Ctnnb1 gene locus to generate a true β-catenin-null mutant mouse strain. Ablation of β-catenin alone, or in combination with its homologue γ-catenin, did not affect thymocyte maturation, survival or proliferation. Deficiency in β/γ-catenin did not detectably affect differentiation of CD4+T follicular helper cells or that of effector and memory CD8+ cytotoxic cells in response to acute viral infection. In an MLL-AF9 AML mouse model, genetic deletion of β-catenin, or even all four Tcf/Lef family transcription factors that interact with β-catenin, did not affect AML onset in primary recipients, or the ability of leukemic stem cells (LSCs) in propagating AML in secondary recipients. Our data thus clarify on a long-standing controversy and indicate that β-catenin is dispensable for T cells and AML LSCs.The β-catenin transcriptional coregulator is involved in various biological and pathological processes; however, its requirements in hematopoietic cells remain controversial. We re-targeted the  Lineage-negative BM cells and peripheral T cells from CreER+Ctnnb1fl/fl mice were cultured in vitro with tamoxifen or its carrier solvent, DMSO, and immunoblotting validated effective ablation of β-catenin protein without generating truncated form(s)  . A band  form(s) . These a+Ctnnb1fl/fl mice exhibited morbidity within 10 days, while CreER+Ctnnb1+/+ mice remained healthy (+ CreER+Ctnnb1+/+ (WT) or CreER+Ctnnb1fl/fl mice into irradiated CD45.1+ recipients  in combination with primer 15R (complementary to undeleted exon 15)  . These cJup), we crossed the Jupfl/fl strain  to CD4+CD8+double positive (DP) followed by further differentiation to CD4+ or CD8+ single positive cells. Each thymic subset was detected at similar frequency among all genotypes examined  was monitored for 16 weeks after tamoxifen treatment, and none of these mice showed signs of malignant transformation of thymocytes or other hematopoietic lineage cells. These analyses showed that complete loss of β-catenin or both β- and γ-catenin did not detectably cause T cell developmental blocks, or alterations in thymocyte proliferative capacity or survival.Following tamoxifen-induced target gene deletion in the chimeras, total thymic cellularity was similar in WT chimeras and those lacking β-catenin alone or both β-catenin and γ-catenin . Thymocyexamined . By intrenotypes . Detectienotypes . In addienotypes . A cohor+ and CD8+ T cells mount protective immune responses by clonal expansion and differentiation into functional effector cells. To test the requirements for β-catenin and γ-catenin in mature T cell responses, we infected tamoxifen-treated BM chimera with lymphocytic choriomeningitis virus Armstrong strain (LCMV-Arm) to elicit acute viral infection. Activated CD4+ T cells predominantly differentiate into CXCR5–SLAMhi T helper 1 (TH1) and CXCR5+SLAMloT follicular helper (TFH) lineage cells   33 epitope  . In addienotypes , suggest T cells , fully date Tcf1 . Consist+T cells . Not sur epitope . The GP3enotypes , suggestH1 and TFH cells  cells, in which HSCs were enriched  recipients  recipients . To fully address functional redundancy among Tcf/Lef family TFs, we used Tcf7l1-targeted murine embryonic stem (ES) cells generated by the International Knockout Mouse Consortium (Tcf7l1fl/fl mouse strain. By crossing with CreER and Tcf7l2fl/fl (+Tcf7l1fl/flTcf7l2fl/fl (CreER+Tcf3/4-dKO) mice. This line was further crossed with our established CreER+Tcf7fl/flLef1fl/fl (CreER+Tcf1/Lef1-dKO) mice . Following treatment with the same tamoxifen regimen as above, the CreER+Tcf-qKO mice did not exhibit early lethality, and Tcf1 and Lef1 proteins were ablated in thymocytes of CreER+Tcf1/Lef1-dKO mice as determined by intracellular staining  mice  to generstaining . While astaining , Tcf3 prstaining . It shoustaining . We furtbundance . Tamoxif numbers . Consist numbers , tamoxifrequency . Within ymocytes , in lineymocytes . In all – BM cells from tamoxifen-treated CreER+ WT or CreER+Tcf-qKO mice directly for the AML studies, without establishing BM chimeras. The 1o recipients of MLL-AF9-infected Tcf-qKO BM cells showed similar survival rate as those of WT BM cells  geFH cell differentiation at effector and memory phases  (Jupfl/fl (Tcf7fl/fl (Lef1fl/fl (Tcf7l2fl/fl (Rosa26Cre-ERT2/+ (oratory) , Jupfl/fJupfl/fl , Tcf7fl/cf7fl/fl , Lef1fl/ef1fl/fl , Tcf7l2f7l2fl/fl , and Rose-ERT2/+ . Transple-ERT2/+ . All mouCtnnb1fl/fl mice were generated using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology by Applied StemCell Inc. In brief, two LoxP cassettes were inserted into introns 1 and 14, flanking exons 2 to 14 of the Ctnnb1 locus  cells were generated by the European Conditional Mouse Mutagenesis Program (EUCOMM), currently part of the International Knockout Mouse Consortium (IKMC) (Tcf7l1tm1a(EUCOMM)Wtsi . Microinjection of ES cells was performed at the Wellcome Trust Sanger Institute (UK), and germline-transmitted mice were bred with Flippase recombinase  to remove the LacZ/Neomycin cassette flanked by FRT sites. For genotyping of the resulting Tcf7l1fl/+ allele, the following primers were used to amplify genomic DNA: 5’-AGCAACCAAATGAAGGCTCAC and 5’-CTGCTGCCCCTCTTTTCATC, which detects Tcf7l1 WT allele at 335 bp, and Tcf7l1-floxed allele at 424 bp, and Tcf7l1-deleted allele at 562 bp. Effective ablation of Tcf3 protein in Tcf7l1fl/fl mice was demonstrated by an independent study using the same ES cell-derived allele  . The alld allele . We alsod exon 6 . Deletiod exon 1 .+WT or CreER+Ctnnb1fl/fl mice were cultured in IMDM medium supplemented with 15% fetal bovine serum (FBS), 20 ng/ml thrombopoietin (TPO), and 50 ng/ml stem cell factor (SCF). Positively selected CD3+ T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 50 ng/ml IL-7, and 50 ng/ml IL-15. On second day of culture, 4-hydroxy-tamoxifen  was added to a final concentration of 250 nM, and cells were harvested 3–5 days later for immunoblotting. For in vivo treatment, the mice were administered with tamoxifen  at 0.2 mg/g body weight via oral gavage for four consecutive days, as illustrated in For ex vivo treatment, lineage-depleted BM cells from CreER+ thymocytes, splenic T cells, BM cells, or GFP+Mac1+ AML cells, resolved on SDS-PAGE, followed by immunoblotting with anti-β-catenin , with anti-β-actin  detection as control for equal loading. Thymocytes, BM and liver cells were detected with anti-γ-catenin  or anti-Tcf4 .Cell lysates were prepared from sorted CD45.2FH cells. For detection of Bcl6 (K112-91) and Ki67 , Tcf1 (C63D9) and Lef1 (C12A5) and corresponding isotype control , surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences/ThermoFisher Scientific), followed by incubation with corresponding fluorochrome-conjugated antibodies. Active Caspsase-3/7 was detected using the Vybrant FAM caspase-3/7 assay kit (Invitrogen/Life Technologies) as described  with 2 × 105 PFU of LCMV-Armstrong strain to elicit acute viral infection  were infected with MLL-AF9-GFP retrovirus. The infected cells containing 1 × 104 GFP+c-Kit+ cells were transplanted into lethally irradiated congenic mice along with 2 × 105 CD45.1+ protector BM cells. The primary recipients were treated with tamoxifen during days 25–27 after BM transplantation, and one day later, the BM cells were harvested and sorted for CD45.2+ AML LSCs following the gating strategy outlined in 5 CD45.1+ protector BM cells and transplanted into secondary recipients, followed by tracking CD45.2+GFP+Mac1+ AML leukemia burden in PBCs. Both primary and secondary recipients were subjected to repeated rounds of tamoxifen treatment for three consecutive days by the end of every 4 weeks, and monitored for survival.The AML mouse model was established following similar protocol in our previous CML/AML studies . In briet-test was used, with a two-tailed distribution assuming equal sample variance. For multiple group comparisons, one-way ANOVA was used to determine whether any of the differences between the means are statistically significant. As post hoc correction, Tukey’s test was used to determine statistically significance between two groups of interest. Comparison of AML mouse survival among different experimental groups was determined by log-rank test using Prism8 software. Statistical parameters, including numbers of samples or recipient mice analyzed (n), descriptive statistics (means and standard deviation) are reported in the figures and figure legends. P values equal to or more than 0.05 are considered not statistically significant (marked as ‘ns’ or unmarked for clarity). P values of less than 0.05 are considered statistically significant, the following asterisk marks are used to indicate the level of significance: *, p<0.05; **, p<0.01; ***, p<0.001.For comparison between two experimental groups, Student’s  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:The findings reported here add to the body of literature, often conflicting, on the role of β-catenin in particular in AML stem cells and T lymphocytes. In particular, the finding that the complete knockout of β-catenin and γ-catenin does not impair AML in primary or secondary recipients is an interesting and important finding with scientific and translational implications.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Utpal Banerjee as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "β-catenin and γ-catenin are dispensable for T lymphocytes and AML leukemic stem cells" 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:In the manuscript by Zhao et al., the authors investigate the requirement for β- and γ-catenin in T cell development, T cell activation, and AML LSCs using a novel β-catenin knockout mouse that eliminates most exons of this gene. Prior studies investigating β-catenin in these processes used mice with limited exon targeting, raising the possibility that these mice actually expressed a truncated protein with potentially novel functions. Using the new mice alone or bred with γ-catenin knockout, the authors show that these proteins are not required for normal T cell development, T cell activation and viral immune responses, and AML development and LSC transplantation using the MLL-AF9 retroviral model. Moreover, deletion of all 4 LEF/TCF transcription factors also had no effect on AML development or LSC transplantation in this same model. In general, the role and requirement of β-catenin in T cell development and AML is of interest – and has been controversial due to confounding reports in the literature. However, there are several issues with the current manuscript.Essential revisions:1) One of the key issues with this manuscript is the discrepancy between the findings here and the results from the prior published studies with the alternative models. Thus, it is key to identify mechanisms that account for the discrepancy. Is there any evidence that the prior β-catenin mice targeted in exons 2/3-6 produce a truncated protein? Independent of these mice, can a truncated β-catenin be generated from the non-targeted exons? what is the phenotype of this truncated protein in relevant assays of β-catenin function?2) In the Western blot in Figure 1D showing absence of β-catenin protein, a band is detected at 50kD labeled as non-specific. However, this band seems to be decreased in the β-catenin KO cells as well, raising the possibility that it is not non-specific. Can the authors comment on the identity of this band? Is it possibly an aberrant form of β-catenin?3) The KO of γ-catenin should be validated with Western blot and DNA/RNA analysis as was done for β-catenin.4) In Figure 4A,B, the authors conclude that normal hematopoiesis and HSCs are unaffected by β- and γ-catenin double KO. However, in order to formally make this conclusion, transplantation assays should be conducted showing no difference compared to WT control.5) What is the efficacy of β- and γ-catenin knockout at time of primary mice analysis and secondary mice analysis as determined by Western blot? It is possible that rare non-deleted cells are growing out, particularly at later time points.6) For the Tcf-qKO cells, Western blot should be shown demonstrating elimination of each protein. What is the effect of Tcf-qKO on T cell development and activation in the experiments shown in Figures 2,3?7) Do the β-catenin and γ-catenin individual or double knockout mice develop T cell lymphoma? Essential revisions:1) One of the key issues with this manuscript is the discrepancy between the findings here and the results from the prior published studies with the alternative models. Thus, it is key to identify mechanisms that account for the discrepancy. Is there any evidence that the prior β-catenin mice targeted in exons 2/3-6 produce a truncated protein? Independent of these mice, can a truncated β-catenin be generated from the non-targeted exons? what is the phenotype of this truncated protein in relevant assays of β-catenin function?These are important points. Production of a truncated β-catenin protein from existing mouse models was previously reported by Held and colleagues . The authors examined bone marrow cells from both mouse models, exons 2-6 floxed model , and exons 3-6 floxed model .As shown in As for the function of truncated forms of β-catenin protein, because the N-terminus of β-catenin contains phosphorylation sites for ubiquitin-dependent degradation, an