Occludin protein expression at cell junctions with beta-catenin overexpression? The transcriptional analysis suggests that they do, but the authors need to show some staining with Occludin antibodies.5) Figures 7 and 8 need to be better structured to illustrate more clearly the partial shift in gene expression in AP and PP blood vessels after beta-catenin activation. Perhaps some of the key BBB genes that are induced or nor induced in CVO blood vessels can be annotated in the numerous scatter plots. The salient points of these figures are difficult to follow without some proper annotation of key BBB genes or pituitary genes. Additionally, the authors may consider replacing some of the scatter plots with Venn Diagrams illustrating changes in gene expression.Reviewer #3:Ctnnb1-exon3 allele to induce stabilization of beta-catenin in endothelial cells and knockouts for the Wnt inhibitors Wif1 and Sostdc1 to show loss of permeability and gain of a BBB-like phenotype in the non-barrier vasculature.The paper by Wang et al. shows, in a nutshell, that inhibition of Wnt-signaling is an important determinant in the maintenance of the fenestrated and permeable features of specific regional vascular beds within or in close contact with the CNS, i.e. the CVOs, choroid plexus, choriocapillaris and the ciliary body vasculature, which are characterized by a non-barrier phenotype, in marked contrast to the blood-brain/retina barrier (BBB) phenotype of the other CNS vasculature. The authors use a floxed All in all this is a beautiful study that deserves publication without additional experimental work. While the mechanistic insights are valuable, they don't come as a surprise given prior literature, in part from the Nathan's lab, demonstrating the importance of Wnt signaling for induction and maintenance of the BBB. What is equally valuable to the field, and what impressed me a lot during the reading was the beautiful morphological analyses and the comprehensive and scholarly overview of the field and its history. The manuscript is clearly written and partly a wonderful read. Perhaps the RNA seq and ATAC Seq section in the Results could be condensed and somewhat simplified to enhance the reading. The conclusions of this part are straightforward and simple and the data description would therefore benefit from some shortening.I have a few suggestions for additional citations:The paper from Phoenix et al. (Gilbertson lab) deserves mentioning, as it beautifully illustrates the power of Wnt-inhibition to reprogram the BBB into a fenestrated non-barrier vasculature: DOI:https://doi.org/10.1016/j.ccell.2016.03.002In conjunction with the discussion of MFSD2A, I think David Silver's ground-breaking work on the lipid-transporting capacity of this molecule should be mentioned along with the reference to Ben-Zvi et al. (Gu lab): doi: 10.1038/nature13241The similarity between the choroid plexus and ciliary body is striking and could be additionally cited by e.g. the work from Maria Lehtinen's lab on tumor development in these structures: doi: 10.1016/j.ajpath.2018.02.009. Summary and essential revisions:Reviewers agreed that the study is interesting and the major conclusions of the paper, once substantiated by additional data as outlined in the reviews, would in principle be sufficient to warrant publication in eLife. The two major aspects to revise are:1) Better characterization of the permeability properties of the CVO endothelial cells;We have performed a permeability analysis using Sulfo-NHS-biotin and the results are shown in panels H and I in the newly expanded Figure 5. Stabilizing beta-catenin in endothelial cells (ECs) results in dramatically reduced vascular permeability in the CVOs and modestly reduced permeability in the choroid plexus. Thank you for suggesting this experiment.2) A more precise characterization of the composition of endothelial cell junctions in the different conditions.We have expanded Figure 3 and added three new supplementary figures showing the induction of ZO1 and Occludin in CVO endothelial cells in response to beta-catenin stabilization. These figures also show the induction of the multidrug resistance transporter MDR1 in CVO endothelial cells in response to beta-catenin stabilization. Multiple figures show induction of CLDN5 in CVO ECs in response to beta-catenin stabilization.Reviewer #1:In the present paper Wang et al. study the role of canonical Wnt /beta-catenin signaling on the permeability properties of the capillaries of the circumventricular organs and choriocapillaris. These vessels present different anatomical and functional characteristics that strongly influence their low control of permeability. Applying excellent imaging analysis and a large set of murine strains of genetically modified mice the authors bring evidence that low beta-catenin signaling is responsible, at least to a significant extent, for low permeability control. This observation is intriguing since the same signaling pathway appears to be required for correct differentiation of the blood brain barrier but should be strongly reduced in the CVO vessels to favor low permeability and dynamic exchanges. This work also shows that brain endothelial cells maintain a certain degree of plasticity in the adult being capable to positively or negatively respond to Wnt signalingThe paper is well done and certainly of interest but some aspects should be better defined to make it more complete:1) Along the manuscript the way to define the blood brain barrier is mostly based on upregulation of claudin-5 expression, increase in GLUT-1 and decreased PLVAP. This may be an oversimplification also considering the extreme variety of the vasculature in the brain or CVO capillaries. I strongly recommend addong staining of other tight junction elements such as cingulin or ZOs and possibly of specific transporters, at least in some key figures.We have added immunostaining for ZO-1, Occludin, and MDR1 in the following new figures or parts of figures: Figure 3E and F, Figure 2—figure supplement 3, Figure 2—figure supplement 4, Figure 3—figure supplement 3. These new data show induction of each of these BBB markers in CVOs in response to beta-catenin stabilization.2) The analysis of chromatin accessibility and RNAseq confirm the fact that beta-catenin is a major player in the maintenance of permeability control in the different regions of the brain. However, beta-catenin concentration should be tightly controlled to avoid pathological reactions. In CVO, as reported in this paper, optimal level of beta-catenin activation is due to the fine balance between activators and inhibitors such as WIF-1 and Sostdc1. These observations are novel and of interest. However, overall, the paper looks essentially descriptive. It would improve its impact by, at least, discussing the physiological and pathological meaning of these profound differences in barrier functions in the different regions of the brain.Thank you for that suggestion regarding an expanded discussion of physiologic and pathologic implications. We have added a new section – titled “Clinical Implications” – to the Discussion to encompass some of our thoughts regarding the implications of the present work for understanding pathologic BBB breakdown and for developing ways to reduce BBB function for therapeutic purposes.Reviewer #2:In this manuscript, Wang et al., address a critical question regarding the mechanisms that prevent blood vessels in circumventricular organs (CVO) to acquire blood-brain barrier (BBB) properties compared to the rest of the central nervous system (CNS) blood vessels. The authors attribute the high permeability in CVO blood vessels to the low levels of beta-catenin signaling in these endothelial cells compared to the rest of the CNS. For example, endothelial cells in area postrema is permeable due to high expression of Wnt inhibitory factor-1 (WIF1) in this region. Moreover, overexpression of a stabilized form of beta-catenin in CVO endothelial cells led to a partial induction of BBB properties both structurally and based on the molecular signatures from the RNA seq and ATAC seq data.Overall, the study is well conceived and executed, and strengthens our understanding of the molecular mechanisms that regulate barrier properties in the CNS endothelial cells versus blood vessels in CVOs as well as the diversity among endothelial cells in distinct CNS regions. However, there are some concerns about the manuscript as follows:1) A major functional feature of the BBB is to exclude various circulating molecules from entering the CNS. However, a functional assessment of barrier properties in CVO blood vessels after beta-catenin stabilization is missing throughout the paper. The authors need to inject intravenously small or large MW tracers and assess their permeability into the CVOs under conditions of Wnt/beta-catenin activation in endothelial cells.Thank you for suggesting this experiment. We have performed a permeability analysis using Sulfo-NHS-biotin and the results are shown in panels H and I in the newly expanded Figure 5. This experiment shows that stabilizing beta-catenin results in a dramatically reduced vascular permeability in the CVOs and modestly reduced permeability in the choroid plexus.2) The transcriptional analysis shown in Figures 6, 7, Figure 7—figure supplement 1, Figure 7—figure supplement 2 and Supplementary files 1, 2 clearly demonstrates a partial induction of BBB-gene expression profile in CVO blood vessels. In my opinion, I do not think that this is only due to an incomplete recombinations which is obvious