EBs derived from ESC with the ablated gene for HIF1α had abnormally increased neuronal characteristics during differentiation. An increased neural phenotype in −/−Hif1α EBs was accompanied by the disruption of β-catenin signaling together with the increased cytoplasmic degradation of β-catenin. The knock-in of Hif1α, as well as β-catenin ectopic overexpression in −/−Hif1α EBs, induced a reduction in neural markers to the levels observed in wild-type EBs. Interestingly, direct interaction between HIF1α and β-catenin was demonstrated by immunoprecipitation analysis of the nuclear fraction of wild-type EBs. Together, these results emphasize the regulatory role of HIF1α in β-catenin stabilization during spontaneous differentiation, which seems to be a crucial mechanism for the natural inhibition of premature neural differentiation.Extensive research in the field of stem cells and developmental biology has revealed evidence of the role of hypoxia as an important factor regulating self-renewal and differentiation. However, comprehensive information about the exact hypoxia-mediated regulatory mechanism of stem cell fate during early embryonic development is still missing. Using a model of embryoid bodies (EBs) derived from murine embryonic stem cells (ESC), we here tried to encrypt the role of hypoxia-inducible factor 1 The imph groups . Intriguh groups –6. Besidh groups –10. Howeα isoform during ESC differentiation is associated with impaired vasculogenesis and cardiomyogenesis [α in stem cell fate and neural differentiation has been suggested, mostly through a direct interaction between HIF1α and regulatory elements of the crucial developmental pathways activated by Wnt or Notch ligands [α and its interaction with other signaling pathways during stem cell differentiation, especially in early neural differentiation.Cellular responses to hypoxia are mainly mediated via hypoxia-inducible factors (HIF). These transcription factors are master regulators of adaptation processes in response to decreased oxygen supply, including angiogenesis, glycolysis, and red blood cell production . Besidesogenesis , 13. A r ligands , 15. Howα in early neural differentiation using a model of EBs derived from murine ESC. In the EB microenvironment, ESC undergo spontaneous differentiation into all three germ layers and recapitulate early mammalian development [α in the EB microenvironment appears to be a crucial regulatory mechanism for the inhibition of premature neural differentiation. We also address the crosstalk between HIF1α and β-catenin signaling and evaluate the impact of this interaction on neural fate in ESC differentiation.In this study, we highlight the role of HIF1elopment , 16. Our−/−Hif1α cells derived from R1 were kindly provided by Peter F. Carmeliet; Vesalius Research Center, VIB, University of Leuven, 3000 Leuven, Belgium) were cultivated on gelatin-coated dishes in Dulbecco's modified Eagle's medium  supplemented with 15% fetal bovine serum , 100 IU/ml penicillin, and 0.1 mg/ml streptomycin , 1x nonessential amino acid , 0.05 mM β-mercaptoethanol , and 1000 U/ml of leukemia inhibitory factor . ESC were differentiated as described previously [6 cells/ml) was seeded on the surface of silicone mold-made microwells in 1.5% agarose (VWR) to form embryoid bodies (EBs). After 24 hours of aggregate formation (day 0), the EBs were transferred onto an agar-coated dish and cultivated without leukemia inhibitory factor supplementation. All transfection procedures were performed on EBs at this point, prior to their transfer to agar plates. For neural differentiation, 5-day-old EBs (5d) were seeded on gelatin-coated dishes in DMEM/F-12 (1 : 1) medium  supplemented with insulin-transferrin-selenium (Gibco) and antibiotics (specification above) and the EBs were grown for 10 days (5 + 10d) in adherent culture.mESC  primers according to the manufacturer's instructions . qPCR reactions were performed in a LightCycler480 instrument using a LightCycler®480 DNA SYBR Green I Master . The program steps were described previously [−∆cq. The sequences of primers used for individual markers were as follows:Total RNA was extracted from ESC and EBs using the UltraClean Tissue & Cells RNA Isolation Kit . 