N-terminal truncated form of β-catenin protein has been shown to have longer half-life, and ectopic expression of the N-terminal truncated β-catenin stimulates proliferation and apoptosis of intestinal crypts . In planarians (flatworms), a C-terminal truncated β-catenin is a naturally occurring paralog and acts as a negative regulator of full-length β-catenin during planarian eye photoreceptor specification . We now cited these papers and described relevant findings in the Introduction.2) In the Western blot in Figure 1D showing absence of β-catenin protein, a band is detected at 50kD labeled as non-specific. However, this band seems to be decreased in the β-catenin KO cells as well, raising the possibility that it is not non-specific. Can the authors comment on the identity of this band? Is it possibly an aberrant form of β-catenin?As described in response to Point #1 and shown in Figure 1—figure supplement 1, the truncated β-catenin resulting from exon 7 translation in the RK model was detected at ~40 kDa. The non-specific band was at ~50 kDa. To substantiate our interpretation that the 50 kDa band results from non-specific reaction to the β-catenin antibody, we added the following two sets of data:First, we treat lineage-negative bone marrow and peripheral T cells with tamoxifen in vitro. As shown in immunoblots in Figure 1B, the lineage-negative BM cells were completely devoid of non-specific bands; while T cells had the non-specific band, the signal intensity was similar between DMSO- and tamoxifen-treated cells. This was included as new Figure 1B, and described in the Results and Discussion first paragraph.Second, we updated original Figure 1D which showed one set of data with the new Figure 1E which now includes two sets of data. The non-specific bands were not always detected (Thymocytes Exp1 and BM cells Exp2), and if detected, the non-specific bands did not always show decreased intensity in β-catenin KO cells .Taking the new Ctnnb1-targeting strategy into consideration, where Ctnnb1 exons 2-14 are floxed, the extensive immunoblot data suggest that the 50 kb band is unlikely a truncated form of β-catenin.3) The KO of γ-catenin should be validated with Western blot and DNA/RNA analysis as was done for β-catenin.As requested, we have designed primers based on the published γ-catenin  targeting strategy as summarized below and in Figure 1—figure supplement 4A.On the genomic DNA level, we validated effective excision of the floxed Jup exons using the F1-R1 and F1-R2 primer combinations .On the mRNA level, we found that Jup transcripts were much less abundant compared with β-catenin transcripts in WT BM cells . Nonetheless, we were able to observe consistent deletion of Jup transcripts upon tamoxifen treatment in the KO bone marrow chimeras, after zooming in to Jup transcripts only .Because of the low abundance of Jup transcripts, the γ-catenin protein was below detection limit by immunoblotting in bone marrow cells . However, γ-catenin protein was more readily detectable in hepatocytes, and we confirmed induced deletion of γ-catenin by tamoxifen in non-BM chimera, KO mice .Overall, these analyses validated effective ablation of γ-catenin in our experimental system.4) In Figure 4A,B, the authors conclude that normal hematopoiesis and HSCs are unaffected by β- and γ-catenin double KO. However, in order to formally make this conclusion, transplantation assays should be conducted showing no difference compared to WT control.We agree with the reviewers that if we were to conclude that ablation of β-/γ-catenin does not affect HSC function and self-renewal, it would be necessary to perform serial transplantation assays. Based on data in Figure 3A,B, we were careful to conclude that “loss of β-/γ-catenin did not perturb HSC homeostasis” but did not comment on HSC self-renewal. The rationale is that previous studies on HSCs using the existing RK or JH β-catenin targeted models did not observe a strong impact of deleting β-catenin on HSC self-renewal, even if truncated β-catenin protein may have been present in those analyses . In contrast, previous studies on AML LSCs using these models reported a requirement for β-catenin for LSC self-renewal . We therefore focused on biological aspects where β-catenin was considered to have a prominent role, and this focus is reflected in manuscript title “T lymphocytes and AML leukemic stem cells”.+Ctnnb1fl/fl HSCs, where the contribution of WT and β-catenin deficient HSCs contributed similarly to blood reconstitution and LSK compartment in secondary recipients when analyzed at 6 weeks after transplantation  What is the efficacy of β- and γ-catenin knockout at time of primary mice analysis and secondary mice analysis as determined by Western blot? It is possible that rare non-deleted cells are growing out, particularly at later time points.We thank the reviewers for raising this point, we are fully aware of the “escapee” cells that may have growth advantage over KO cells. We therefore have laid out an experiment plan , where the tamoxifen was administered repeatedly to ensure that the rare non-deleted cells were eliminated for the duration of the entire study.As depicted in Figure 3C in the AML studies, the BM chimeras were treated with tamoxifen for the initial induced deletion, which were then retrovirally transduced with MLL-AF9 and transplanted into the primary recipients. The primary recipients were treated with another round of tamoxifen on days 25, 26 and 27 after transplantation, so as to be sure that AML LSCs contained minimal, if any, non-deleted cells before transplanted into secondary recipients. Both primary and secondary recipients were continuously treated every 4 weeks.+GFP+Mac1+ AML cells on day 28 after BM transplantation , and validated absence of β-catenin protein in the KO AML cells . Although we did not perform the same experiment on AML cells from the secondary recipients, we deduce that the standardized tamoxifen regimen should have consistent effect in both primary and secondary recipients under the same experimental system.It is a fine balance to weed out “non-deleted” cells and avoid unwanted side-effects resulting from too frequent tamoxifen treatments. The regimen of treatment at 4-weeks interval was optimized at the early stages of the studies. In fact, from the primary recipients, we sorted CD45.2We refined the description of the tamoxifen regimen and its purpose in the text and figure legends.6) For the Tcf-qKO cells, Western blot should be shown demonstrating elimination of each protein. What is the effect of Tcf-qKO on T cell development and activation in the experiments shown in Figures 2,3?As requested, we performed the following analyses:1) To demonstrate efficiency of protein ablation, we used intracellular staining for Tcf1 and Lef1 in thymocytes, where both proteins are mostly abundantly expressed. As shown in Figure 4B, both Tcf1 and Lef1 proteins were greatly reduced to a level similar to isotype controls.Immunoblotting also showed that Tcf4 protein was substantially reduced in thymocytes upon induced deletion. On the other hand, Tcf3 protein were not reliably detected in thymocytes and BM cells from WT or Tcf3/4 dKO mice. This is likely due to low abundance of Tcf7l1 expression in hematopoietic cells, as reflected in RT-PCR analysis shown in Figure 4C, where Tcf7l1 transcripts were about 1/25th of Tcf7l2 transcripts in Flt3–LSK cells. In spite of low expression, Tcf7l1 transcripts were ablated to an undetectable level in Tcf-qKO cells . It is also important to note that the same Tcf7l1-targeted ES clone was used in another independent study to ablate Tcf3 in mouse embryonic fibroblasts , corroborating the efficacy of Tcf3 deletion using the same model. Because Tcf1, Lef1 and Tcf4 were deleted effectively in our experimental system, we respectfully suggest that it is reasonable to deduce similar capacity of Tcf3 protein in the same cells.2) We analyzed T cell development in Tcf-qKO mice, along with CreER+ Tcf1/Lef1 KO and CreER+ Tcf3/Tcf4 KO mice. Tamoxifen treatment resulted in over 90% reduction in thymic cellularity in CreER+ Tcf1/Lef1 KO and Tcf-qKO mice, but did not detectably affect thymocyte numbers in CreER+ Tcf3/Tcf4 KO mice.In CreER+ Tcf1/Lef1 KO and Tcf-qKO thymuses, the DP and DN2 thymocytes showed more pronounced reduction, with DN1 thymocytes showing premature CD25 upregulation and relative accumulation compared with CreER+ WT mice. These aspects are consistent with well-established requirements for Tcf1 in promoting T cell lineage commitment and DP thymocyte survival. In addition, none of these changes was evident in CreER+ Tcf3/Tcf4 KO mice, and the defects in CreER+ Tcf1/Lef1 KO mice were not further exacerbated in Tcf-qKO mice. These observations suggest that Tcf3 and Tcf4 are not required for thymopoiesis and further validate critical function of Tcf1 and Lef1 among the Tcf/Lef family. We now included all these data as Figure 4E and 4F, and described in the Results and Discussion.The analysis of T cell development also validated the effectiveness of induced deletion of Tcf/Lef proteins in our experimental system, and thus lent additional support to the conclusion that Tcf/Lef factors and β-catenin are dispensable for AML LSC self-renewal. This was also the primary reason and purpose for inclusion of the Tcf-qKO model.As for T cell activation in the Tcf-qKO model, we agree that is an important scientific question, but respectfully suggest it is beyond the focus on β/γ catenin in this manuscript. As shown in Figure 4E and F, induced deletion of Tcf/Lef proteins (primarily due to deletion of Tcf1) greatly diminished thymic output. As a result, the precursor frequency of antigen-specific T cells was reduced in the peripheral lymphoid organs when challenged with viral infection, making it a less ideal model for investigation of T cell activation and differentiation. This was not a major issue for induced deletion of β- and γ-catenin , because thymic development was not detectably affected. As such, analysis of the contribution of Tcf3/Tcf4 and their possible cooperativity with Tcf1/Lef1 should be performed using a model where these proteins are specifically deleted in mature T cells, like we did for Tcf1 and Lef1 in previous studies . We hope that the reviewers/editors would allow us to address those questions in a separate, dedicated project.7) Do the β-catenin and γ-catenin individual or double knockout mice develop T cell lymphoma?+Ctnnb1fl/flJupfl/fl BM chimeras for 16 weeks after tamoxifen treatment and did not observed signs of malignant transformation of thymocytes or other hematopoietic lineages. We included a statement on this observation.We did monitor a cohort of WT and CreER


Banana is a climacteric fruit and its ripening process is greatly influenced by presence of ethylene. This physiological climacteric characteristic of banana fruit leads to a fast ripening and a short shelf-life. Application of edible coating such as chitosan aims to prolong fruit shelf life. The knowledge on gene expression will help to understand the fruit ripening process itself and chitosan effect on global gene expression. Global gene expression data of chitosan treated and control of Cavendish banana during fruit ripening were provided. Total RNA was isolated from banana pulp for differential gene expression analysis. The RNA-sequencing generated ranged from 16,155,947 to 23,587,110 total reads, with 75.8%–83.8% of reads were mapped against the genome reference. In total, 33,797–35,944 transcripts were detected. The transcriptomics data discussed in this publication are accessible through NCBI's Gene Expression Omnibus with GEO Series accession number GSE139457. These data provide information to identify candidate genes involved in fruit ripening in response to chitosan coating to design a better banana postharvest management. Previous study by Lustriane et al. [22.1Ethylene-treated banana fingers were used as samples for fruit ripening process. Three biological replicates of banana pulp samples were taken on 1st and 7th day from each untreated and 1.25% chitosan treated banana, as previously described by Pratiwi et al. , Lustria2.2Total RNA was extracted from fruit pulp of each sample using Cordeiro's method . The RNA2.3The TruSeq RNA Sample Prep KIT v2 was then used to construct RNA library from each sample and sequenced using Ilumina platform HiSeq 2000 with HCS V2.2 software.2.4FastQC program to examine low base score, adapter, and PCR contaminations in order to obtain clean reads [Musa acuminata DH Pahang Version 2 Genome Sequences (https://banana-genome-hub.southgreen.fr/sites/banana-genome-hub.southgreen.fr/files/data/fasta/version2/musa_acuminata_v2_pseudochromosome.fna) [TopHat software [Cufflinks V2.2.1 [Each sample was checked for quality control using an reads . Then, tome.fna)  using Tosoftware ,9 , Supervision. Husna Nugrahapraja: Resources, Validation, Analysis, Investigation, Data Curation, Writing , Visualization. Eiichiro Fukusaki: Writing . Sastia Prama Putri: Writing . Cindy Novianti: Analysis, Investigation, Writing , Visualization. Septhy Kusuma Radjasa: Analysis, Investigation, Writing , Visualization. Tessa Fauziah: Analysis, Investigation, Writing , Visualization. Lutfi Dewi Nurmala Sari: Analysis, Investigation, Writing , Visualization.