in the variability of the induction of Wnt downstream target genes  . Several key transcriptional regulators of the BBB  are completely missing in CVO blood vessels after beta-catenin overexpression after examining carefully Supplementary file 1. There seems to be a complete absence of induction of these genes which likely reflects permanent changes in chromatin accessibility. This has not been discussed or addressed in detail in the paper. The data are there, but they are buried in supplementary files. This is a critical point that needs to be highlighted in greater detail.Thank you for pointing this out. We agree. We have expanded the Results text to discuss more fully the fact that the gene expression changes induced by beta-catenin stabilization represent only a partial recapitulation of the full BBB gene expression program and we have added two supplementary figures . In the Results, we have added the following sentence: “As seen in Figure 6A, Figure 6A—figure supplement 1, and in the figures that follow, a consistent feature of BBB transcript levels and ATAC peak areas in beta-catenin stabilized pituitary ECs is that, even when induced, the majority are substantially lower than their counterparts in cerebellar ECs.” In the Discussion, we have added the following sentence: “The ATAC analyses represents one step in defining this plasticity at the level of the chromatin state, and – like the transcriptome changes that accompany beta-catenin stabilization – they show that changes in accessible chromatin do not fully recapitulate the BBB pattern, implying a requirement for additional signals or an irreversibility to some chromatin features of the high permeability EC gene expression program.”3) In Figure 5, it would be important to show TEM images of the structural changes in ciliary body blood vessels following beta-catenin stabilization in this organ.We have these TEM images, but they are not as aesthetically pleasing as the ones from the pituitary, choroid plexus, and choriocapillaris because, in our hands, cardiac perfusion does not efficiently flush out the contents of the ciliary body capillaries. The result is a background haze of serum proteins in the vessel lumen. This does not affect our ability to see and quantify the fenestrae, but it makes for an image that is not aesthetically pleasing.4) Do the blood vessels in CVO acquire Occludin protein expression at cell junctions with beta-catenin overexpression? The transcriptional analysis suggests that they do, but the authors need to show some staining with Occludin antibodies.Yes, Occludin and ZO1 are both induced in CVO endothelial cells in response to beta-catenin stabilization and they both cluster at cell junctions, as does Claudin5. These data have been added in a set of new panels and new supplementary figures .5) Figures 7 and 8 need to be better structured to illustrate more clearly the partial shift in gene expression in AP and PP blood vessels after beta-catenin activation. Perhaps some of the key BBB genes that are induced or nor induced in CVO blood vessels can be annotated in the numerous scatter plots. The salient points of these figures are difficult to follow without some proper annotation of key BBB genes or pituitary genes. Additionally, the authors may consider replacing some of the scatter plots with Venn Diagrams illustrating changes in gene expression.Thank you for that comment. We have annotated the same set of 7 key BBB genes in each of the scatterplots in Figure 7 so that the reader can follow the abundances of those transcripts across the different comparisons.Reviewer #3:[…] All in all this is a beautiful study that deserves publication without additional experimental work. While the mechanistic insights are valuable, they don't come as a surprise given prior literature, in part from the Nathan's lab, demonstrating the importance of Wnt signaling for induction and maintenance of the BBB. What is equally valuable to the field, and what impressed me a lot during the reading was the beautiful morphological analyses and the comprehensive and scholarly overview of the field and its history. The manuscript is clearly written and partly a wonderful read. Perhaps the RNA seq and ATAC Seq section in the Results could be condensed and somewhat simplified to enhance the reading. The conclusions of this part are straightforward and simple and the data description would therefore benefit from some shortening.Thank you for the positive assessment. In considering the description of the RNAseq and ATAC-seq data, we don’t think that we can shorten it without making it difficult for the reader to follow the logic of the analyses and the conclusions. Also, not all readers will be fully familiar with the “omics” literature in the context of vascular endothelial cells. Some extra explanatory text will help make the work accessible to a broader audience. We have broken this up into an additional section with a new heading, to improve the organization.I have a few suggestions for additional citations:The paper from Phoenix et al. (Gilbertson lab) deserves mentioning, as it beautifully illustrates the power of Wnt-inhibition to reprogram the BBB into a fenestrated non-barrier vasculature: DOI:https://doi.org/10.1016/j.ccell.2016.03.002In conjunction with the discussion of MFSD2A, I think David Silver's ground-breaking work on the lipid-transporting capacity of this molecule should be mentioned along with the reference to Ben-Zvi et al. (Gu lab): doi: 10.1038/nature13241The similarity between the choroid plexus and ciliary body is striking and could be additionally cited by e.g. the work from Maria Lehtinen's lab on tumor development in these structures: doi: 10.1016/j.ajpath.2018.02.009.Thank you for these excellent suggestions. We have incorporated all three references into the text and we have expanded the Discussion  to discuss, among other things, the Phoenix et al. study of vascular permeability in medulloblastoma.


Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential β-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/β-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases β-catenin Kat3 coactivator usage and enhances β-catenin/CBP transcription at the expense of β-catenin/p300 transcription. Biased β-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced β-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/β-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of β-catenin Kat3 coactivator and the mode of stem cell division. Adult somatic stem cells (SSCs) ensure homeostatic maintenance and provide regenerative capacity after insult. Long-term repopulating SSCs, although generally quiescent, can reenter the cell cycle and subsequently, undergo mitosis. These mitotic divisions can be asymmetric, whereby one of the daughter cells remains in the niche as an SSC, while the other daughter proceeds to differentiate. Alternatively, SSCs can undergo symmetric divisions, whereby either both daughter cells remain as SSCs, or both differentiate. The decision to undergo an asymmetric versus a symmetric division is critical both during embryogenesis and in the adult organism. Improper regulation of this decision likely underlies a large array of diseases including cancer, neurodevelopmental and neurodegenerative diseases, and more generally, is associated with the aging process ,2,3,4.We have extensively examined the effects and therapeutic potential of selectively antagonizing the CBP/β-catenin interaction, utilizing the specific-small-molecule antagonist ICG-001, in a variety of preclinical models. We have demonstrated the ability to safely eliminate drug-resistant cancer stem/tumor-initiating cells (CSC/TIC), both in solid tumors and leukemias, without any deleterious effects to the normal endogenous stem cell populations ,9,10,11.Normal long-term repopulating SSCs preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue both during homeostatic maintenance and repair processes A, whereaTo further investigate this model, we chose a system wherein precise regulation of the SSC’s capacity to self-renew and differentiate is necessary for harmonious histogenesis . In thisMCPH1) are linked to microcephaly. p73, a member of the p53 family, is a target of MCPH1 [Subtle changes in the number of progenitors present at the onset of neurogenesis can have a dramatic impact on the expansion of the cortical surface and ultimately, brain size ,26,27. Iof MCPH1 . Interesof MCPH1 ,36,37. pof MCPH1 ,39,40. Eof MCPH1 ,39,41. Iof MCPH1 . We, theCrosstalk between Wnt- and p73-dependent signaling has previously been examined in both stem cells and more differentiated progenitors ,44. We nThe role of p73 during neuronal development has been previously investigated in vitro, implicating p73 in the long-term maintenance of the NSC pool ,40. In oTo examine if the p73KO phenotype is due to premature neuronal differentiation, we monitored neurogenic status during early corticogenesis using NeuN immunostaining. We first assessed the impact of p73 loss on the post-mitotic neurons present at E13.5. Although the overall cellularity of the cerebral cortex is not affected , E13.5 pSSCs self-renew through asymmetric divisions, and can amplify themselves through symmetric proliferative divisions. On the contrary, symmetric differentiative divisions can lead to the exhaustion of the SSC pool . The impTwo additional phenomena could potentially contribute to the depletion of NSCs: (i) a decrease in proliferative potential, pushing progenitors toward premature differentiation ; (ii) orWe previously proposed that