0.5 eviously . Data weNestin:F-CATACAGGACTCTGCTGGAGG, R-GAGAAGGATGTTGGGCTGAG;Ascl1/Mash1:F-GGTCTCGTCCTACTCCTCCG, R-GCTGCCATCCTGCTTCCAAA;Hif1α:F-TTCTGGATGCCGGTGGTCTA, R-AAACCATGTCGCCGTCATCT;Tcf1/Tcf7:F-CAGCTCCCCCATACTGTGAG, R-TGCTGTCTATATCCGCAGGAA;Lef1:F-TCCTGAAATCCCCACCTTC, R-ACCCGTGATGGGATAAACAG;Tcf3/Tcf7like1:F-CTGAGCAGCCCGTACCTCT, R-AGGGGCCATTTCATCTGTAG;Tcf4:F-ATTTGTGGCCATTGAAGGTT, R-GTCCCTAAGGCAGCCATTC;Axin2:F-GCGCTTTGATAAGGTCCTGG, R-TCATGTGAGCCTCCTCTCTTTT;NeuroD1:F-AACCTTTTAACAACAGGAAGTGGA, R-CTCATCTGTCCAGCTTGGGG;Gapdh:F-AAGGGCTCATGACCACAGTC, R-CATACTTGGCAGGTTTCTCCA.α , mouse monoclonal anti-vinculin , mouse monoclonal anti-β-catenin , rabbit polyclonal anti-phospho-β-catenin , goat polyclonal anti-β-actin (Santa Cruz), goat polyclonal anti-laminB (Santa Cruz), rabbit monoclonal anti-AKT , and mouse monoclonal anti-LewisX/Forse1 . The following secondary antibodies  conjugated with peroxidase were used: goat anti-mouse (A4416), goat anti-rabbit (A0545), and rabbit anti-goat (A4174). The protein levels of neural markers were normalized to the vinculin signal; the level of phosphorylated β-catenin was normalized to the total β-catenin signal. The optical densities of protein bands were quantified by ImageJ software  using the Gel analysis feature, and extrapolated values from the peak area were subjected to statistical analysis.Cells were lysed using SDS buffer  at pH 7.4) at particular time intervals . Proteins were separated using an 8% or 10% SDS-PAGE gel, then transferred onto a polyvinyl difluoride membrane , and blocked in 5% low-fat milk in TBS-T buffer . The detection of selected proteins was then performed using the following specific primary antibodies: mouse monoclonal anti-TUJ1 , mouse monoclonal anti-PAX6 , rabbit polyclonal anti-BLBP , rabbit polyclonal anti-HIF1g to separate the cytoplasmic fraction in the supernatant. The resulting pellet was dissolved in lysis buffer, sonicated for 6 s (low intensity), and centrifuged for 10 minutes to separate the nuclear protein fraction in the supernatant. Fractions with antigen samples were incubated with anti-HIF1α antibody , added to washed magnetic beads covered with A/G protein mixture, and incubated overnight at 4°C. Next day, all samples were washed thoroughly to remove the unbound protein fraction, the beads were boiled for 5 minutes in Laemmli buffer, and the denaturated samples were analyzed by western blot. Each fraction was sampled prior to the addition of magnetic beads for subsequent WB analysis and total protein estimation.For immunoprecipitation analysis, we used a modified protocol employed by the research group of Dr. Bryja. Tris buffer ) was used for gentle cell lysis and protein complex preservation. Two-day-old EBs were lysed for 45 minutes on ice, mixed, and centrifuged for 10 minutes at 1500Prior to immunohistochemistry analysis, cells/EB colonies attached to glass surface were washed 2 times in PBS, fixed in 4% (wt/wt) paraformaldehyde (PFA) for 15 min/4°C, again washed in PBS, and incubated with 1% BSA/0.3% Triton X for 1 hr/RT. After blocking reaction, primary antibodies were dissolved in 1% BSA/0.3% Triton X at 4°C overnight. Next day, samples were washed 3 times in PBS and incubated with secondary antibodies for 1 hr/RT. After this, samples were washed 3 times in PBS and samples were mounted using VectaShield H1000 . Immunofluorescence was visualized with an Olympus CLSM confocal microscope. Primary antibody against TUJ1 was the same as for western blotting; secondary antibody was Alexa Fluor 568 donkey anti-rabbit.μg/well of a 12-well plate), either separately or by cotransfection, if desired: plasmid expressing nondegradable β-catenin mutated on S33 , plasmid overexpressing mouse Hif1α , and empty pcDNA3 plasmid for control transfections. Luciferase reporter assay was used to quantify the β-catenin-mediated transcriptional activation of TCF/LEF binding sites in 2-day-old EBs. Aggregates cultivated in agar microwells (12-well plate) were transfected on day 0 with 0.8 μg of Super8XTOPflash/Super8XFOPflash luciferase plasmid  together with 0.4 μg Renilla luciferase reporter plasmid . 