Although transdermal drug delivery systems (DDS) offer numerous benefits for patients, including the avoidance of both gastric irritation and first-pass metabolism effect, as well as improved patient compliance, only a limited number of active pharmaceutical ingredients (APIs) can be delivered accordingly. Microneedles (MNs) represent one of the most promising concepts for effective transdermal drug delivery that penetrate the protective skin barrier in a minimally invasive and painless manner. The first MNs were produced in the 90s, and since then, this field has been continually evolving. Therefore, different manufacturing methods, not only for MNs but also MN molds, are introduced, which allows for the cost-effective production of MNs for drug and vaccine delivery and even diagnostic/monitoring purposes. The focus of this review is to give a brief overview of MN characteristics, material composition, as well as the production and commercial development of MN-based systems. The application of various chemical agents on the skin dates back thousands of years, when they were applied to treat diseases, protect the skin, or for cosmetic reasons . The ancstratum corneum (SC) represents the main barrier for the penetration and permeation of active pharmaceutical ingredients (APIs) ,4342,43]In the second case, solid MNs are not only used for piercing the skin but also serve as drug reservoirs when MNs are coated with the suitable drug formulation. Although this approach allows for the rapid delivery of the therapeutic API dose, a limited drug amount can be applied on the base and shaft of the MN  . FurtherCoated MNs are manufactured by coating solid MNs with a drug formulation before application on the skin C. Once tBesides APIs, dissolving MNs consist of soluble matrix containing biocompatible polymers or sugars. MN tips dissolve after contact with the interstitial fluid, followed by API release (poke and release approach) D. As theNowadays, there is growing interest in dissolving MNs made of biodegradable materials, as they enable API delivery without creating sharp, bio-contaminated, and non-degradable waste . MoreoveA relatively new type of MNs is fabricated from a hydrogel-forming matrix and was first described in 2010 . This nePhase transition MNs are a subcategory of hydrogel MNs. After the absorption of the body fluid, the API from the MN matrix is released due to swelling of the polymer. This MN type leaves some or no residue after application . Althouggentle manufacturing without damaging sensitive and unstable molecules;controlled or immediate drug release; andsufficient mechanical strength for skin penetration.The advent of microfabrication manufacturing technology in recent decades has enabled the development of MNs in research laboratories and pharmaceutical companies . TherefoThe first solid MNs were made of silicon , as induThe selected material determines the manufacturing MN method that should be accurate, reproducible, robust, and precise . The manSolid and hollow MNs, as well as molds for dissolving MNs, have been manufactured directly from a suitable material substrate using MEMS methods . The proIn the first step, a film with a thickness between a few nanometers and 100 μm is formed on a substrate by a chemical (CVD) or physical vapor deposition (PVD) ,116,117.Then, a two-dimensional master pattern of the desired material is transferred from the original photomask to the photosensitive-coated substrate during the second phase of the process, called patterning. In most cases, a silicon wafer is used as a substrate, and the transferring process is made using a radiation source with one of the lithography process , while others such as glass are transparent. In this process, an optic mask, an opaque template for generating the desired pattern in a wafer, is created  (PDMS) is followed by the application of the chosen material .The etching is achieved by applying a strong acid or caustic agent to etch out the uncovered parts of the substrate to form a design on the surface of the material. Two types of etching can be distinguished: wet and dry etching . In the On the other hand, the dry etching process is achieved by using a vapor phase or plasma etcher. Two main types of dry etching are distinguished: reactive ion etching (RIE) and ion-beam milling (IBM). In the RIE process, the gas excitation into a reactive state enables a reaction between the gas and the substrate. The number of ions that influence the degree of isotropy can be adjusted by controlling the gas pressure. The electric field can accelerate ions and further increase the direction of etching. In the case of the IBM process, inert ions are speeded up from a source to physically remove the material to be etched . AlthougHenry et al. used DRIE to produce silicone, out-of-plane MNs. First, the chromium masking material was applied to a silicon wafer and formed into dots of a diameter equal to the base of the desired MNs. Subsequently, the wafers are exposed to RIE, so that the regions protected by the metal mask are preserved to allow the MNs to form . Wang etPaik et al. fabricated an in-plane single, crystal-silicon MN array integrated with PDMS microfluid chip for blood extraction in point-of-care devices and DDS. The MNs were produced by anisotropic dry etching, isotropic dry etching, and the trench-refilling process. They concluded that the MNs are stiff enough to penetrate animal skin models without creating damage . Ma et aMetal MNs can be manufactured by 3D laser cutting ,72,93,94Arrays of solid MNs are produced by cutting stainless steel or titanium sheets in the shape of MNs with an infrared laser . The desλ = 248 nm) was used to make holes from the side of PLA sheets, which had previously been manufactured using a micromolding technique . I. I153]. −2) MNs .Solid silicon microprocjections were coated with a thin layer of gold. The solution, with ideal surface tension and viscosity, including methylcellulose, surfactant and model drug, is coated on the whole length of the microprojection. Drying started with a gas jet at 6–8 m/s and incident angle of 20° horizontally to direct the coating liquid onto the MNs and away from the base. The thickness of the coated layer on microprojections was 5 µm and increased rapidly to allow the coated material to dry instead of relocating on the base substrate. This method offers several advantages including uniform distribution and fast drying of the coating solution, more or less constant viscosity of the bottom layer of the solution, and the possibility of removing the excess coating solution from the base substrate . The improved delivery of large vaccine molecules, through the SC, can be achieved by modifying the gas jet method for coating MNs. Raising the incident angle from 20° to 70°, removing the patch edge, and rotating the patches during the coating process ensured the uniformity and relocation of the drug from the whole MNs only to the tips. Much lower doses provided an equivalent protective immune response as the intramuscular injection. These MN patches also contributed to extended and improved vaccine stability .Spray coating implies using fluid pressure to create droplets. An intact film-coat is formed from fine droplets (<280 µm), which are deposited on MN array and then, outspread and coalesced. The first step is atomization, which generates fine droplets . Then coThe nozzle design, concentration, input ray, physicochemical characteristics of the coating solution , and processing parameters like air-to-liquid mass ratio, the duration of spraying, atomization air pressure, gun-to-surface distance, and air cap setting determine the droplet size. The deposition of droplets on the surface is determined by spray velocity and spray density . The sprMcGrath et al. used a nozzle, connected to a compressed air source and coating solution, for the production of an atomized spray. They fixed silicon MN patches to the adjustable stage by tape and used a syringe driver and peristaltic pump to control the rate of liquid input. A coating solution made of HPMC, CMC, and surfactant provided fast film-forming and enhancement of the coalescence of droplets on the MN surface .Electrohydrodynamic atomization (EHDA) is a production method of atomized droplets by a moving liquid where charge inside the droplets is generated by the electrical field. When the critical voltage is achieved, liquid spurts out of a nozzle in the form of droplets. Then, it is deposited onto a grounded collector positioned below the nozzle tip  157]. T. T157]. The EHDA process can be single needled (formulation is injected into a single nozzle by syringe pump), coaxial (two or more immiscible liquids are put in separate nozzles), and multiplexed . The coaxial system protects the drug from direct exposure to the environment and enables sustained and controlled drug release . Flow raThis method is used for the delivery of insulin, folic acid, titanium dioxide antimicrobial agent, gold used in gene delivery, and sensitive biomolecules like peptides and proteins, which are unstable when administrated orally ,159. AliAngkawinitwong et al. used the EHDA process to coat ovalbumin loaded-PLGA nanoparticles onto hydrogel-forming MN arrays. The extended-release of ovalbumin over 28 days was observed. Uniform particle coating on the MNs was perceived by microscopic analysis. The mechanical and insertion properties of the coated MNs were the same as the uncoated MN, suggesting that the coating did not harm the application of MNs .Piezoelectric inkjet printing provides a controlled and precise MN coating with liquid droplets (1–100 pl), which is followed by solidification. The method is compatible with different aqueous and organic solvents. The low viscosity of the formulation is preferable to prevent clogging the small jetting nozzle. The voltage supplied material connected to a piezoelectric transducer produce vibrations to eject drops from the nozzle .A modified method called thermal inkjet printing implies drop production by increasing the formulation temperature a bit higher than its boiling point  162]. T. T162]. This method provides coating MNs with poorly soluble drugs. Biodegradable PGA MNs were coated with voriconazole and showed antifungal activity . The tra®, Dermapen®). Recently, MNs were investigated as a part of the monitoring/diagnosis system to provide the ability for at transdermal sampling of body fluids in a completely painless manner [Up to now, MNs with a broad range of geometries, with or without a MN application device, have been fabricated using different manufacturing methods from a variety of materials. Although MNs have been extensively studied for transdermal drug delivery and vaccine delivery, these systems can also be designed for delivery targeted to other tissues such as oral mucosa, vaginal mucosa, anal sphincter muscles, and hair follicles . Nowadays manner . During It is necessary to adequately select the type of MNs and their geometry in order to successfully developed the final product with sufficient and reproducible penetration for transdermal drug delivery. Usually, in order to obtain the possibility of self-administration a MN application device  is necessary. Many pharmaceutical companies and research laboratories are involved in the MN-based product development including 3M, Zosano Pharma, Alza Corporation, Becton-Dickinson Technologies, Valeritas, Vaxxas, Microneedle Therapy System, Nanopass Technologies Lohmann, Therapie- Systeme AG, and others.®  (3Mem (MTS) C includiTM dermal drug delivery could play a significant role in the modern healthcare system in the future and the success of these minimally-invasive devices would also open up a wide range of therapeutic opportunities for buccal, oral, vaginal, rectal, and ocular drug delivery.