differential β-catenin/Kat3 coactivator association plays a key role in the maintenance of SSC pools ,2,4,19. Importantly, based upon Par3 distribution, ICG-001 treatment is also sufficient to correct the proportion of asymmetric divisions to the normal level . Taken tOur results demonstrate that p73KO disrupts the mode of division of early NSCs. Mitotic spindle orientation appears to play a decisive role in the choice between symmetric and asymmetric division in NSCs during neurogenesis ,57,58. IPP4 has previously been shown to mediate the localization of the Miranda complex during Drosophila neuroblast asymmetric differentiation . AdditioTo further demonstrate the causal relationship between the modulation of β-catenin Kat3 coactivator interactions and the mode of division of embryonic cortical NSCs, we utilized two previously well-characterized specific-small-molecule p300/β-catenin antagonists, YH249 and IQ1 ,66,67,68+ precursors have been previously shown to undergo asymmetric divisions [+ mitotic figures present in the E12.5 ventricular surface underwent asymmetric mitotic division, based upon Par3 distribution  , which mivisions . We confn 65.6%;  last rown 65.6%; . This suTo assess the consequences of direct perturbation of the mode of division in NSCs on the rate of neurogenesis, we again investigated NeuN and ngn2 expression by IF (immunofluorescence). Treatment with YH249 or IQ-1 increased the percentage of post-mitotic neurons, as well as the percentage of neurogenic precursors, similarly to p73KO A,B. WherFinally, we monitored the long-term impact of antagonizing the β-catenin/p300 interaction on NSC pool maintenance. E9.5 pregnant wild-type Bl6 females were treated daily with YH249 for 8 days. Recapitulating the effects seen with p73KO embryos, YH249 treatment resulted in significant depletion of the NSC pool in the GZ at E17.5 . Daily tWe conclude that direct disruption of the mode of NSC division via modulation of the β-catenin interaction with p300 affects the rate of neurogenesis at the early stages of cerebral cortex development, which is likely coupled to PP4 regulation of the phosphorylation status of Ndel1 thus modulating its interaction with Lis1, thereby affecting mitotic spindle orientation and the accurate control of cell division asymmetry associated with normal neuronal differentiation . The lonTimely activation and proper control of the mode of division of the SSC pool are critical decisions required during development and for both normal adult homeostasis and repair after injury. We have previously demonstrated that the potent specific-small-molecule CBP/β-catenin antagonist ICG-001, can safely eliminate drug-resistant cancer stem cells (CSCs), yet have beneficial effects on the normal endogenous stem cell populations ,9,10,11.We have now examined this model during mouse embryonic neurogenesis . PreviouThe data presented here at first glance may appear contradictory to previously published in vitro data. In vitro studies have concluded that the pharmacologic disruption of p300/β-catenin interaction is sufficient to maintain pluripotency and the self-renewal of both mouse and human ES (embryonic stem cell) cells ,52,53,66Tight control over SSC self-renewal capacity is essential to prevent oncogenesis ,80,81. RAnimal studies were approved by the USC  institutional  IACUC as per protocol #11158 . Wild-type (WT), p73 knock-out (KO)  and Tis2After unmasking treatment with DAKO Target Retrieval Solution (#S1700) and blocking with normal serum, the following antibodies were used: Activated Caspase-3 ; GFP ; GFP ; mNeuN ; mNgn2 ; Par3 ; PCNA ; phospho-Histone H3 ; phospho-Vimentin . DAPI was used as a counter-stain. All pictures were taken using a Zeiss LSM5 confocal system and further analysis of intensity levels was processed using Adobe Photoshop software .Co-immunoprecipitation of CBP or p300 with β-catenin was performed as described elsewhere . For WesAfter immunostaining for Par3 and Ph-Vimentin, z-stacks of phospho-vimentin+ mitosis were taken with a Zeiss LSM5 confocal. Using Adobe Photoshop software, the level of Par3 and DAPI signals in both daughter cells were quantified in at least 4 consecutive plans. The normalized ratios of fluorescent intensity for both Par3 and DAPI were calculated as described by Bultje et al. . Mitosis-ddCt method.Using PerfectPure RNA Fibrous Tissue Kit (Fisher Scientific), RNA was extracted from the telencephalon of E13.5 WT and p73KO embryos, which had undergone treatment for 4 days with either DMSO vehicle or ICG-001 (660 mg/kg). Reverse transcription was performed using a Quanta Biosciences qScript cDNA Synthesis kit. qPCR was run using Quanta PerfeCTa SYBR Green super Mix for iQ on BioRad Cycler. Primer sequences: PPP4c: 5′-CTTGGTAGAAGAGAGCAACGTG-3′, 5′-CGCCACCTACTCTGAACAGC-3′; SMEK1: 5′-TATGACTTGGCCCTTAGCTTTCA-3′, 5′-ACCTGGTGAGGACATATCATCA-3′. Data were normalized to the reference gene, mouse Gapdh. Relative expression levels were calculated using the 2t-test or One-way ANOVA with a post-hoc Newman–Keuls multiple comparison test was performed as appropriate. p-values < 0.05 were considered significant.Numerical data were expressed as the means ± SEM, unless otherwise noted. Student’s Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required to generate new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. Even when treated with the CBP/β-catenin antagonist ICG-001, normal SSCs that divide asymmetrically will always retain one of the dividing daughter cells in the stem cell niche. However, when treated with ICG-001, CSCs will be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases β-catenin Kat3 coactivator usage and enhances β-catenin/CBP transcription at the expense of β-catenin/p300 transcription. Biased β-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced β-catenin/CBP interaction and transcription leads to an increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by KO of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/β-catenin antagonist, ICG-001. Taken together, our results demonstrate the causal relationship between the choice of β-catenin Kat3 coactivator and the mode of stem cell division, with major implications for how to therapeutically target and eliminate CSCs, as well as enhance the repair potential of normal SSCs, without damaging or depleting the normal endogenous stem cell populations.


We reported the preparation of multilayer Ti3C2 MXene by HF etching method and obtained few-layer Ti3C2 MXene by simple ultrasonication. Meanwhile, Ti3C2/epoxy composites were prepared by a solution blending method. The results show that the thermal properties of the composites are improved in comparison with the neat epoxy. Thermal conductivity value (0.587 W/mK) of epoxy composite with only 1.0 wt% Ti3C2 MXene fillers, is increased by 141.3% compared with that of neat epoxy. In addition, the composite presents an increased glass transition temperature, high thermal stability and lower coefficient of thermal expansion. This work is of great significance for the research of high-performance composite materials.With the development of electronic devices such as integrated circuits toward the continual increase in power density and consumption, the efficient heat dissipation and low thermal expansion of materials become one of the most important issue. However, conventional polymers have the problem of poor thermal dissipation performance, which hinder application for electronic devices. In this work, the two-dimensional material, MXene (Ti Meanwhile, in the field of electronic packaging, it’s necessary for polymer composites to improve the thermal conductivity meanwhile lower thermal expansion coefficient4. In order to improve the thermal conductivity of polymers, a method of adding high content of fillers into the polymer matrix is employed in most previous works. The method often results in poor mechanical properties, high density and high costs5. These adverse consequences make it difficult for polymer composites to meet the requirements of industry application. Therefore, it is very meaningful to research on the preparation of polymer composites with high thermal conductivity and ultralow fillers loading.As electronic devices towards more integrated and lightweight, efficient heat dissipation is critical to enhance the longevity of the device6. Two-dimensional materials possess the greatly high aspect ratios and the thicknesses corresponding to a few atomic layers10. As a novel type of two-dimensional transition metal carbides or carbonitrides, MXenes possess a graphene-like two-dimensional structure and synthesized by HF selectively etching away A atom layers of the MAX phases12. And they possess a unique two-dimensional layered structure, high specific surface area, glorious thermal properties, outstanding adsorption properties, excellent mechanical properties and electrical properties. Thus, MXenes have been used in many fields such as energy storage, catalysis, adsorption, hydrogen storage, sensors and so on27. Up to now, many reported literatures explore the use of MXenes in energy storage applications, conductivity enhancers of polymers and reinforcement additives for the mechanical properties of polymers32. While, so far, few studies have considered MXenes as a material ever to optimize the thermal properties of polymers33.Nowadays, two-dimensional materials have attracted more and more attention since the discovery of graphene35. However, the intrinsically low thermal conductivity and relatively inferior coefficient of thermal expansion limit its