48 hours after transfection, reporter activity was measured using the Dual-Luciferase Reporter Assay System Kit (Promega). Analyses of each experimental condition were performed in triplicates. The luciferase activity of SuperTOP/FOPflash was normalized to the Renilla luciferase signal.EBs were transfected on day 0 using polyethyleneimine . The following plasmid DNAs were used , and statistically significant differences were defined as follows: p < .05 (∗), p < .01 (∗∗), and p < .001 (∗∗∗). Results are presented as mean + SEM.The results were subjected to statistical analyses  revealed the substantially increased expression of genes involved in early neurogenesis, such as Nestin, Neurogenin 1, and ASCL1 . We also checked the morphology of differentiated EB colonies using a light microscope and observed many −/−Hif1α colonies sprouting multiple neurite projections from the periphery, in contrast to wt colonies, where we detected almost no neurites at all (B). These results emphasize the fact that EBs derived from ESC lacking the Hif1α gene generally prefer a neural lineage fate during spontaneous differentiation.We first explored the developmental fate preferred by nd ASCL1 . A neuraons TUJ1 . Immunoh−/−Hif1 EBs compared to wt . Interestingly, we observed changes in the neural phenotype in 5-day-old −/−Hif1α EBs. Using western blot analysis, we found a significant increase in the transcription factor PAX6 in −/−Hif1α EBs, an important and specific regulator of early neurogenesis, and the same increasing trend was also shown by the neural markers BLBP and TUJ1 , a potent angiogenic and neurotrophic factor, and downstream target of HIF1α signaling [−/−Hif1α EBs, especially the level of LewisX, a marker of neural stem/progenitor cells; however, we hardly saw decrease in neural markers in −/−Hif1α EBs when VEGF was added during the formation of EBs . This led us to the conviction that there must be another signaling pathway dependent on HIF1α, which accounts for the blockade of the neural phenotype during EB formation.After one day of cellular aggregate formation , A, aggrand TUJ1 . Importan levels . This clignaling , 19. We β-catenin pathway in spontaneous differentiation and early neurogenesis has already been evidenced by several authors , we observed significantly higher luciferase activity compared to ESC cultivated in normoxic conditions , where we clearly detected the stabilization of HIF1α protein, suggesting hypoxic environment . Howevepromoter . First, nditions ; more imironment . Naturalol level . We alsoet genes . We obseα in neural lineage shift as well as in the upstream regulation of β-catenin, we transfected growing EBs with plasmid containing construct for constitutive expression of β-catenin and analyzed the proteins involved in neural differentiation in 5-day-old EBs. We observed the overall suppression of neurogenesis in −/−Hif1α EBs after β-catenin ectopic overexpression (−/−Hif1α EBs. Also, BLBP protein decreased significantly in −/−Hif1α EBs after β-catenin transfection. The level of TUJ1, a clear indicator of immature neurons, decreased more than 5 times after β-catenin transfection and was, unlike BLBP and PAX6, noticeably lower than after HIF1α transfection, suggesting the relevant impact of β-catenin on neuronal fate. This also confirmed that β-catenin alone, in the absence of stabilized HIF1, is capable of attenuating the neurophenotype during spontaneous differentiation.Since previous results suggested the involvement of HIF1pression . PAX6 prα and β-catenin in spontaneous differentiation by addressing the degradation status of β-catenin in −/−Hif1α EBs. Surprisingly, we found that −/−Hif1α EBs had a markedly higher level of β-catenin phosphorylated on Ser33/37/Thr41, a modification which targets the protein for ubiquitination and thus degradation in the cytoplasm [Hif1α in −/−Hif1α EBs diminished the level of pβ-catenin back to, and even below, the control level (α directly binds to β-catenin, we performed immunoprecipitation analysis from the nuclear fraction of 2-day-old EBs, where stabilized HIF1α was already proven to be involved in β-catenin signaling (α and β-catenin compared to the