The present study aims to provide a comparative analysis of the etiologies of female infertility between Dehong, on the Yunnan Frontier, and Kunming.A retrospective study, which included 941 infertile females in Kunming who were treated in the First People’s Hospital of Yunnan Province and infertile females who were treated in the local hospital in Dehong from January 2016 to November 2018, was conducted. A comparative analysis of the etiologies of infertility in the two regions was then carried out.In patients with primary infertility, ovulation disorder (15.03%) was the main cause of infertility in Kunming, and pelvic inflammatory disease (25.59%) was the main cause in Dehong. With regard to secondary infertility, although pelvic inflammatory disease was the main cause of infertility in both regions, the incidence of intrauterine adhesions in Kunming was significantly higher than in Dehong.The etiology of infertility showed different epidemiological characteristics depending on the region, hence individualized treatment should be given accordingly According to criteria issued by the World Health Organization (WHO), those who are married, have a normal sex life without the use of contraception, and have lived with their partner for one year without conception are called infertile . Due to Infertility is regarded as a global problem that affects the health and economic situations of sufferers and their families , 4. The Previous studies have revealed that differences in the etiologies of infertility are mainly due to differences in culture, socioeconomics, health care practices, policies, and environmental conditions . The preThis is a cross-sectional study. The random sample selection was used. From January 2016 to November 2018, 941 infertile Han females in Kunming who were treated in the First People's Hospital of Yunnan Province, and 813 infertile females who were treated in Dehong Dai and Jingpo Autonomous Prefecture Hospital, were enrolled in the study.All the patients met the WHO criteria for the diagnosis of female infertility: having been sexually active for at least 12 months without using contraception without becoming pregnant. Among them, those who have never been pregnant previously fall under the category of primary infertility; those who have been pregnant in the past but who have not been pregnant for 12 consecutive months without contraception fall under the category of secondary infertility.All patients had normal cognitive function and participated in the study voluntarily with signed informed consent.Exclusion criteria for this study were infertility due to the male component, less than one year of menarche or entrance into the perimenopausal period as verified by the detection of sex hormones, malignant tumors, severe cardiovascular, cerebrovascular, hepatic, or renal disease, mental disease or cognitive impairment, unwillingness to sign informed consent, and relocation from other places to the survey sites.Medical records were obtained from doctors in the two hospitals. A proforma used to extract information from the patient records. The contents of clinical data included age, educational background, nationality, residence, body mass index (BMI), marriage age, duration of disease, menstrual history, previous history of infertility, and reproductive history. The actual etiologies of infertility were obtained from the operations themselves, and the etiologies and related factors of infertility in the two regions were compared and analyzed.An excel spreadsheet was formulated and the collected medical history data were input by recruiting and reexamination. For those cases that required surgery, the intraoperative conditions were recorded. Double entry was adopted for data input. In case of any discrepancy in data entry, the original inquiry questionnaire and medical record data were rechecked.χ2 test was used for comparing proportions, and logistic regression analysis was adopted for the analysis of the influencing factors of infertility. P value < 0.05 was regarded as statistically significant.SPSS 22.0 software was used for data analysis, the Pearson 2 = 7.42, P value = 0.192). The mean age of infertile patients in Kunming was 29.09 ± 4.81 years (range 20–41 years), and the mean age of infertile patients in Delong was 28.87 ± 5.02 years (range 19–44 years); the difference of age between the two regions was not statistically significant . The difference in the distribution of educational background, however, was statistically significant ; there were more infertile patients with a high educational background (college degree or above) in Kunming than in Dehong. Statistical significance was also found in the distribution of residence in the two regions ; the majority of the infertile patients (75.24%) in Kunming lived in urban areas, whereas the majority in Dehong (77.49%) lived in rural areas. Statistical analysis showed that there was a significant difference in the distribution of marriage age between the two regions ; the percentage of patients with a marriage age of ≤ 25 years in the Dehong region (66.18%) was significantly higher than in Kunming (48.54%). There was no significant difference in the number of sexual encounters per week between the two regions . In addition, among the patients in Kunming, the number of patients who knew their most fertile days of the month was higher than in the Dehong area; the difference was statistically significant .From the 1,754 patients analyzed in the present study, 941 were in Kunming and 813 in Dehong. There was no statistical significance in the age distribution of infertile patients between the two regions .The majority of infertile patients in both regions had a BMI within the normal range, but the BMI of patients in Kunming was higher than in Dehong. There were more lean patients BMI < 18.5) in Dehong than that in Kunming, and there were more overweight patients (BMI ≥ 25) in Kunming than that in Dehong  areas. Then, based on the data of infertile patients in Kunming, the regression equation was used for the comparison between Dehong and non-Dehong (Kunming) areas. The results were showed in Table In Kunming region, patients with older marriage age , higher BMI , irregular menstruation , and less menstrual volume  were more likely to be infertile compared to those without. In Dehong region, patients who had pelvic inflammatory disease were almost four times likely to be infertile compared to those without .Among the 1,754 infertile patients included in the present study, 1,257 (71.66%) had undergone laparoscopy combined with hysteroscopy. Among them, pathologies were found in 1,234 patients, accounting for 98.17% of the total number of infertile patients; 432 cases (33.41%) had pelvic inflammatory disease, 311 cases (24.74%) had hydrosalpinx, 251 cases (19.97%) had an ovarian cyst, 237 cases (18.85%) had hysteromyoma, 165 cases (13.13%) had intrauterine adhesions, and 147 cases (11.69%) had PCOS. Of these 1,257 cases of infertility, 366 (20.05%) possessed two or more of the factors that contribute to infertility. Details are shown in Table Among the etiologies of primary infertility in the two regions, ovulation disorder 36.71%) was the main cause in Kunming, while pelvic inflammatory disease (25.59%) was the main cause in Dehong. Additionally, the proportions of tubal factor  and pelvic tuberculosis  in Dehong were much higher than in Kunming, as can be seen in Table .71% was As shown in Table The present study revealed that infertile patients in Kunming were mainly affected by such risk factors as late marriage age, being overweight, irregular menstruation, and less menstrual volume, while the infertile patients in Dehong were mainly influenced by the risk factor of previous pelvic inflammatory disease. The differences in etiologies between the two regions were mainly reflected in four factors, including pelvic inflammatory disease, hydrosalpinx, PCOS, and pelvic tuberculosis.Pelvic inflammation includes the infection and inflammation of the upper genital tract  –9, whichPelvic inflammatory disease causes infertility by changing the topography of the pelvic cavity and the internal structure of the fallopian tubes, and ovulation disorders are caused by inflammation around the ovary . In addiIn the current study, from the 1,754 infertile patients, 432 had pelvic inflammatory disease, accounting for 24.63% of the total number of patients. The proportion of infertile patients with previous pelvic inflammatory disease in Dehong (15.62%) was higher than in Kunming (5.95%) (P < 0.05). This difference may be correlated with the medical and health conditions, the poor sexual health consciousness, and the younger marriage age in Dehong. Thus, we should note the levels and prevalence of sexual health and hygiene education for childbearing-age couples in Dehong.Common diseases leading to ovulation disorders in females of childbearing age are PCOS and hyperprolactinemia. Among such females, the incidence of PCOS ranges from 5 to 10%, and the incidence in infertile patients is as high as 30–60% . The pheTubal infertility, of which hydrosalpinx is a major cause, accounts for 30–50% of patients with infertility . InflammIt has been reported that the incidence of infertility in patients with endometrial adhesions is as high as 90% . UterineThis study has several limitations. First, the cross-sectional nature of the study design might not show the cause and effect relationships between study variables. Second, the samples for this study were taken from the only one teaching hospital of each area. Finally, the sample size of this study was not calculated based on a certain equation.In conclusion, there were marked differences in the etiologies and risk factors of infertility between women living in Kunming and Dehong.