application37. Therefore, it is necessary to improve the thermal properties for epoxy resin. As one of the useful strategies for improving the thermal properties of polymers, adding reinforcement additives into polymers is often used. Among reinforcement additives, nanomaterials are advantageous for improving some properties of polymers due to large specific surface area and other unique properties26. By far, a lot of nanomaterials have been researched as fillers and reported in previous works, such as graphene39, boron nitride43, and silicon carbide nanowires45. These works demonstrate that the thermal, mechanical and electrical property can be significantly enhanced by addition of nanomaterials. In recent years, numerous two-dimensional materials have aroused a great deal of interest and triggered many works on improving thermal properties of polymers50. However, there is no study yet that using Ti3C2 MXene as additives to improve the thermal properties of epoxy resin. Hence, the studies on epoxy composites with Ti3C2 MXene are of theoretical and practical significance.Epoxy resin is high-performance engineering thermosetting plastic and possesses various outstanding properties, such as good chemical inertness, high wear resistance, excellent adhesive property, outstanding mechanical properties and so on. Hence, it is widely utilized in the fields of electronic products, high-voltage electrical equipment, petrochemicals and anti-corrosion pipeline valves3C2 MXene was prepared by directly etching MAX phase (Ti3AlC2) with HF. Afterwards, few-layer Ti3C2 MXene was obtained from multilayer Ti3C2 MXene by simple ultrasonication and was added into epoxy matrix to fabricate Ti3C2/epoxy composites by a solution blending method. As a result, the fabricated composites with ultralow loading showed higher thermal conductivity, glass transition temperature (Tg), thermal stability and lower coefficient of thermal expansion (CTE) compared with that of neat epoxy.In this work, multilayer Ti3AlC2 powder was obtained from Forsman Scientific Co., Ltd. . Cycloaliphatic epoxy resin  used in the study was purchased from Shanghai Liyi Science & Technology Development, China. The curing agent, Methylhexahydrophthalic anhydride, was brought from Zhejiang Alpharm Chemical Technology Co., Ltd. . Neodymium(III) 2,4-pentanedionate (Nd(III)acac) was provided by Aldrich Chemicals.Hydrogen fluoride  and anhydrous ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. . Ti3C2 MXene was synthesized by exfoliating the MAX phases with hydrogen fluoride solution. The specific steps are showing as follows: first of all, 10.0 g Ti3AlC2 powder was slowly added into 100 ml 40% hydrogen fluoride solution in a polytetrafluoroethylene container. At room temperature, the mixture was kept being stirred for 3 h. Afterward, 300 ml deionized water was slowly added into the resulting mixture to get the diluted suspension. Subsequently, the suspension was centrifuged at 4000 rpm for 15 min and the supernatant was discarded to collect the sediment. The sediment was washed with deionized water until the pH was close to 752. And then, the washed powder was dried under vacuum conditions at 100 °C for 6 h. To obtain few-layer Ti3C2 MXene, 2.5 g dried powder was added to 400 ml distilled water in a 500 ml glass beaker and was ultrasonicated for 2 h. The obtained suspension was centrifuged at 2000 rpm for 15 min. The supernatant was separated from the sediment powders and a black colloidal suspension was obtained. Finally, the colloidal suspension was filtered onto a 0.22 mm pore-size polypropylene membrane and was dried under vacuum conditions at 100 °C for 24 h to obtain few-layer Ti3C2 MXene powder.Ti3C2 MXene fillers were prepared as following steps. Firstly, Nd(III)acac was added to the epoxy resin, with the weight ratio to be 1:1000 (Nd(III)acac: epoxy resin). And the mixture was kept being stirred at 80 °C for 3 h. The resulting homogeneous solution was then cooled to room temperature. Secondly, 0.2, 0.4, 0.6, 0.8 and 1.0 wt% of Ti3C2 MXene (the weight ratio means the weight of Ti3C2 MXene to the weight of the epoxy composite) was added respectively into a certain amount of ethanol (1 mg of Ti3C2 MXene per 1 ml ethanol) and was then ultrasonicated for 1 h to form a homogeneously dispersed suspension. Subsequently, the pre-prepared epoxy resin was added into the suspension. Meanwhile, the mass of the mixture was measured and recorded. The mixture was then kept being stirred with an electric stirrer at 80 °C for 4 h. After that, the mixture was weighed every ten minutes and the mass difference was calculated. When the mass difference is equal to the mass of the added ethanol (the mass error ≤0.4%) and remains unchanged, the ethanol is considered to be evaporated completely. The real photos of the obtain mixture are shown in Fig. 3C2/epoxy composites were used to represent the epoxy composites with Ti3C2 MXene fillers. The real photos of Ti3C2/epoxy composites and neat epoxy are exhibited in Fig. The epoxy composites with varying contents of Ti3C2 MXene and the fractured surfaces of epoxy samples, with an accelerating voltage of 10 kV. To observe the nature of Ti3C2 MXene bonding to epoxy, samples were quenched in liquid nitrogen and then broken to obtain the fracture surfaces. Besides, to avoid the charge accumulation, a thin layer of gold was sprayed on the fracture surfaces. JEOL JEM2100 transmission electron microscopy (TEM) with an accelerating voltage of 200 kV was used to observe the microstructures of ultrathin Ti3C2 MXene nanosheets. The Ti3C2 MXene suspension was dropt onto carbon-coated copper grids and dried in a vacuum oven to fabricate the samples for TEM. A scanning probe microscope  was used to determine the thickness of exfoliated Ti3C2 MXene. A powder diffractometer  with CuKα radiation was used to acquire the X-ray diffraction (XRD) patterns of Ti3C2 MXene. The Raman spectra was performed on a Raman spectrometer . Fourier transform infrared (FT-IR) spectra were obtained using a FTIR  between 400 and 4000 cm−1. X-ray photoelectron spectra (XPS) was conducted by means of an X-ray photoelectron spectrometer . A flash thermal conductivity meter  was used for measurements of the thermal diffusivity  and the size of each measured sample is 10 (length) × 10 (width) × 0.8 mm (thickness). The thermal conductivity  of Ti3C2/epoxy composites and neat epoxy was calculated through the Eq. . The density  was measured through liquid displacement method. The specific heat capacity  and the glass transition temperature (Tg) were obtained by differential scanning calorimetry (DSC). And the testing equipment is a differential scanning calorimeter . Thermal gravimetric analysis (TGA) was conducted by using a TGA 209 F3 . Composites and Ti3C2 MXene were measured in the temperature range of 50–800 and 50–1000 °C, respectively. Besides, all the measurements were conducted under a nitrogen atmosphere and the heating rate is 10 °C/min. The CTE measurements were conducted on a thermal mechanical analyzer  from 40 to 180 °C at a heating rate of 5 °C/min. An infrared camera  was used to take IR-photos.A field emission scanning electron microscope  equipped with energy dispersive spectrometer was used to characterize the surface morphology of the Ti3C2 MXene was observed by FE-SEM. Figure 3C2 MXene53. It is found that Ti3C2 MXene obtained by HF directly etching MAX phase has stacked layers and the thickness of Ti3C2 MXene is approximately 10 μm. From the inserted higher magnification image, the gaps between layers are of different sizes and the thickness of 1 μm corresponds to approximately 11 layers. Figure 3C2 MXene is mainly composed of ultrathin Ti3C2 MXene nanosheets and the lateral size of the Ti3C2 MXene nanosheets is approximately 2 μm. The results indicate that ultrasonication is effective for converting multi-layered Ti3C2 MXene into few-layer Ti3C2 MXene. Besides, the energy dispersive spectroscopy (EDS) of Ti3C2 Mxene was shown in Fig. 3C2 MXene. And the Fig. 3C2 MXene nanosheets are quite thin and transparent. From a high-resolution TEM image, Fig. 3C2 can be observed clearly, which illustrates that Ti3C2 MXene is a kind of crystal. The inset of Fig. 3C2 MXene nanosheet and it obviously signifies that Ti3C2 MXene has hexagonal structure, which is the same with Ti3AlC2 (MAX phase)54. An AFM image of Ti3C2 MXene nanosheets is shown in the Fig. 3C2 MXene nanosheet is about 3.9 nm. In addition, the AFM image also proves that Ti3C2 MXene nanosheets are quite thin and flat, which is consistent with previous analysis of SEM and TEM. As shown in Fig. 3C2 MXene nanosheets is 5.1 nm.As shown in Fig. 3C2 MXene is shown in Fig. −1 are the characteristic peaks of Ti3C2 MXene55. The peaks at 215 and 705 cm−1 are assigned to the Ti-C and C-C vibrations (A1g symmetry) of the oxygen-terminated Ti3C2O2. The peak at 387 cm−1 is attributed to the O atoms Eg vibrations. The peak at 620 cm−1 comes mostly from Eg vibrations of the C atoms in the OH-terminated Ti3C2. The above results illustrate that there are some surface terminations on the surface of Ti3C2, such as -OH and -O. Additionally, the incompletely etched Ti3AlC2 brings about a sharp peak at around 135 cm−1 56.Raman spectrum of the Ti3C2 MXene is shown in Fig. 3AlC2 are at 9.7° and 19.4°, respectively. However, it is obvious that the (002) peak and (004) peak shift to low angle, 9.1° and 18.3°, which is attributed to the newly synthesized Ti3C2 MXene51. In addition, the peaks appearing at 37° and 61.5° belong to Ti3C2 MXene. The shift and broadening of (002) peak