IgG input control (β-catenin level after HIF1α knockout suggesting that the observed changes in neural phenotype are associated rather with nuclear β-catenin function (α in cells within growing EBs is able to stabilize and bind to β-catenin, which results in enhanced nuclear β-catenin signaling during spontaneous differentiation.We then aimed to search deeper into the connection between HIF1ytoplasm . Importaol level . To checignaling . Nuclear control , A suggefunction , B. Fromα plays a fundamental role in neural fate during the spontaneous differentiation of ESC. We found that EBs derived from ESC with the ablated Hif1α gene underwent an abnormal developmental shift towards the neural lineage after ten days of differentiation in an adherent culture. Importantly, increased neural differentiation appeared in −/−Hif1α EBs after only 5 days of cultivation and the knock-in of Hif1α during EB formation returned the neural phenotype back to that observed in wild-type EBs. Research by Professor Simon's group demonstrated that EBs with a disrupted HIF1 complex exhibited inappropriate mesodermal and hemangioblast differentiation [Hif1α gene exhibit the deregulation of important endodermal and mesodermal lineage markers and impaired cardiomyogenesis [Hif1α abolished this lineage shift [α is a crucial transcription factor involved at the decision crossroad of ESC during spontaneous differentiation and that its stabilization leads to preferential endodermal/mesodermal fate while suppressing differentiation into neuroectoderm.In this study, we present evidence that transcription factor HIF1ogenesis . Lee et ge shift . Overall−/−Hif1α ESC during EB formation. VEGF was described as the main HIF1α-dependent effector responsible for vascular-lineage commitment in EBs primed in 1% hypoxia [α and subsequently induce the strong expression of VEGF, which in turn could have a higher impact on vascular-lineage commitment, unlike our experimental conditions, which reflected the natural “semihypoxic” microenvironment of EBs.We did not observe the return of the abnormal neural phenotype back to normal physiological conditions after VEGF treatment of  hypoxia . We assuα and β-catenin in the regulation of neural lineage blockade during EB maturation. We demonstrated that stabilized HIF1α in EBs naturally protected β-catenin from degradation and increased its nuclear activity which was associated with neural lineage fate blockade. Moreover, immunoblots clearly identified β-catenin-HIF1α protein interaction in the nuclear fraction of wt EBs. There is much evidence in developmental and cancer biology that HIF1α under hypoxic conditions coincides with the Wnt signaling pathway through β-catenin, which has substantial consequences for cellular fate and survival. In colorectal and hepatocellular carcinoma, HIF1α was found to compete with TCF-4 for direct binding to β-catenin enhancing transcriptional HIF1α activity [α negatively regulated skeletal myogenesis through the inhibition of canonical Wnt signaling [α signaling and Wnt canonical target expression and the colocalization of both HIF1α and β-catenin in the nuclear fraction [α was found to enhance Wnt/β-catenin signaling through binding to HRE elements in promoter regions of the β-catenin downstream effectors LEF-1 and TCF-1 [β-catenin signaling activity rather emerged from increased β-catenin nuclear translocation after its stabilization by HIF1α protein in the cytoplasm. These results indicate that the cellular outcome of β-catenin and HIF1α interaction is highly dependent on tissue type, developmental stage, and pathophysiological conditions, as well as oxygen level. One can even speculate that HIF1α seems to have an inhibitory effect on Wnt canonical signaling in carcinogenesis, whereas it rather supports β-catenin signaling and downstream targets during early embryogenesis and neurogenesis.In this study, we show a clear connection between HIF1activity , 23. Durignaling . Howeverfraction . In ESC nd TCF-1 . Unlike,β-catenin in the neural phenotype shift during EB differentiation was demonstrated by several of our results. The knock-out of the Hif1α gene preceded the induction of the neural