In this article, amino functionalized multiwalled carbon nanotubes (MWCNTs) were prepared by chemical modification of the surface of a MWCNTs using γ-aminopropyltriethoxysilane (APTES) and dispersed into the epoxy composition. The modifying agent (APTES) was directly deposited on the MWCNTs surfaces. For the functionalization of MWCNTs, was used not the APTES concentrate, as it had been described in previous works, but its freshly prepared aqueous solution. Properties of APTES-treated MWCNTs were characterized by transmission electron microscope (TEM), Raman spectroscopy and FT-IR. The results showed that the functionalization and chemical compatibility of APTES-treated MWCNTs with epoxy composition provides their best dispersion in the epoxy composition, had important influence on curing behavior, structure and physicochemical properties of the epoxy composites plasticized with trichloroethyl phosphate. The results showed that the functionalization and chemical compatibility of APTES-treated MWCNTs with epoxy composition provides increased of physicomechanical properties of epoxy composites: bending stress increases by 194% and bending modulus increases by 137%, the tensile strength increases by 108% and the tensile elastic modulus increases by 52%, impact strength increases by 300%, in comparison with plasticized epoxy composite that does not contain MWCNTs. Research in the field of polymer modification with functional additives is widely used to develop polymer composites with a given set of consumer characteristics ,2,3. So Currently, nanocomposites are often used in various industries, due to their unique physicomechanical characteristics, which are due to the fact that nanodispersed particles are not stress concentrators in polymer composites ,13,14,15Taking into account excellent physical and mechanical properties, as well as high aspect ratio of carbon nanotubes (CNTs), they are believed to be a promising reinforcement phase for polymer composites among inorganic nanofillers . AnalysiTo achieve the maximum effect from the addition of CNTs into polymer composites, the surface of CNTs is functionalized by various methods, such as fluorination , silaniz2) with n-butyl glycidylether (BuGE) and benzyl glycidylether (BeGE). The results showed that the functionalization of MWCNTs provides their best dispersion in the epoxy composition, had important influence on structure, physicochemical and mechanical properties: MWCNT-BeGE/epoxy nanocomposites exhibited increasing flexural strength and modulus of 22.9% and 37.8%, respectively compared with the neat epoxy.In paper , a surfaIn most modern studies, the influence of functionalized CNTs on the processes of structure formation during the curing the epoxy composites is noted. Choi et al.  found thBesides, the gain effects from the addition of various amino-functionalized CNTs into epoxy nanocomposites were analyzed, and their positive effect on the operational characteristics of polymer composites was proved ,17,18.However, the amino groups on the surface of CNTs, as reactive chemical functional groups, can have an effect on CNTs dispersion, the curing reaction, and interfacial interaction in epoxy composites ,24. SincIn this article, amino-functionalized MWCNTs were prepared by chemical modification of the surface of a MWCNTs using γ-aminopropyltriethoxysilane (APTES) and dispersed into the epoxy composition. The novelty of this work lies in a new approach to the modification of MWCNTs. It is proposed to use an aqueous solution of the modifier (APTES), because the use of a concentrate of APTES is inconvenient due to the fact that a small amount of it is very difficult to distribute on MWCNTs, which are characterized by a high specific surface area. The use of an aqueous solution avoids this difficulty and uniformly to applies the modifier to the MWCNTs surface. In addition, researchers often recommended that the modifier be added directly to the epoxy composition ,26, whicProperties of APTES-treated MWCNTs were characterized by transmission electron microscope (TEM), Raman spectroscopy, and FT-IR. The results showed that the functionalization and chemical compatibility of APTES-treated MWCNTs with epoxy composition provides their best dispersion in the epoxy composition, had important influence on curing behavior, structure, physicochemical and mechanical properties of the epoxy composites plasticized with trichloroethyl phosphate. This work shows an important reference value for modification, optimizing, designing of epoxy compositions for production of high performance MWCNTs/epoxy nanocomposites.Epoxy resin ED-20 (GOST 10587-93) manufactured by CHIMEX Limited  was used as a polymer matrix. Its chemical structures is shown in The qualitative characteristics of ED-20 are presented in 2 N(–CH2CH2NH–)nH2; n = 1–4. The qualitative characteristics of PEPA are presented in Polyethylene polyamine (PEPA) (TS 6-02-594-85) manufactured by CHIMEX Limited (Russia) was used as a hardener of an epoxy compositions. Its chemical formula: HTo plasticize epoxy composites and making them flame-resistant, we used tri-2-chloroethyl ether of orthophosphoric acid (TCEP) with purity of 95.99%, which is an orthophosphoric acid and ethylene chlorohydrin ester, manufactured by PJSC “Khimprom” .The choice of TCEP is due to the presence of combustion inhibitors-phosphorus and chlorine, which reduce the flammability of the epoxy composite ,28. The Multi-walled carboxylated carbon nanotubes (MWCNTs) manufactured by "Nanotechcenter, Ltd."  were used as a nanostructural additive. The qualitative characteristics of MWCNTs are presented in 2O–APTES (95–5) solution with an ultrasonic homogenizer for 10 min. The suspension was kept under reflux at 80 °C under constant low-speed stirring at 100 rpm for 12 h. The pH of the mixture was adjusted to 5. We selected an acidic media (CH3COOH) in order to increase the level of silanol formation and to decrease self-condensation reactions between the hydrolyzed silanol groups. To remove the remaining silane compound around the MWCNTs particles the resulting suspension was centrifuged and washed twice with H2O. Then, the product was dried in the laboratory oven [The surface of MWCNTs was functionalized with a γ-aminopropyltriethoxysilane (APTES) coupling agent, manufactured by the Penta-91 company . For this purpose, 0.5 g of MWCNTs was dispersed in 100 mL of the Hory oven .Previously, we determined experimentally the ratio of epoxy oligomer, plasticizer and hardener: 100 parts by mass of ED-20, 40 parts by mass of TCEP and 15 parts by mass of PEPA ,28. MWCNBending stress and flexural modulus was determined according to ISO 178: 2010, strength and modulus of tensile elasticity was determined according to ISO 527-2: 2012, impact strength was determined according to ISO 179-1: 2010, surface morphology of the samples was studied using a Tescan VEGA 3 SBH scanning electron microscope , FT-IR spectroscopy was carried out using the Shimadzu IRTracer-100 , Raman spectra were measured by DXR Raman Microscope  with laser excitation at 532 nm, the MWCNTs structure was studied by transmitting electron microscope JEOL JEM-1400 . The dispersion of MWCNTs was tested by dynamic light scattering method and Nicomp 380 ZLS  was applied. Three parallel studies were done, the coefficient of variation was 7%. Nicomp 380 ZLS is a fully automatic particle size and zeta potential analyzer. Measurement of the zeta potential, or the potential of the double layer, allows you to determine the strength of the interaction between the particles, which is a characteristic of the stability of the system and the ability of particles to agglomerate. Electrophoresis is used to determine the zeta potential in the Nicomp 380 ZLS analyzer. A small amount of sample is placed in a measuring cell with palladium electrodes that create an electromagnetic field. The specific surface area of the samples was determined by means of the Quantachrome Nova 2200 specific surface area and porosity analyzer  using the initial portion of the isotherm of physical nitrogen sorption (99.999%). The studied samples were placed in a cell preliminarily calibrated with respect to internal volume at a temperature of liquid nitrogen (about 78 K), which was degassed in vacuum to a constant mass at a given temperature (150 °C) for 3 h. After that, the cell was installed in the device and the isotherm of nitrogen adsorption was measured in the pressure range 0.03–0.3 P/Po. The specific surface of the test sample was calculated by the BET method with the Quantachrome Nova 2200 software .As a modifying agent (0.01–0.50 parts by mass) pristine MWCNTs were added into plasticized epoxy composition . Previous studies have shown that the addition of TCEP into epoxy composition provides not only an increase in mechanical characteristics (bending stress increases by 32% and impact strength is 2.6 times higher), but also reduces the flammability of the epoxy composite (oxygen index (OI) increases from 19% to 27% by volume) ,30.The conducted research has shown that the most rational content of pristine MWCNTs as a structuring additive is 0.1 parts by mass, which provides maximum values of physical and mechanical properties, To ensure the chemical interaction of the filler with the polymer matrix and to increase physicomechanical characteristics of epoxy composites based on them, the MWCNTs surface was treated with the APTES sizing additive.The effect of the APTES treatment on the structure and degree of MWCNTs defectiveness was studied using Raman spectroscopy and transmission electron microscopy (TEM). Analysis of Raman spectra was shown that the R (the ratio of peak D and peak G) value of (1) is significantly smaller than (2), which infers a more regular structure of carbon atoms of (1), suggesting that the modification increased the number of defects on MWCNTs, 2/g.According to TEM, a decrease in aggregation and a degree of particle confusion is observed, which is confirmed by an increase in the distance between individual nanotubes due to the APTES treatment, To study the dispersion of MWCNTs in an epoxy binder, a dynamic light scattering technique was used to estimate the effective particle size of the dispersed phase that would be the true size if these particles were spherical. Since MWCNTs themselves and the agglomerates formed by them are not spherical, the measurement results allow us to evaluate only the relative changes in the dispersed composition of the analyzed suspensions. As the analysis of the diagram of the suspension particles distribution by effective sizes shows, The average effective size of the smallest agglomerates is 418 nm. The second peak shows an effective particle size of about 7 μm, large agglomerates slightly prevailing over smaller ones in terms of quantity. For a suspension with APTES-treated MWCNTs, the diagram of the suspension particles distribution by effective sizes is monodisperse and the average effective size of the agglomerates is in the region of 900 nm, with no larger agglomerates being observed. Thus, the APTES treatment of MWCNTs surface provides their best dispersion in the epoxy composition.−1), which are involved in the formation of the APTES layer, decreases. One can also see a broad maximum of about 1000 cm−1, which indicates the presence of Si–O bonds in the sample. In addition, peaks appears at 1040 cm−1 and 630 cm−1, confirming the formation of the Si–O–C bond.The formation of strong bonds between MWCNTs and APTES was proved by IR Fourier spectroscopy, The chemical interaction of the APTES functional groups and the epoxy oligomer was proved in .Based on this, it can be assumed that the reactions shown in The results showed that the functionalization and chemical compatibility of APTES-treated MWCNTs with epoxy composition provides increased of physicomechanical properties of epoxy composites: bending stress increases by 194% and bending modulus increases by 137%, the tensile strength increases by 108% and the tensile elastic modulus increases by 52%, impact strength increases by 300%, in comparison with plasticized epoxy composite that does not contain MWCNTs, In Nanodispersed fillers have a significant effect on the kinetic characteristics of the curing process of epoxy composites ,34,35,36The study of the curing kinetics of epoxy compounds, The addition of APTES-treated MWCNTs into the epoxy composition accelerates the curing process, which can be seen in the reduction of the duration of the gelation process from 65 to 52 min, and the duration of the curing process from 78 to 61 min, with an increase in the maximum curing temperature from 132 to 172 °C. The results of measuring the coefficients of heat and thermal diffusivity are given in The conducted research has proven that it is possible to regulate directly operational properties of epoxy composites using small MWCNTs additives that allow to develop epoxy composites with high performance properties. The effectiveness of the surface modification of MWCNTs with γ-aminopropyltriethoxysilane and the formation of strong chemical bonds at the polymer matrix/filler interface have been proved, which ensured an increase in the physicomechanical characteristics of epoxy composites: bending stress increases by 194% and bending modulus increases by 137%, the tensile strength increases by 108% and the tensile elastic modulus increases by 52%, impact strength increases by 300%, in comparison with plasticized epoxy composite that does not contain MWCNTs.The effect on the structure and structure formation processes during curing of epoxy compositions containing both pristine MWCNTs and APTES-treated MWCNTs has been established. The addition of APTES-treated MWCNTs into the epoxy composition accelerates the curing process, which can be seen in the reduction of the duration of the gelation process from 65 to 52 min, and the duration of the curing process from 78 to 61 min, with an increase in the maximum curing temperature from 132 to 172 °C in comparison with a composition containing pristine MWCNTs, which further confirms the participation of APTES functional groups in the curing process.As a result of the studies, it has been proved that the addition of 0.05 to 0.5 parts by weight of both pristine MWCNTs and APTES-treated MWCNTs into the epoxy composite increases the thermal diffusivity by 20–42% and the thermal conductivity by 73–87%.