mean that the d-spacing of Ti3C2 MXene is increased and the thickness of Ti3C2 MXene layers is declined58. The XRD peak at 23.4° is corresponding to the (100) peak of TiOF2, which is formed from a reaction between Ti3C2 MXene with -F and -OH/-O terminating groups59. The (111) peak at 36° and (200) peak at 41.7° are assigned to TiC impurity60. Besides, the peak at 41.7° partly testifies the presence of residual Ti3AlC2. It is notable that the characteristic (104) peak (39.2°) of Ti3AlC2 disappears, which indicates that the HF etch of Ti3AlC2 (MAX phase) is efficient52.The XRD pattern of theTi3C2 MXene, the FTIR spectrum of theTi3C2 MXene is shown in Fig. −1 demonstrate the existence of crystal water. The peak at 1645 cm−1 reveals the existence of liquid water13. There are four peaks at 1110, 1055, 1020 and 990 cm−1, due to the stretching vibrations of C-OH62. The peaks at 1147 and 1160 cm−1 are respectively assigned to the symmetric stretching of CH3 and CH2. The peaks appear at 1210 and 1278 cm−1, corresponding to the antisymmetric stretching of CH2 and CH3. Additionally, there are some characteristic peaks at 1315, 1340, 1370 and 2900 cm−1, which are associated with the C-H band of CHx63. Particularly, the characteristic peak at 2900 cm−1 is assigned to the stretching vibration of C-H. The FTIR results agree well with the foregoing Raman spectrum and XRD.In order to characterize the functional groups on the surface of Ti3C2 MXene, the TGA and DTG curve of Ti3C2 MXene are exhibited in Fig. 3C2 MXene surface67. Due to the loss of OH groups attached on Ti3C2 MXene surface, the weight loss is approximately 4.9% in the second stage (200–780 °C). In the third stage (780–1000 °C), the weight loss is about 5.2%, which is caused by the loss of chemically bonded F and O groups68.For the research of the thermal stability of Ti3C2 MXene in detail, XPS analysis was conducted and shown in Fig. 69. In particular, from Fig. 3C2 MXene, it is known that the peaks at around 284, 455, 532 and 685 eV respectively correspond to C 1s, O 1s, Ti 2p and F 1s. The region C 1s of Ti3C2 MXene is shown in Fig. x, such as Ti3C2Ox, Ti3C2(OH)x, Ti3C2Fx and Ti3C2OH-H2O71. The peak at 284.7 eV is assigned to graphitic C-C and the peak at 287.6 eV is assigned to CHx or C-O72. Figure 3C2 MXene. The peaks at binding energy values of 455, 455.8, 457.2, 458.6 and 459.3 eV are assigned to Ti-C, Ti(II), Ti(III), TiO2 and TiO2-xFx, respectively74. The presence of Ti(II) and Ti(III) is due to the formation of Ti3C2OX, Ti3C2(OH)x and Ti3C2OH-H2O. Figure 3C2 MXene sample, which is divided into two peaks. The peak at 529.9 eV corresponds to TiO2 and the peak at 532 eV is assigned to C-Ti-Ox or C-Ti-(OH)x75. The region F 1s of Ti3C2 MXene sample is shown in Fig. 76. The other occurring at 686.4 eV is assigned to Al-F74. The above results indicate that the terminations of the Ti3C2 MXene surface are various and also demonstrate the existence of some surface terminations, such as -O, -OH and -F.To study the surface chemical compositions of Ti3C2 MXene in the epoxy matrix and its interfacial adhesion with epoxy matrix, a FE-SEM was used to investigate the microcosmic morphologies of the samples. The SEM images were shown in Fig. 3C2 MXene fillers, respectively. Figure 3C2/epoxy composites possess rougher fracture surfaces compared with neat epoxy. Furthermore, it can be obviously observed that few-layer Ti3C2 MXene fillers are homogeneously distributed and inserted into the matrix, which reveals the strong interaction between few-layer Ti3C2 MXene fillers and the matrix. Meanwhile, as the content of the filler increases, more and more few-layer Ti3C2 MXene fillers appear on the fracture surfaces.To further study the dispersion of Ti3C2/epoxy composites and neat epoxy. It can be clearly known that the heat conduction performance of these composites is gradually improved with an increase in the amount of Ti3C2 MXene fillers. The thermal diffusivity value and thermal conductivity value of neat epoxy are 0.110 mm2/s and 0.243 W/mK, respectively. The thermal diffusivity values of Ti3C2/epoxy composites with 0.2, 0.4, 0.6, 0.8 and 1.0 wt% Ti3C2 MXene fillers are 0.122, 0.132, 0.145, 0.168 and 0.261 mm2/s, respectively. And the corresponding thermal conductivity values of Ti3C2/epoxy composites are 0.270, 0.293, 0.323, 0.374 and 0.587 W/mK. The thermal conductivity enhancement (TCE) was calculated and shown in the inset of Fig. 3C2 MXene filler is improved by 141.3% compared with that of neat epoxy. To properly evaluate heat conduction performance of the samples in this work, some thermal conductivity values of epoxy composites, which are reported in previous works. As shown in Fig. 89. In addition, this work possesses comparable or even higher the thermal conductivity values compared with previous works, where filler loading is higher. It should be noted that the thermal conductivity value of the epoxy composite with 20 wt% few-layer boron nitride nanosheets is 0.58 W/mK and that of the epoxy composite with 5.0 wt% modified graphene nanosheets is 0.56 W/mK89. The results of those two works are lower than that of this wok (0.587 W/mK). In order to reasonably explain the improvement of thermal conductivity for this work, the influencing factors are analyzed as follows. The neat epoxy and its composites are amorphous polymers. Phonons, a quantum mode of vibration, play a key role in heat conduction of amorphous polymers. In this mode of heat conduction, the scattering of phonons determines the thermal resistance at low temperatures and influences the heat conduction performance. As known to all, neat epoxy is poor in crystal structure. And the molecular chains of the epoxy resin are randomly entangled, which causes a severe phonon scattering phenomenon90. Therefore, neat epoxy possesses low thermal conductivity. Addition of high thermal conductivity fillers, scattering of phonons and interfacial thermal resistance between fillers and matrix significantly affect the heat conduction performance of polymer composites. Ti3C2 MXene fillers are added into the epoxy matrix as reinforcement phase. As a bridge between epoxy molecular chains, Ti3C2 MXene promotes the transfer of heat between molecular chains, which could efficiently improve the heat conduction performance of epoxy composites92. Furthermore, the addition of Ti3C2 MXene fillers could reduce the randomness of the molecular chains around fillers and weaken the scattering of phonons in a degree. In addition, Ti3C2 MXene fillers homogeneously disperse in the epoxy matrix and surface terminations of Ti3C2 MXene are beneficial to forming the good interface compatibility between fillers and epoxy matrix, which reduce the interfacial thermal resistance.Figure 93. Hence, the phenomenon of increased thermal conductivity values is explained by using Eq.  is only related to the density and elastic mechanics properties of the material itself, and it can be considered as a constant. The specific heat capacity (c) increases with temperature. And as the temperature rises, the volume of the composites and segmental mobility of molecular chains increases, resulting in a reduction in the degree of interaction or entanglement between the molecular chains, which facilitates the increase of the mean free path (l) of the phonons. Furthermore, the molecular chains around the fillers become more ordered with a rise of test temperature, which also leads to a slight increase in the free path. Hence, the thermal conductivity values of the composites may increase with temperature.In order to study the effect of temperature on heat conduction performance of the samples, the tests of thermal conductivity were conducted at 25, 50, 75, 100 and 125 °C, respectively, as shown in Fig. 3C2/epoxy composite with 1.0 wt% Ti3C2 MXene fillers are shown in Fig. 94.Moreover, the heating and cooling cycles of neat epoxy and Ti3C2 MXene fillers and neat epoxy, the surface temperatures of the samples were recorded by an infrared camera and the results are presented in Fig. 3C2/epoxy composite is always above that of neat epoxy, demonstrating that it possess better heat conduction performance. Figure 3C2/epoxy composite, indicating that the surface temperature of the neat epoxy is lower. Besides, it can also be seen that the color of the composite approximates that of the heater at 210 s.For the purpose of visually comparing the heat conduction performance of epoxy composites with 1.0 wt% Tig) of the samples. It can be observed that the Tgs of these composites are gradually improved upon the addition of Ti3C2 MXene fillers. The Tg of neat epoxy is 192.8 °C. Meanwhile, those of the Ti3C2/epoxy composites with 0.2, 0.4, 0.6, 0.8 and 1.0 wt% Ti3C2 MXene fillers are 201.6, 205.5, 207.3, 211.1 and 219.1 °C, respectively. About the improvements of the Tgs, some factors are considered as follows. The Ti3C2 MXene fillers added into the epoxy matrix can serve as physical interlock points and be intertwined by epoxy chains, which is conducive to restricting epoxy chains and reducing the motion of them27. Furthermore, the hydroxy groups of the Ti3C2 MXene fillers take part in the curing reaction so as to improve the crosslinking density95.Figure 3C2 MXene fillers are 46.3, 46.0, 45.1, 41.0 and 40.7 ppm/K at 40 °C, respectively. In addition, the thermal strain curves are obtained and shown in Fig. 3C2 MXene fillers into epoxy matrix can restrict epoxy chains and reduce the motion of them96. Therefore, the prepared epoxy composites exhibit lower CTE and thermal strain values compared with neat epoxy. In this work, TDP