phenotype as well as the downregulation of β-catenin signaling. Importantly, the transient increase in β-catenin expression was a sufficient signal for the inhibition of the neural differentiation in −/−Hif1α EBs. It appears that the impact of β-catenin overexpression on the reduction of the neuronal marker TUJ1 seems to be higher than after HIF1α reintroduction. These results rather favour β-catenin over HIF1α as the final regulatory effector in neural fate blockade. The involvement of β-catenin in neural fate blockade during ESC differentiation and development has already been described by several authors. The prevention of β-catenin signaling after the loss of the Wnt1/pLRP6 receptor or after the addition of the Wnt/β-catenin pathway inhibitor DKK1 resulted in increased neuroectodermal differentiation and a population positive for TUJ1 cells in differentiating ESC [Pax6 or Ngn2, as well as the emergence of cell immunoreactive for the TUJ1 protein during EB formation; treatment with Wnt1 ligand or GSK3 inhibitors inhibited this neuronal differentiation [β-catenin in neural precursors of the embryonic cortical ventricular zone in vivo caused premature neuronal differentiation [β-catenin and neural differentiation in both EBs and mouse development. EB formation from ESC with abrogated nuclear β-catenin signaling was accompanied by increased differentiation into neurons, as shown by an abundance of TUJ1-positive aggregates. Moreover, embryos originating from this ESC line were abnormally developed with increased markers of neural precursors and neurons such as PAX6 and N-CAM in the inner embryonic layer [β-catenin is in line with our results where −/−Hif1α EBs with downregulated β-catenin signaling exhibited a marked increase in several neuronal markers during EB formation as well as later during adherent differentiation. We assume that, in our case, the observed effect of β-catenin on neural fate blockade during early spontaneous differentiation was rather due to its acting on an endogenous/physiological level and that the attenuation of β-catenin signaling in −/−Hif1α EBs led to increased neurogenesis.The role of ting ESC . Treatmentiation . The focntiation . Other ric layer . The indα results in blockade of proneural genes, including NeuroD1, as well as in the enhancement of β-catenin downstream effector Lef1. A similar regulation was discovered in hypoxic pancreatic β-cells, where the expression of the NeuroD1 gene was suppressed by stabilized HIF1α, while the knockdown of Hif1α led to its upregulation [β-catenin may positively regulate HIF1α signaling by direct interaction, which was also documented elsewhere [β-catenin alone has the potential to regulate stem cell fate in EBs lacking HIF1α. The upregulation of transcription factor PAX6 in early neural progenitors is associated with the increased expression of important proneuronal genes, including Ascl1 or NeuroD1 [β-catenin was documented by several other authors [β-catenin/LEF1 fusion protein in the cortical neural precursors of mouse embryo delayed the expression of several neurogenic genes, including PAX6, while the conditional ablation of β-catenin accelerated the expression of PAX6 [Lef1 gene expression regulated by β-catenin seems to be the crucial upstream stimulus inhibiting the activation of the neuronal program in EBs. Still, the exact molecular mechanism underlying the neural inhibitory effect of HIF1α and/or β-catenin in EBs should be clarified by further research.Overall, our data clearly show that the stabilization of HIF1gulation . Additiolsewhere –24. Howe NeuroD1 . Therefo authors , 28. Mor of PAX6 . Thus, aα blocks the degradation of β-catenin in the cytoplasm and increases β-catenin nuclear activity, which results in the natural developmental blockade of neural differentiation in ESC (α and β-catenin are fundamental factors in developmental as well as cancer biology, we believe that these in vitro findings can contribute to understanding the mechanisms of neural differentiation under both physiological and pathophysiological conditions.In summary, we hereby demonstrate that the stabilization of HIF1n in ESC . These r