Human cardiac stem cells isolated from atrial appendages based on aldehyde dehydrogenase activity (CASCs) can be expanded in vitro and differentiate into mature cardiomyocytes. In this study, we assess whether Wnt activation stimulates human CASC proliferation, whereas Wnt inhibition induces cardiac maturation. CASCs were cultured as described before. Conventional PCR confirmed the presence of the Frizzled receptors. Small-molecule inhibitors  and activator (CHIR99021) of the Wnt/β -catenin signaling pathway were applied, and the effect on β-catenin and target genes for proliferation and differentiation was assessed by Western blot and RT-qPCR. CASCs express multiple early cardiac differentiation markers and are committed toward myocardial differentiation. They express several Frizzled receptors, suggesting a role for Wnt signaling in clonogenicity, proliferation, and differentiation. Wnt activation increases total and active β-catenin levels. However, this does not affect CASC proliferation or clonogenicity. Wnt inhibition upregulated early cardiac markers but could not induce mature myocardial differentiation. When CASCs are committed toward myocardial differentiation, the Wnt pathway is active and can be modulated. However, despite its role in cardiogenesis and myocardial differentiation of pluripotent stem-cell populations, our data indicate that Wnt signaling has limited effects on CASC clonogenicity, proliferation, and differentiation. Heart failure after myocardial infarction (MI) is the leading cause of mortality worldwide and results from the loss of cardiomyocytes and remodeling of the remote cardiac tissue due to coronary occlusion. While heart transplantation is currently the only available therapy to fully restore cardiac functionality after an MI, its major drawbacks, such as life-long immunosuppressive therapy and the paucity of donor hearts, do not allow it to be a realistic option for every patient . In that+ CSCs are the most studied, but recently also the most challenged CSC type. Beltrami et al. were the first to describe a c-kit+ CSC population in rat hearts which gave rise to cardiomyocytes, smooth muscle cells, and endothelial cells, and which improved cardiac function after injection in a rat MI model . Du. DuCyclibr cells within various compartments of the heart. Within the same patient, the proportion of ALDHbr cells was significantly higher in the right atrial appendage (RAA) (5.1% ± 4.4%) compared to the left atrial appendage (LAA)   . To analthe apex . In genebr cells (dim population could not be cultured after isolation [KDR) was expressed in CASCs, as well as in adult atrial and ventricular heart muscle, while Brachyury (T) was absent. Furthermore, the early cardiac transcription markers such as GATA binding protein 4 (GATA4), T-box 5 (TBX5), T-box 18 (TBX18), and NK2 homeobox 5 (NKX2.5) were expressed in human CASC cultures, as well as in adult heart muscle. From the mature cardiac markers typically found in adult cardiomyocytes, only cardiac type troponin T2 (TNNT2) and myosin light chain 2  were expressed in human CASC cultures, although at lower levels as compared to adult atrial and ventricular tissue. Myosin light chain 7  and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) were present in adult heart muscle but were completely absent in CASCs. These data suggest that human CASCs display a cardiac progenitor-like phenotype rather than a mature cardiac phenotype during expansion.To identify the cardiac differentiation stadium of human CASCs during expansion, a number of early- and late-stage cardiac specific markers were evaluated in ALDHbr cells . As prevsolation . For theFZD1, 2, 4, 6, and 7 was already detected after 25 cycles, indicating abundant expression levels of these FZD subtypes.When expanding CASCs for clinical use, it would be beneficial to reduce the expansion time by stimulating CASC proliferation. This might be achieved by interfering with the canonical Wnt pathway. Since binding of the Wnt ligand to the FZD receptor is essential for the activation of the downstream Wnt/β-catenin pathway, we firstly analyzed the expression pattern of several FZD receptors in CASCs by conventional PCR. As shown in To test if the Wnt/β-catenin pathway could be modulated in CASCs, we investigated whether the levels of total and active β-catenin (dephosphorylated on Ser37 or Thr41) could be modified by CHIR99021  or C59, IWP2, XAV939, and IWR1-endo .p < 0.05). 293T cells, used as a positive control, showed a 23-fold and 26-fold increase in total and active β-catenin levels. As expected, CHIR99021 treatment did not upregulate total or active β-catenin levels in the SW480 cell line, due to an adenomatous polyposis coli (APC) mutation which inhibits β-catenin ubiquitination [As shown in tination . Finallyp < 0.05), indicating a potent inhibition of Wnt/β-catenin signaling in CASCs. Total β-catenin levels remained unchanged in CASCs (p < 0.05), after XAV939 treatment and by 91% and 100%, respectively (p < 0.05), after IWR1-endo treatment  increase in Cyclin D1 and cJUN expression (Cyclin D1 in CASCs (cJUN expression in CASCs only decreased by 25% (p < 0.05) upon XAV939 treatment (p = 0.0188) in Cyclin D1 expression after XAV939 and IWR1-endo treatment, respectively (cMYC expression decreased by 39% (p < 0.05) in SW480 cells after IWR1-endo treatment  after CHIR99021 treatment (p < 0.05) after XAV939 treatment  were examined. As shown in GATA4 expression increased after XAV939 treatment, but this could not be reproduced with IWR-1endo treatment. IWR1-endo significantly increased TBX5 expression and decreased TNNT2 expression, but this could not be reproduced with XAV939 treatment. NKX2.5, TBX18, and MYL2 expression in CASCs was not affected by Wnt inhibition. These data demonstrate that Wnt inhibition alone is insufficient to induce mature cardiomyocyte differentiation in CASCs.Finally, a potential effect of Wnt inhibition on CASC differentiation was investigated. To this end, CASCs were treated with 2 µM XAV939 or 4 µM IWR1-endo, which effectively inhibited canonical Wnt signaling as shown above. Subsequently, changes in mRNA expression of different cardiac differentiation markers  progenitors, are also expressed in CASCs. Recently, early expression of HCN4 was also reported as a marker for FHF progenitors [TBX5 was [When studying the cardiac differentiation stadium of CASCs during expansion, qPCR analysis demonstrated expression of of CASCs . Howevergenitors . HoweverTBX5 was . These rTBX5 was . HoweverNKX2.5, although lower than in the atrium and ventricle, might suggest a limited endothelial differentiation potential of CASCs. Wu et al. demonstrated that NKX2.5 expression comprises endothelial differentiation in cardiac-specific NKX2.5-positive cells from the developing mouse embryo [TNNT2 and MYL2 in CASCs suggests that CASCs can be considered as an intermediate already committed toward myocardial differentiation. The profound myocardial differentiation potential was already confirmed both in vitro and in vivo. Indeed, co-culturing CASCs with neonatal rat cardiomyocytes induced sarcomeric organization of cTnT and cTnI while transplanting CASCs in the left ventricle of a Göttingen minipig model for acute MI-induced sarcomere formation and protein expression of cTnT, cTnI, connexin 43 (CX43), and MLC-2v [The expression of e embryo . This wae embryo . Despitee embryo . The expd MLC-2v ,12.FZD1, 2, 4, 6, and 7. According to human gene database GeneCards, these specific FZD receptors are also known to be expressed in adult heart. Furthermore, CHIR99021 treatment upregulated total and active β catenin, while XAV939 and IWR1-endo reduced active β-catenin expression. However, CHIR99021 treatment resulted in a slight but significant decrease in cell viability. This is most likely caused by a toxic effect of the product and not the result of Wnt stimulation, since cell toxicity was also observed in SW480 cells where Wnt signaling was not further upregulated. This cytotoxic effect of CHIR99021 was also observed in mouse embryonic stem cells [Cyclin D1, cMYC, and cJUN was detected in CASCs after Wnt activation. Instead, we observed a decrease in cMYC expression levels, both in CASCs and in 293T cells. However, a similar decrease in cMYC expression was also seen after inhibiting canonical Wnt signaling in CASCs and SW480 cells. The reason why both Wnt upregulation and downregulation induce a decrease in cMYC expression is unknown, but a negative effect of cMYC on cardiac function by inducing cardiomyocyte hypertrophy was already described [Although CASCs can efficiently differentiate into cardiomyocytes, we observe a high variability between patients with respect to their proliferation potential. In addition, it is known that myocardial differentiation is only obtained when CASCs are in close contact with functional cardiomyocytes ,12,19. Tem cells . Since Cescribed . Furtherescribed . Indeed,escribed .GATA4 and TBX5, while downregulating the mature cardiac differentiation marker TNNT2. The upregulation of GATA4 after Wnt inhibition is in line with the findings of Afouda et al., showing that canonical Wnt signaling inhibits GATA4 expression and that this restricts cardiogenesis in Xenopus animal cap and cardiac mesoderm explant assays [GATA4 and TBX5 expression after β-catenin depletion in mouse Sca-1-positive cardiac progenitor cells. However, as opposed to our study, this was also accompanied by an increase in the number of cTnT-positive cells [TBX5 and NkX2.5, but not the mature myocardial differentiation markers cTnI and myosin heavy chain (MHC)α [Finally, the effect of Wnt inhibition on CASC differentiation was examined. Current differentiation protocols for PSCs use a sequential incubation with small-molecule Wnt activators and inhibitors to drive cardiomyocyte differentiation . In CASCt assays . A studyve cells . This din (MHC)α . Finallyn (MHC)α . These fimmediately analyzed. However, we have strong evidence that the obtained results would not have been different had the cells been analyzed at a later point in time.In this study, cells were incubated for 48 h with these various treatments, after which gene expression was l-ascorbic acid 2-phosphate, and 500 µg/mL rice-derived recombinant human albumin), with the Wnt-inhibitor IWP-2 for two days, and then replaced by the differentiation media alone for the remainder of the process. Cells were analyzed after five and 15 days in media alone for cardiomyogenic-specific gene expression, including Nkx2.5, MEF2C, TNNT2, and cardiac actin. This protocol was unable to induce significant variations of cardiac gene expressions at five and 15 days as compared to control, indirectly indicating that analyzing the β-catenin activation/inhibition or gene expression at a later stage after Wnt inhibition would not have altered the result (data not shown).While developing an “in vitro” potency test for cardiomyogenic differentiation, CASCs (50.000 cells/well) were seeded in mesenchymal stem cell (MSC)-brew good manufacturing practice (GMP) medium in six-well plates. After 24 h, the medium was replaced by a differentiation medium  1640, 213 µg/mL All procedures were carried out in accordance with the principles set forth in the Helsinki Declaration. Approval by the Jessa Institutional Review Board and informed consent from each patient were obtained. Human CASCs were isolated from atrial appendages obtained from ischemic heart disease patients undergoing routine cardiac surgery, as previously described by Koninckx et al. . In brieThe characteristics of the patients used to isolate and characterize CASCs are shown in 2.Isolated CASCs were cultured in X-vivo 15 medium  supplemented with 2% penicillin/streptomycin  and 10% fetal bovine serum . 