is introduced and used to indicate the comprehensive performance of engineering materials4. It is defined as the following Eq.  were selected as characteristic thermal parameters to clearly compare the thermal stability of the samples. Meanwhile, the curves over the temperature range (250~350 °C) were amplified and shown in the inset. T5%-0, T5%-1, T5%-2, T5%-3, T5%-4 and T5%-5 respectively represent the characteristic thermal parameters of the epoxy composites with 0, 0.2, 0.4, 0.6, 0.8 and 1.0 wt% Ti3C2 MXene fillers. The values of the characteristic thermal parameters are 329.8, 333.8, 337.1, 338.6, 339.8 and 342.3 °C, respectively. The results show that the epoxy composites possess better thermal stability than neat epoxy resins. Because the molecular chains of the epoxy matrix are restricted by Ti3C2 MXene fillers and the thermal movement of them are weakened. In addition, during composite degradation, a Ti3C2 MXene char acts as a mass transport barrier and an isolator between the bulk polymer matrix and surface, where combustion occurs98. Therefore, the thermal stability of the epoxy composites is enhanced.In order to further study the thermal stability of the samples, the thermal gravimetric analysis (TGA) was conducted and the TGA curves are shown in Fig. 3C2 MXene was prepared by a simple and effective ultrasonic method to obtain. And adding the Ti3C2 MXene fillers into the epoxy matrix is proposed to prepare the epoxy composites possessing excellent thermal properties. The thermal conductivity of epoxy composite with 1.0 wt% Ti3C2 MXene is 0.587 W/mK and increased by 141.3% compared with that of neat epoxy. Meanwhile, the Tg and decomposing temperatures of epoxy composites are improved with the increasing Ti3C2 MXene fillers. Particularly, the Tg of the epoxy composite with 1.0 wt% Ti3C2 MXene is 219.1 °C, corresponds to a 13.7% improvement compared with that of neat epoxy. Moreover, the CTE and TDP values decrease with the increase of fillers content. Our work will pave the way for novel fundamental and application studies of MXene.In this work, few-layer TiSUPPORTING INFORMATION-Enhanced Thermal Conductivity of Epoxy Composites Filled with 2D Transition Metal Carbides (MXenes) with Ultralow Loading


Dual-specificity phosphatase (DUSP)14  inhibits T-cell receptor (TCR) signaling and T-cell–mediated immune responses by inactivation of the TGF-β activated kinase 1 binding protein (TAB1)–TGF-β activated kinase 1 (TAK1) complex and ERK. DUSP14 phosphatase activity is induced by the E3 ligase TNF receptor associated factor (TRAF)2-mediated Lys63-linked ubiquitination. Here we report an interaction between DUSP14 and protein arginine methyltransferase (PRMT)5 by proximity ligation assay; similarly, DUSP14 directly interacted with TAB1 but not TAK1. DUSP14 is methylated by PRMT5 at arginine 17, 38, and 45 residues. The DUSP14 triple-methylation mutant was impaired in PRMT5-mediated arginine methylation, TRAF2-mediated lysine ubiquitination, and DUSP14 phosphatase activity. Consistently, DUSP14 methylation, TRAF2 binding, and DUSP14 ubiquitination were attenuated by PRMT5 short hairpin RNA knockdown. Furthermore, DUSP14 was inducibly interacted with PRMT5 and was methylated during TCR signaling in T cells. Together, these findings reveal a novel regulatory mechanism of DUSP14 by which PRMT5-mediated arginine methylation may sequentially stimulate TRAF2-mediated DUSP14 ubiquitination and phosphatase activity, leading to inhibition of TCR signaling.—Yang, C.-Y., Chiu, L.-L., Chang, C.-C., Chuang, H.-C., Tan, T.-H. Induction of DUSP14 ubiquitination by PRMT5-mediated arginine methylation. Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the transfer of methyl groups from S-adenosyl methionine to the guanidine nitrogen of arginine residues . Proteinin vitro (in situ proximity ligation assay (PLA) (in this report). DUSP14 is also reported to directly interact with TAK1 in the pull-down assay using immunopurified proteins  remove phosphate groups from phosphoserine/phosphothreonine and/or phosphotyrosine residues from their substrates . The DUS12in vitro . DUSP14 in vitro , 17 and in vitro . If TAB1in vitro  as well proteins ; howeverproteins . Thus, Dproteins . We furtproteins . Here weMyc-DUSP14, Flag-DUSP14, Flag-DUSP14 (C111S) mutant, Flag-TAB1, Flag-TAK1 , Flag-DU181IPDVYEKESRHLMPYWGI198 (corresponding to human DUSP14 protein sequence 181VPDVYEKESRHLMPYWGI198) (Anti-DUSP14 antibody was generated by immunization of rabbits with the murine DUSP14 peptide YWGI198) . Anti–phYWGI198) . Anti–ph2, 25 mM NaF, 1 mM Na3VO4). The cell lysates were incubated with anti-Myc beads for 60 min and then incubated with anti-Flag beads for another 60 min. The Alpha signals from each pair of anti-Flag and anti-Myc beads in proximity (<200 nm)   were detIn Situ Red Starter Kit (MilliporeSigma) according to the manufacturer’s instructions. Briefly, Flag-DUSP14 plus Myc-PRMT5, Myc-DUSP14 plus Flag-TAK1, Myc-DUSP14 plus Flag-TAB1, or Flag-TAK1 plus Myc-TAB1 coexpressing HEK293T cells were incubated with anti-Flag  and anti-Myc (MilliporeSigma) antibodies, followed by rabbit- and mouse-specific secondary antibodies conjugated with oligonucleotides (PLA probes). After ligation and amplification reactions, the PLA signals from each pair of PLA probes in proximity (<40 nm) were visualized as individual red dots by a fluorescence microscope .PLA assays  were perMethylation assays were carried out at 37°C for 1 h in 30 µl of buffer containing 50 mM Tris (pH 7.5), 0.1 mM EDTA, 50 mM NaC1, 0.8 mM S-adenosyl methionine, purified Flag-PRMT5 proteins, and purified Flag-DUSP14 wild-type or mutant proteins. Reactions were stopped by boiling in SDS sample buffer; proteins were resolved by SDS-PAGE and then immunoblotted with specific antibodies.Sample preparation and liquid chromatography–tandem mass spectrometry (MS) analysis were performed as previously described , 23.These DUSP14 experiments were used or performed as previously described . Cell lisem; the mean ± sem values were calculated using SPSS v.19 software .All experiments were repeated at least 3 times. Bar graphs are presented as mean ± Mr of about 70 kDa coimmunoprecipitated with DUSP14; this protein was identified as the protein arginine methyltransferase PRMT5 by MS  in cells with paired antibody-conjugated probes  of DUSP14 were identified to be methylated A and dimin vitro by methylation assays using purified PRMT5 and DUSP14 proteins  was generated in which Arg17, Arg38, and Arg45 residues were altered to lysine residues. Flag-tagged DUSP14 wild-type or mutant (3R→K) was transfected into HEK293T cells. The methylation of immunoprecipitated DUSP14 was detected by immunoblotting using an anti–methyl-arginine antibody, which has been successfully used for detecting the arginine methylation of proteins . The metproteins E. When wproteins E, lane 2in vitro phosphatase assays. HEK293T cells were transfected with Flag-DUSP14 wild-type, C111S mutant, or 3R→K mutant. The cell lysates were immunoprecipitated with anti-Flag antibody. The anti–Flag-DUSP14 immunoprecipitates were subjected to in vitro phosphatase assays using p-nitrophenyl phosphate as the substrate. For DUSP14 mutant (C111S), the Cys-to-Ser substitution resulted in a reduction of phosphatase activity  was immunoprecipitated with anti-Flag antibody from transfected HEK293T cells, followed by immunoblotting with anti–methyl-arginine antibody. We found that DUSP14 wild-type and ubiquitination-defective mutant (K103R) were methylated equally well in cells , we cannWe next studied whether DUSP14 methylation regulates its ubiquitination. HEK293T cells were transfected with HA-ubiquitin plus either Flag-DUSP14 wild-type or methylation-defective mutant (3R→K). Flag-tagged DUSP14 proteins were immunoprecipitated with anti-Flag antibody from the transfected cells and then immunoblotted with anti-HA antibody for detecting ubiquitinated proteins. Wild-type DUSP14 was ubiquitinated, whereas methylation-defective mutant (3R→K) was only slightly ubiquitinated B, lane 527IAQIT31, near the putative DUSP14 methylation sites. Thus, we studied whether PRMT5-mediated DUSP14 methylation regulates TRAF2 binding to DUSP14 using PRMT5 shRNA and coimmunoprecipitation assays. HEK293T cells were cotransfected with Myc-TRAF2, Flag-DUSP14, and either PRMT5 shRNA #2 or empty vector. The data showed that the interaction between DUSP14 and TRAF2 was reduced by PRMT5 shRNA knockdown  did not interact with TRAF2  T cells were transfected with either Flag-DUSP14 wild-type or mutant (3R→K). Stimulation with anti-CD3 antibody induced the interaction between DUSP14 and PRMT5 in transfected Jurkat T cells B, lane 327I-A-Q-I-T31, which is close to 3 putative methylation sites . Our data also showed that double mutations  on the putative TRAF2-binding motif of DUSP14 abolished its TRAF2 binding. Given the above observations, we propose a model (Here we report that DUSP14 was methylated at arginine 17, 38, and 45 residues by the arginine methyltransferase PRMT5. Furthermore, PRMT5-mediated arginine methylation of DUSP14 promotes its ubiquitination, which may lead to DUSP14 activation during TCR signaling. The regulation of DUSP14 ubiquitination by its methylation may be due to enhancement of the interaction between the methylated DUSP14 and the E3 ligase TRAF2. Notably, DUSP14 contains the putative TRAF2-binding motif  a model  that PRM a model , 16. ColArginine methylation can modulate protein-protein interactions by disrupting hydrogen bonding or by increasing hydrophobicity . PRMT1-mProtein methylation also regulates protein stability through inducing Lys48-linked ubiquitination –29. For The activity of PRMT5 is regulated by phosphorylation . The ser


Nanocellulose has been subjected to a wide range of chemical modifications towards increasing its potential in certain fields of interest. These modifications either modulated the chemistry of the nanocellulose itself or introduced certain functional groups onto its surface, which varied from simple molecules to polymers. Among many, aliphatic and aromatic mono- and di-isocyanates are a group of chemicals that have been used for a century to modify cellulose. Despite only being used recently with nanocellulose, they have shown great potential as surface modifiers and chemical linkers to graft certain functional chemicals and polymers onto the nanocellulose surface. This review discusses the modification of cellulose and nanocellulose using isocyanates including phenyl isocyanate (PI), octadecyl isocyanate (OI), toluene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), hexamethylene diisocyanate (HMDI), and their derivatives and polymers. It also presents the most commonly used nanocellulose modification strategies including their advantages and disadvantages. It finally discusses the challenges of using isocyanates, in general, for nanocellulose modification. Cellulose is Earth’s most abundant biopolymer. It represents 40–60% of wood mass and can be extracted in the form of 20–40 µm thick fibers upon pulping ,2. ApartThe surface modification of nanocellulose through its hydroxyl groups has significantly increased its potential. A wide range of chemical functionalities has been placed on nanocellulose surface through simple reactions such as oxidation and acetylation ,17. SomeAliphatic and aromatic mono-isocyanates such as phenyl isocyanate (PI) and n-octadecyl isocyanate (OI), and di-isocyanates such as toluene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), hexamethylene diisocyanate (HMDI), and their polymeric forms have all been used for the modification of cellulose and nanocellulose  25,26,2,28. PI aTDI is a very interesting di-isocyanate as the isocyanates of its 2,4-isomer differ in their reactivity. The ortho isocyanate is 5–10 time less reactive than the para isocyanate due to the steric hindrance from the neighboring methyl group ,34. ThisTo facilitate the reaction between isocyanates and the hydroxyl groups of nanocellulose and the resultant formation of polyurethane bonds, amines such as triethylamine were found to be excellent catalysts although they could also facilitate the self-polymerization of isocyanates as a side reaction .This review discusses the use of aliphatic and aromatic mono- and di-isocyanates in their molecular and polymeric forms for the surface modification of both cellulose and nanocellulose. For each material, the literature reports will be categorized based on the goal of the surface modification. Some of these reports followed application-oriented approaches, which focused on modifying cellulose and nanocellulose to function in certain applications while other reports were property-oriented, which aimed at improving the properties of cellulose and nanocellulose in general, such as hydrophobicity and thermal stability, without targeting a specific application. Most of the reports; however, focused on the use of isocyanates to facilitate the processing of cellulose and nanocellulose in nonpolar thermoplastic and thermoset matrices to prepare reinforced composites with improved interfacial adhesion.Most of the reported reactions between nanocellulose and isocyanates took place through one of four scenarios. The first scenario represents the reaction of nanocellulose with a mono-isocyanate carrying either a hydrophobic  ,37 or a Di-isocyanates were mainly used to (1) bind functional groups onto the nanocellulose surface or to (2) support its processing with thermoset matrices. In the first case, two scenarios were used . In ScenThe first report on the use of isocyanates with cellulose seems to go back to 1920 by Charles  followedCellulose and its derivatives have been functionalized for a variety of applications including water treatment, chromatography, and biotechnological applications. In terms of the biotechnological applications, Gemeiner et al. used in 1977 the reaction between cellulose and 2,4-TDI to prepare cellulose isothiocyanates, whose binding capacity to thiol and amine compounds was studied . The aimThere are two reports on cellulose functionalization for water filtration. In 1983 Sato et al. used the reaction between cellulose and 2,4-TDI to graft amino acids, such as glycine and serine, onto the cellulose to produce a powder capable of adsorbing heavy metals such as copper, zinc, and cadmium ,63. ReceTo developed chiral stationary phases for liquid chromatography, cotton spheres were reacted with HMDI, 2,4-TDI, 1,4-MDI, or substituted PIs ,27,28. CIn this category, cellulose was treated with isocyanates to mainly reduce its hydrophilicity or improve its biocompatibility. To reduce it hydrophilicity, cellulose was reacted with PI, cyclohexyl isocyanate, HMDI, and oxime-blocked isocyanate oligomers ,66,67. FIsocyanates were also used to improve cellulose biocompatibility with human blood. For instance, cellulose sheets were reacted with HMDI through one of its isocyanates followed by the reaction of the other isocyanate with betaine-containing molecules . SimilarIn this category, cellulose was either reacted with the isocyanate prior to mixing/compounding with the nonpolar matrix or the reaction took place during compounding by relying on the heat used in the process. Mono-isocyanates, di-isocyanates, and their polymers have all been used in this category while the matrices were mainly petroleum-based thermoplastic polymers such as polystyrene (PS), polypropylene (PP), and polyethylene (PE). The modification led in most of these reports to a significant improvement in the mechanical properties and a drop in the water uptake of the composite. Cellulose was modified using poly(methylene(polyphenyl isocyanate)) and MDI to improve the interfacial adhesion between cellulose and PS ,78,79 whCellulose has also been modified before being used to reinforce bio-based matrices such as natural rubber , epoxidiThe reports in this category used di-isocyanates  for cross-linking cellulose with the matrix. In some cases, the matrix was reacted with one of the isocyanates of the di-isocyanate then the cellulose was reacted with the free isocyanate (Scenario 3). For instance, poly(caprolactone) (PCL) was reacted with 2,4-TDI then cellulose was reacted with the free isocyanate to form the composite . CopolymDespite their use for cellulose modification for almost a century, isocyanates were used for the first time in 2008 to modify nanocellulose . Two yeaThe reports on nanocellulose modification fell in the same four categories mentioned earlier for cellulose but with more focus on nanocellulose processing with thermoplastic and thermoset matrices :There are only three reports on nanocellulose functionalization. Similar to previous reports using cellulose, CNCs were reacted with 3,5-dimethylphenyl isocyanate to function as a stationary phase in liquid chromatography with chiral recognition abilities . CNCs weAlmost all the reports here focused on nanocellulose hydrophobization. CNFs were hydrophobized following Scenario 1 by reacting with OI  while CNTo be processed with PCL as a matrix, nanocellulose was modified using different approaches. In one approach, nanocellulose was reacted with OI following Scenario 1 and then mixed with PCL ,114,115.The reports in this category cover the use of nanocellulose for reinforcing thermosets of different forms , which were made of lignin, castor oil, poly(ethylene glycol) (PEG), polyether polyols, or other polyols. In some of these reports, the nanocellulose was added to the polyol/di-isocyanate mixture to crosslink all together ,49,119. It is clear that mono- and di-isocyanates have great potential for nanocellulose modification. However, in addition to their reported toxicity, many challenges exist limiting their use ,129. OneAnother issue is the difficulty of controlling the reaction between di-isocyanates and nanocellulose. Di-isocyanates are supposed to react with the surface hydroxyl groups of nanocellulose only through one of their two isocyanates  to reduce the hydrophilicity of the nanocellulose. Sometimes, a di-isocyanate was used for the same purpose. In the second scenario, the nanocellulose was reacted with one of the isocyanates of a di-isocyanate then the free isocyanate was reacted with a functional material or a matrix. This scenario suffers the possibility of the di-isocyanate to react with the nanocellulose using both of its isocyanates becoming dysfunctional for the following step. The third scenario overcame this issue by reacting the di-isocyanate at first with the functional material or matrix using one of its isocyanates then the free isocyanate was reacted with the nanocellulose. The fourth scenario represents mainly the use of nanocellulose to reinforce nonpolar thermosets. In this scenario, the di-isocyanate was reacted with both the nanocellulose and the polyol at the same time.Despite the reported potential of isocyanate compounds as chemical linkers and surface modifiers, their reactions with nanocellulose face many challenges including the need for moisture-free environment, nanocellulose aggregation during solvent-exchange, reaction controllability, and self-polymerization of isocyanates.