293T and SW480 cell lines were cultured in Dulbecco’s modified Eagle medium/Nutrient Mixture F-12  medium  supplemented with 2% PS and 10% FBS. All cell cultures were expanded at 37 °C in a humidified atmosphere containing 5% COdim, CD73, CD90, and CD105. For CD90 in particular, two subpopulations were demonstrated to be present, CD90dim and CD90+ [The immunophenotype of CASCs was continuously monitored at different passages throughout the culture period and remained stable without any specific alteration. Expanded CASCs remained CD34-, CD45-, and CD117-negative. In contrast, the cells stably expressed CD29, CD55nd CD90+ .n = 3). For human samples, LAA and RAA samples of the same patient were used (n = 33). In addition to the routinely isolated RAA, the LAA was obtained as a precaution to avoid thrombus formation in patients at risk of developing atrial fibrillation. Subsequently the percentage of ALDHbr cells was determined by Aldefluor staining as previously described and expressed as the percentage of ALDHbr cells with respect to total cells.Equal amounts of tissue (3 g) were taken from left and right atrial appendage (LAA and RAA), left and right atrium, left and right ventricle, septum, and apex of slaughterhouse pig hearts , IWP2 , C59 , XAV939 , and IWR1-endo . For all Wnt-related experiments, except the serial clonogenic assays, low-glucose DMEM  with 2% PS and 2% FBS was used. Equal concentrations of DMSO were used as control. Cells were incubated for 48 h to the various treatments. For CHIR 99021, a prolonged incubation period was also tested , but this resulted in a clear decrease in cell viability.For all Wnt inhibitors , a five-fold higher concentration was tested on SW480 cells, but this did not result in a more pronounced effect on total and β-catenin expression. Furthermore, a two-fold higher concentration of the Wnt activator CHIR 99021 was tested for CASC viability, but this led to cell death due to high toxicity.Total RNA was extracted using the RNeasy mini kit  and equal amounts of RNA were reverse-transcribed with the SuperScript III Reverse Transcriptase Kit  primed with a random hexamer primer according to the manufacturer’s instructions.2, 250 µM dNTPs, 50 mU/µL Taq DNA Polymerase , 500 nM primer , and 1.5 µL of cDNA. Primers sequences with corresponding annealing temperature and expected fragment size are listed in β-actin was used as an internal control.PCR reactions were performed on a C1000 Touch thermal cycler . The final reaction master mix (26 µL) was composed of 1× PCR buffer, 2.5 mM MgCl® SYBR® Green qPCR SuperMix-UDG , 2 mM extra MgCl2 , and 2 µL of cDNA diluted 10-fold with DNase/RNase-free water. Primer sequences with corresponding annealing temperature and concentration are listed in MYL7), an additional step of 80 °C for 30 s was added to each cycle. SW480 cells were used as a positive control for Brachyury transcription factor (T). The most stable reference genes for each experimental set-up were identified with NormFinder .Real-time PCR reactions were carried out in duplicate with the Rotor-Gene Q  according to the MIQE guidelines . The fin® 800 CW  and goat anti-rabbit IRDye® 680 RD . Detection and quantification were accomplished using the Odyssey Infrared Imaging System and the LI-COR Odyssey Imaging software 2.1 . Data are shown as ratios normalized to the loading control GAPDH and compared with their respective control condition.For total cellular protein, cells were immediately lysed with NP-40 lysis buffer  supplemented with Halt Protease inhibitor cocktail  and 1 mM sodium orthovanadate. Equal amounts of protein, as measured by NanoDrop , were electrophoresed on 4–15% Mini-PROTEAN TGX gels and transferred to 0.2-µm nitrocellulose membranes using the Trans-Blot Turbo Transfer System . Blots were blocked in Odyssey blocking buffer  for 1 h and probed with primary and secondary antibodies for 2 h and 45 min, respectively. The primary antibodies used were rabbit anti-β-catenin (D1048) mAb , mouse anti-active β-catenin mAb clone 8E7 , mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mAb , and rabbit anti-GAPDH mAb . The following infrared fluorescently labeled secondary antibodies were used: goat anti-mouse IRDyeCASC viability was measured with the fluorescein isothiocyanate (FITC) annexin V Apoptosis Detection Kit II  according to the manufacturer’s manual. Early apoptotic cells were defined as annexin V-positive and propidium iodide (PI)-negative, while cells positive for both annexin V and PI were defined as late apoptotic cells. Viable cells were negative for both annexin V and PI. The percentage of viable cells was measured by a FACSAria and expressed as the percentage annexin V- and PI-negative cells of total cells.The clonogenic and proliferative potentials of CASCs were assessed by serial cloning. CASCs were cultured in X-vivo 15 medium with 2% PS and 20% FBS, supplemented with or without 6 µM CHIR99021 for the treatment and control condition, respectively. In the treatment group, continuous exposure to CHIR 99021 was used.Prior to assessing the clonogenic character, CASCs were labeled with green fluorescent protein (GFP). Production of GFP-containing lentiviruses and transduction of CASCs was performed as previously described . In briebr cells in LAA and RAA of patients, a paired parametric t-test was used. A value of p < 0.05 was considered significant.All quantitative results are presented as medians ± IQR. When d’Agostino and Pearson testing revealed that the data were not normally distributed, comparisons between two paired groups were performed in GraphPad version 6.01  using the non-parametric Wilcoxon signed rank test. To compare more than two paired groups, the non-parametric Friedman test with Dunn’s post hoc test was used. For the comparison of the percentage of ALDHIn conclusion, these results indicate that CASCs are predominantly present in the atrial appendages, are committed toward myocardial differentiation, and express several FZD receptors. However, despite the crucial role of Wnt signaling in cardiogenesis and differentiation of PSCs towards cardiac lineages, Wnt signaling is functional but has limited effects on CASCs. Further research is, therefore, necessary to identify the molecular mechanisms that are involved in CASC proliferation and differentiation.The technology for isolating CASCs is patented: EP 2 258 833 B1


Emerging evidence indicates the role of cancer stem cells (CSCs) in tumor relapse and therapeutic resistance in patients with hepatocellular carcinoma (HCC). To identify novel targets against liver CSCs, an integrative analysis of publicly available datasets involving HCC clinical and stemness-related data was employed to select genes that play crucial roles in HCC via regulation of liver CSCs. We revealed an enrichment of an interstrand cross-link repair pathway, in which ubiquitin-conjugating enzyme E2 T (UBE2T) was the most significantly upregulated. Consistently, our data showed that UBE2T was upregulated in enriched liver CSC populations. Clinically, UBE2T overexpression in HCC was further confirmed at mRNA and protein levels and was correlated with advanced tumor stage and poor patient survival. UBE2T was found to be critically involved in the regulation of liver CSCs, as evidenced by increases in self-renewal, drug resistance, tumorigenicity, and metastasis abilities. Mule, an E3 ubiquitin ligase, was identified to be the direct protein binding partner of UBE2T. Rather than the canonical role of acting as a mediator to transfer ubiquitin to E3 ligases, UBE2T is surprisingly able to physically bind and regulate the protein expression of Mule via ubiquitination. Mule was found to directly degrade β-catenin protein, and UBE2T was found to mediate liver CSC functions through direct regulation of Mule-mediated β-catenin degradation; this effect was abolished when the E2 activity of UBE2T was impaired. In conclusion, we revealed a novel UBE2T/Mule/β-catenin signaling cascade that is involved in the regulation of liver CSCs, which provides an attractive potential therapeutic target for HCC. Treatment modalities of HCC commonly include surgical resection, transarterial chemoembolization, chemotherapy, and molecularly targeted drugs2. However, the survival benefits are modest, attributed to frequent tumor recurrence and the development of chemoresistance. Compelling evidence has emerged in support of the vast contribution of cancer stem cells  to the poor survival of cancer patients as they promote chemoresistance, tumorigenicity, and metastasis7. Therefore, it is critical to identify new therapeutic targets against CSCs for the benefit of HCC patients. Integrative comparative genomic analysis has established molecular similarities between liver CSCs and normal liver stem cells8, suggesting the importance of liver CSCs in hepatocarcinogenesis. Identification of critical molecules/pathways involved in the regulation of liver CSCs may prolong the survival of HCC patients. For this purpose, we have employed an integrative analysis of publicly available datasets involving HCC clinical and stemness-related data to select genes that play crucial roles in HCC via the regulation of liver CSCs. This analysis showed that ubiquitin-conjugating enzyme E2 T (UBE2T), among other genes in the interstrand cross-link repair pathway, was drastically upregulated and activated in self-renewing HCC cells compared to control cells.Liver cancer  is one of the deadliest diseases and is the fourth leading cause of cancer-related mortality worldwide9. UBE2T was first identified in CD34+ hematopoietic stem cells, suggesting its regulatory role in the stemness properties of these cells10. More recently, UBE2T was found to regulate the progression of stomach and nasopharyngeal cancers at least in part through modulation of the Wnt signaling cascade12. Although these findings suggest a potential role of UBE2T in regulating stemness, the molecular mechanism by which UBE2T regulates stemness properties is poorly understood. Whether UBE2T regulates liver CSCs also remains unexplored. In this study, we found that UBE2T plays a crucial role in the regulation of liver CSC functions. First, we found that overexpression of UBE2T at both the mRNA and protein levels was associated with advanced tumor stages and poor patient survival. UBE2T was found to regulate the phenotypes of liver CSCs, including self-renewal, drug resistance, tumorigenicity, and metastasis. By tandem affinity purification coupled with mass spectrometry, we identified Mcl-1 ubiquitin ligase E3 (Mule) as the direct binding partner of UBE2T, which regulates ubiquitination of β-catenin. Collectively, our study identified a novel noncanonical Wnt/β-catenin signaling cascade mediated by the UBE2T/Mule complex. Targeting the UBE2T/Mule/β-catenin signaling cascade is a new potential therapeutic approach for HCC.UBE2T is the E2 ubiquitin-conjugating enzyme of the (Fanconi anemia) FA DNA repair pathway, which binds with different E3 ligases to promote the ubiquitination of target proteinsl-glutamine  supplemented with 10% heat-inactivated fetal bovine serum , 100 mg/mL penicillin G, and 50 µg/mL streptomycin  at 37 °C in a humidified chamber containing 5% CO2. Lentiviral infected cells were cultured in complete DMEM medium supplemented with 1 µg/mL puromycin. Culture medium was refreshed every 2 days. All cell lines used in this study were obtained between 2013 and 2016, regularly authenticated by morphologic observation and AuthentiFiler STR (Invitrogen) and tested for absence of mycoplasma contamination . Cells were used within 20 passages after thawing.Human HCC cell lines MHCC-97L , Hep3B (ATCC), Huh7 and PLC/PRF/5 (Japan Cancer Research Bank), MIHA  and HEK293T (ATTC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose and Paired patient HCC and adjacent noncancerous liver tissue specimens were collected at the time of surgical resection from patients at Queen Mary Hospital, Hong Kong, from 1991 to 2013. The use of human clinical specimens was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Consent from patients was obtained. Sample size of human HCC samples was chosen based on G-power calculation.Stable clones of HEK293T cells expressing UBE2T with N-terminally tagged with SFB were harvested and lysed in NETN buffer for 30 mins on ice. After removal of cell debris by centrifugation at 14K rpm for 15 mins, supernatant containing UBE2T-protein complexes was first immunoprecipitated by streptavidin bead slurry (Amersham) for 4 h at 4 °C. The precipitated protein complexes were then eluted by incubation with NETN buffer containing 2 mg/ml biotin (Sigma-Aldrich). The biotin-eluate was then incubated with S protein agarose (Novagen) for 3 h to perform a second round of immunoprecipitation. The protein complexes were eluted by heating at 95 °C for 5 mins. The eluate was subjected to mass spectrometric analysis (LC/MS/MS) at Taplin Mass Spectrometry Facility (Harvard).6 luciferase-labeled MHCC-97L cells (NTC vs shUBE2T) resuspended in matrigel (DMEM: matrigel 50:50) were injected into the left lobes of livers of 5–7-week-old male BALB/C nude mice. Six weeks after implantation, mice were administered with 100 mg/kg D-luciferin via peritoneal injection 5 mins before bioluminescent imaging . Lungs and livers were harvested for ex vivo imaging. The study protocol was approved by and performed in accordance with the guidelines for the Use of Live Animals in Teaching and Research at Hong Kong Polytechnic University. No specific randomization method was used. Sample size of animals was chosen based on significant p values.A total of 1 × 10p values.In vivo evaluation of tumorigenicity was performed with 5–7-week-old NOD-SCID mice by induction of tumor xenografts. Cells were suspended in 1:1 culture medium and BD Matrigel Matrix (BD Biosciences) and subcutaneously injected into the flanks of the NOD-SCID mice, which were kept under observation. Briefly, each mouse received two injections of cells in both flanks, and cells from each experimental group  were injected into different mice. Tumors were harvested at the end of the experiment for documentation. Tumor-initiating cell frequency was calculated using Extreme Limiting Dilution Analysis (ELDA) software. The study protocol was approved by and performed in accordance with the guidelines for the Use of Live Animals in Teaching and Research at Hong Kong Polytechnic University. No specific randomization method was used. Sample size of animals was chosen based on significant t test using Microsoft Office Excel software (Microsoft Corporation). The displayed results showed the means and the standard deviations, and those with p values less than 0.05 were considered statistically significant . All tests are two-sided. Data point is excluded if it deviates from mean with more than three standard deviations. Investigators were not blinded to the group allocation during experiment and when assessing the outcome in all experiments including animal experiments. There is no estimate of variation within each group of data. Variance is similar between the groups that are being statistically compared. Chi-square test was used to evaluate correlations between clinico-pathological parameters and UBE2T expression. Kaplan–Meier survival analysis was used to analyze overall survival and disease-free survival and a log-rank test was used to determine the statistical significance; these analyses were carried out using SPSS 20 software.Statistical significance of qPCR, sphere formation assay, flow cytometry analysis, migration assay and invasion assay results were determined by Student’s Additional experimental procedures are provided in the 14. First, we searched for genes that were upregulated in HCC that were related to liver CSC signatures and poor differentiation. By this means, we were able to identify 2026 genes that were commonly upregulated  and disease-free survival  than patients with low UBE2T expression , venous invasion (p = 0.049), and advanced tumor stage (p = 0.045) -LIHC) data and stemness-related data (GSE5975 and CSCdb))ted Fig. . Gene onted Fig. , among wted Fig. . Out of ted Fig. . In parated Fig. . In the ted Fig. . In addiion Fig. . To furtion Fig. . StatistTo further examine whether UBE2T functionally contributes to the traits of liver CSCs, we performed UBE2T-knockdown and UBE2T-overexpression experiments using a lentivirus-based approach. By Western blotting, we found high expression of UBE2T in a panel of HCC cell lines, including MHCC-97L and PLC/PRF/5 , which is independent of tumor staging  through the establishment of SFB-UBE2T stable clones   Fig. . To furtst) Fig. . Consistst) Fig. . This re10. UBE2T is the E2 ubiquitin-conjugating enzyme of the FA DNA repair pathway, which is reported to participate in the mitomycin-C-induced DNA repair pathway and activate monoubiquitination of FANCD2, which is essential for the activation of the FA core pathway19. Consistently, we found that UBE2T was regulated in HCC at both the mRNA and protein levels and was correlated with poor survival in patients. In concordance with our finding, previous studies on UBE2T reported upregulation of UBE2T in many cancer types, such as liver, prostate, lung, nasopharyngeal, and gastric cancer22, and UBE2T overexpression has been demonstrated to be related to cell proliferation, migration and invasion, and drug resistance23.In an attempt to identify critical molecules/pathways involved in the maintenance and promotion of liver CSCs, we employed an integrative analysis of three publicly available datasets involving HCC clinical and stemness-related data to identify genes that play crucial roles in HCC via the regulation of liver CSCs. Upon analysis, we found enrichment of the interstrand cross-link repair pathway, in which UBE2T was the most upregulated. This result echoed the previous finding that UBE2T was first identified in normal hematopoietic stem cells24. In addition, we revealed that UBE2T also promoted in vitro invasiveness and induced metastasis in a spontaneous metastasis model. We also demonstrated the putative role of UBE2T in regulating the drug sensitivity of HCC cells to doxorubicin. Taken together, our data strongly support the crucial role of UBE2T in maintaining stem cell-like features in HCC.By analyzing the publicly available dataset, we consistently found that UBE2T was upregulated in enriched liver CSCs marked by EPCAM. By using overexpression and knockdown approaches, we found that UBE2T regulates liver CSCs, as evidenced by its effect on self-renewal ability and liver CSC marker expression. Furthermore, we demonstrated that UBE2T regulates CSC frequency and in vivo tumorigenicity in an in vivo limiting dilution assay. This result is consistent with other studies showing the effect of UBE2T on HCC cell growth, probably via regulation of cell cycle regulatory genes, such as p53 and CDK125. However, the exact mechanism of how UBE2T regulates Akt signaling has yet to be elucidated. Given the physiological role of the E2 enzyme, UBE2T was found to promote ubiquitination of target proteins, including FANCD118, BRCA115, and p5316. To understand the molecular mechanism of how UBE2T regulates CSC properties and tumor behavior, it is crucial to identify its direct interacting partner. Based on TAP-MS analysis, we identified Mule as one of the two E3 ubiquitin ligases that interacts with UBE2T among a list of potential interacting partners. Consistently, we found that BARD1 was one of the potential candidate partners of UBE2T15. However, we did not observe changes in BRCA1 expression upon alteration of UBE2T levels. Mule has been reported as a tumor suppressor in HCC that degrades oncogenic PREX2 proteins via ubiquitination26. In addition, Mule expression is suppressed in obesity-induced HCC27. We confirmed the role of Mule as a tumor suppressor, as there was a reciprocal correlation between UBE2T and Mule expression in HCC cells upon overexpression and repression of UBE2T. Furthermore, we demonstrated that UBE2T suppressed the expression of Mule in HCC cells by facilitating degradation of Mule via ubiquitination, and this effect was dependent on its E2 enzymatic activity at the conserved cysteine residue C86. Based on this finding, UBE2T may ubiquitinate Mule directly instead of acting as a mediator to transfer ubiquitin to E3 ligases. The direct action of E2 enzymes in the regulation of substrates via ubiquitination is not impossible. For example, UBE2O was found to regulate the direct degradation of AMPK and BMAL129. This result may suggest a new noncanonical role of UBE2T in cancer. However, the exact interaction between UBE2T and Mule will be further investigated in the future. E2-E3 interactions are important because the interacting E2s dictate the specific type of inter-Ub linkage either at K48 or K63 and thereby determine the fate of the substrate30. We found that UBE2T specifically regulated Mule expression via ubiquitin linkage at K48 and induced ubiquitin-mediated degradation. However, based on our existing data, we cannot exclude the possibility that UBE2T binds to Mule and promote Mule autoubiquitination and degradation. Further investigation is needed to understand how Mule is degraded via interacting with UBE2T.Although an increasing number of reports have shown UBE2T overexpression and its oncogenic functions in various malignancies, the underlying mechanism remains unclear. UBE2T has been found to be associated with the Akt signaling cascade31. In this study, we focused on how Mule regulates β-catenin expression, as the role of β-catenin in the regulation of stemness in HCC has been well reported33. We found that β-catenin expression was increased in Mule-repressed HCC cells compared to control cells. Furthermore, we found that there is a positive correlation between UBE2T and β-catenin in UBE2T-altered HCC cells and that this relationship was abolished when the E2 activity of UBE2T was impaired. The effect of UBE2T on the activation of the Wnt/β-catenin signaling pathway was further confirmed by the localization and activation of β-catenin transcription. Furthermore, we found that GSK3β inhibitor restored the inhibitory effects of UBE2T knockdown on sphere formation and CSC markers expression. This study suggests that β-catenin is an important downstream effector of UBE2T-mediated CSC functions. However, we could not demonstrate that the degradation of β-catenin is mule-dependent using this approach. In addition, we also observed that the effect of CHIR99021 on spheroid growth is more obvious than the expression of liver CSC markers. The discrepancy may be due to the difference in the time points we assayed for spheroid growth and expression of liver CSC markers. These in vitro results were further confirmed in a cohort of HCC clinical samples that showed a positive correlation between UBE2T and β-catenin. This result is consistent with the correlation between UBE2T and β-catenin in nasopharyngeal carcinoma12. Notably, our study has provided more direct mechanistic insight into how UBE2T regulates β-catenin via the degradation of Mule. Finally, we showed that UBE2T may regulate liver CSC function through the regulation of the Wnt/β-catenin signaling cascade.Mule was previously reported to regulate Wnt signaling in colorectal cancer via degradation of β-catenin and EPHB3In conclusion, we demonstrated that UBE2T was upregulated in both liver CSCs and HCCs compared to control cells. The interplay of UBE2T and Mule was found to be critically involved in the regulation of the Wnt/β-catenin signaling cascade (Supplementary Fig. Supplementary MethodsSupplementary Figure LegendsSupplementary Fig. S1Supplementary Fig. S2Supplementary Fig. S3Supplementary Fig. S4Supplementary Fig. S5Supplementary Fig. S6Supplementary Fig. S7Supplementary Fig. S8Supplementary Fig. S9Supplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5