N-methyl-2-pyrrolidone (NMP) solvent. During this reaction, increased viscosity and formation of amic acid could be confirmed. The chemical reactions were monitored and evidenced using 1H-NMR spectroscopy, FT-IR spectroscopy, water gel-phase chromatography (GPC) analysis, amine value determination and acid value determination. We also studied the effect of additives on thermomechanical properties using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), dynamical mechanical analysis (DMA), thermomechanical analysis (TMA) and by measuring mechanical properties. The BTCP-containing epoxy resin exhibited high mechanical strength and adhesion strength proportional to the amount of BTCP. Furthermore, field-emission scanning electron microscopy images were obtained for examining the cross-sectional morphology changes of the epoxy resin specimens with varying amounts of BTCP.Epoxy resins have found various industrial applications in high-performance thermosetting resins, high-performance composites, electronic-packaging materials, adhesives, protective coatings, etc., due to their outstanding performance, including high toughness, high-temperature performance, chemical and environmental resistance, versatile processability and adhesive properties. However, cured epoxy resins are very brittle, which limits their applications. In this work, we attempted to enhance the toughness of cured epoxy resins by introducing benzene tetracarboxamide polyamine (BTCP), synthesized from pyromellitic dianhydride (PMDA) and diamines in  Epoxy resins are widely used as industrial thermosetting plastics in composites, electronics, adhesives and coatings because of their low price, high toughness, high heat resistance, chemical resistance and excellent mechanical properties ,11,12,13Therefore, the enhancement of toughness and the mechanical and thermal properties of epoxy polymers has been studied by adding soft polymers, such as amine-terminated butadiene-co-acrylonitrile (ATBN), carboxyl-terminated butadiene-co-acrylonitrile (CTBN), epoxidized natural rubber (ENR), hydroxyl-terminated butadiene-co-acrylonitrile (HTBN), polyethersulfone (PES), poly(etherimide) (PEI), poly(acrylonitrile-butadiene-styrene) (ABS), etc. ,21,22,23Herein we prepared a new amine hardener polymer called benzene tetracarboxamide polyamine (BTCP), to compensate for the brittleness of epoxy polymers comprising rigid amine hardeners, like dicyandiamide (DICY) and bisphenol A epoxy resin. We added variable amounts of BTCP (0–20 parts per hundred resin (phr)) in the epoxy compositions and measured the effects on mechanical and thermal properties of the epoxy resin. Furthermore, the fractured surface morphology of the thermosetting polymers was observed using field-emission scanning electron microscopy (FE-SEM).N-methyl-2-pyrrolidone (NMP) was obtained from Sigma-Aldrich Co. . Liquid Jeffamine D-400  = 115 g/eq) was provided by Huntsman . Diglycidylether of bisphenol A  = 187 g/eq) was obtained from Momentive Co. . Latent curing agent, dicyandiamide  and a urea-based reaction promoter (Amicure UR 7/10) were purchased from Air Products . The structures of the epoxy resin, DICY, and BTCP are shown in Pyromellitic dianhydride (PMDA) was purchased from TCI , and 2 gas. PMDA  was slowly added to the flask and stirred for 30 min to prepare amic acid at room temperature. A small amount of amic acid was obtained for acid value measurement using Equation (1). Based on the calculation of acid value, three equivalents of Jeffamine D-400 were added to the flask and stirred for 1 h at 100–130 °C. After the solution was cooled, NMP was removed by vacuum evaporation. The obtained solid was dried for 6 h at 80 °C. The solid BTCP was processed into fine powder using a freezer mill for use as an associative curing agent for epoxy compositions.The reaction scheme for the preparation of BTCP is shown in The detailed formulation ratios of epoxy resin, curing agent and BTCP are shown in The prepared epoxy composition was placed in a metallic mold and heated at 150 °C for 1 h followed by heating at 170 °C for 1 h and at 190 °C for 1 h for measuring thermal and mechanical properties. The cured epoxy polymer network structure and the BTCP-modified polymer network are shown in 1H-NMR spectroscopy  at room temperature. The sample was dissolved in DMSO-d6 with trimethylsilane (TMS) as the internal standard. Furthermore, FT-IR spectrophotometry  was used to observe the change in functional groups of both reactants and products. Moreover, the acid value of the amic acid was calculated using Equation (1). Acid value is the number of milligrams of KOH required to neutralize the acid in 1 g of sample. The mass of Jeffamine D-400 required to neutralize amic acid is determined by titrating amic acid.AV is the acid value (mg KOH/g), c(KOH), The structure of amic acid was confirmed using Tonset), of epoxy compositions was collected using differential scanning calorimetry  performed in the temperature range of 25–300 °C at a heating rate of 10 °C. The glass transition temperature (Tg) was measured by the second DSC run. Dynamic mechanical analysis (DMA) to measure viscoelastic properties was performed on a Q800 instrument . The processed test specimens of dimensions 60 mm δ. Thermomechanical analysis  was used to analyze the linear coefficient of thermal expansion (CTE) and the Tg of cured epoxy polymers processed to dimensions of 5 mm × 5 mm × 3 mm. The test specimens were placed on the expansion-type probe followed by an increase in temperature by 2 °C/min from 30 to 250 °C. The effects of different amounts of BTCP in epoxy compositions on the mechanical properties of the epoxy resin were studied by measuring the flexural, tensile and impact strengths. The tensile strength was measured using the ASTM D-638 method by employing a universal testing machine . The test specimen was processed to dimensions of 150 mm × 13 mm × 3 mm. The test was repeated five times to obtain an averaged value. The flexural strength was measured using the ASTM D 790M method by employing using UTM with test specimens having dimensions of 60 mm × 25 mm × 3 mm. The test was repeated five times to obtain an averaged value. The impact strength was measured with an Izode pendulum impact tester  by the ASTM D256 method. Test samples were processed to dimensions of 63.5 mm × 12.7 mm × 3 mm with a 2.54 mm notch. Five cured polymers were tested for each epoxy composition to obtain an averaged value.Thermal information, such as the onset temperature  after coating with Pt.1H-NMR, FT-IR and measurement of the acid value.The reaction products of both amic acid and BTCP were analyzed with 1H-NMR  (δ (ppm) = 10.3–9.4 , 8.7–8.1 , 8.0–7.5 .Amic acid: DMSO-d6) a: δ  (δ (ppm) = 8.6–8.0 , 7.3–7.6 , 7.6–8.0 .BTCP: DMSO-d6) b: δ ; the acid value decreased dramatically from 0.070 g KOH/g for amic acid to 0.019 g KOH/g for the reaction product to form BTCP.nM) was 463,200 g/mol, and the weight average molecular weight (wM) was 572,640 g/mol. The measured melting point of BTCP was 190 °C.The molecular weight of the produced BTCP was measured by gel permeation chromatography (GPC), and the obtained data are provided in Tpeak and Tg obtained from the DSC experiments are presented in Tpeak are proportional with the amount of BTCP, while Tg is inversely proportional to the amount of BTCP. The Tg of neat epoxy observed at 125.81 °C decreased to 108.74 °C when the amount of BTCP in the epoxy composition reached 20 phr. The addition of BTCP led to a slight decrease in Tg, which is attributed to an increase in the amount of dissolved BTCP within the epoxy matrix. This behavior was also observed in other rubber- or elastomer-modified epoxy systems [Thermal data including the onset temperature,  systems ,27.Tg. α1 represents a CTE below Tg and α2 implies a CTE above Tg. The CTE value of α1 tends to increase with the amount of BTCP, whereas the CTE of α2 decreases with increasing amount of BTCP. In general, when the CTE value increases, the internal stress also increases, causing a crack in the polymer matrix, which, in turn, decreases the adhesive strength. Therefore, as other studies have also shown, it is preferable to lower the CTE value [TE value .δ and the storage modulus (E’) of each epoxy composition, are shown in E’) of the epoxy compositions including BTCP were lower than that of the neat epoxy composition system. A sharp decrease in the storage modulus was obtained in all epoxy compositions near Tg. The temperature at which the sharp decrease begins tends to decrease in proportion to the amount of BTCP. The decrease of tan δ follows a similar trend to that shown in E’) and tan δ of the epoxy compositions. The height of tan δ for the epoxy compositions, including BTCP, is lower than that of the neat epoxy composition, because of the size decrease in the epoxy phase resulting from the increase of the BTCP content, which forms the rubber-rich phase [The dynamic mechanical analysis (DMA) data, including the values of tan ch phase .Tg than that of sample 4. Therefore, the epoxy compositions with BTCP are predicted to compensate for the brittleness of the epoxy polymer due to the lower crosslinking density.The polymer chains of the epoxy composition with 20 phr of BTCP (sample 4) started to move at a lower temperature, as compared to the neat epoxy resin. As the fluidity of the polymer chain is inversely proportional to the crosslinking density, the fluidity of the polymer chains decreases as the crosslinking density increases . In factThe mechanical properties of the cured epoxy compositions were analyzed by measuring their tensile strength, flexural strength, and Izod impact strength. 1H-NMR and acid value measurements. The tensile strength of the epoxy compositions increased with increasing amounts of BTCP, from 74.4 MPa for neat epoxy to 76.96 MPa for the composition with 20 phr BTCP.The structure of BTCP was determined by GPC, FT-IR, Although most toughening agents with rubbery parts reduce the flexural strength, BTCP in varying contents in the compositions did not deteriorate the flexural strength. Moreover, the impact strength of the compositions increased by 48.3% with the amount of BTCP, from 34.28 J/m for neat epoxy to 50.84 J/m for sample 4. In addition, FE-SEM images showed that the fracture surface in the modified system had a rough and irregular appearance. The degree of roughness also increased with the amount of additive BTCP. Therefore, based on the results of the impact strength and FE-SEM experiments, BTCP is promising as an epoxy toughener.