Paenibacillus larvae). Overall, our results indicate that levels of GOX and Def1 are not induced in response to pathogenic infections. We therefore conclude that GOX and Def1 are highly constitutive and co-opted as mechanisms of social immunity, and these factors should be considered when investigating immunity at the individual and colony level in social insects.Honeybees use a variety of defence mechanisms to reduce disease infection and spread throughout the colony. Many of these defences rely on the collective action of multiple individuals to prevent, reduce or eradicate pathogens—often referred to as ‘social immunity’. Glucose oxidase (GOX) and some antimicrobial peptides (e.g. defensin-1 or Def1) are secreted by the hypopharyngeal gland of adult bees on larval food for their antiseptic properties. Because workers secrete these compounds to protect larvae, they have been used as ‘biomarkers’ for social immunity. The aim of this study was to investigate if GOX and Def1 are induced after pathogen exposure to determine whether its production by workers is the result of a collective effort to protect the brood and colony in response to a pathogen challenge. Specifically, we quantified GOX and Def1 in honeybee adults before and after colony-level bacterial infection by American foulbrood ((AFB),  When immunity ,5. Theseimmunity ). For eximmunity  and hygiimmunity , while wimmunity . In termimmunity ,11, whilimmunity .Figure One set of mechanisms of social immunity is based on the use of antimicrobial compounds to reduce the probability of pathogens entering the nest or spreading among individuals . These ad-glucose into d-gluconic acid and hydrogen peroxide (H2O2) through an oxidation reaction [2O2 can prevent bacterial and fungal growth [2O2 production are positively correlated with the inhibition of pathogen development in larval food of honeybees  and defensin-1 (Def1) ,24,25. Greaction . The antl growth ,27. Eveng. honey  and royaPaenibacillus larvae). Specifically, we quantified levels of enzymatic activity of GOX and gene expression of GOX and Def1 for nurse bees in healthy and AFB-infected honeybee colonies to test whether or not these compounds are induced in nurse bees after colony-level exposure to pathogens. Honeybee larvae become infected with AFB by the transfer of spores via contaminated food, which come from spores left behind in re-used cells or from adult bees carrying them and then contaminating brood food [In this study, we investigate whether GOX activity and Def1 expression are activated in nurse bees after a colony-level challenge with the causative agent of the bacterial brood disease American foulbrood ((AFB), ood food . Therefoood food . The alt2.2.1.A. mellifera for eight experimental colonies (five for AFB treatment and three controls), each containing three brood frames and one honey frame, a laying queen and equally sized adult bee populations. The queen of one of the control colonies died during the first week of the experiment, we therefore used two colonies as controls, and five colonies for the bacterial treatment using the AFB solution. Hives were located in an isolated area to prevent transmission of P. larvae–a.k.a. AFB–to healthy colonies. For bacterial infection treatment, we collected ca 30 AFB ‘scales’ or the dried remnants of symptomatic, dead larvae from an infected colony in Mocksville, NC, USA that was obtained from the North Carolina Department of Agriculture. Following standard protocols, scales were macerated with sterile deionized water, heat-shocked for 10 min at 80°C, and diluted in a 10% sucrose solution at a final concentration of 1.5 × 107 spores ml−1. Three control colonies just received the sucrose solution. To infect the five treatment colonies, bees were shaken off each frame and combs were sprayed with a 5 ml solution on each side . AFB infections were quantified weekly throughout the experiment by counting the total number of symptomatic cells in the three brood frames for each colony.We performed the experiment in Raleigh, NC, USA, using five frame nucleus colonies of ecipe in ). Beforen week 4 . We repe2.2.2O2). In the presence of peroxidase, resorufin is produced in a 1 : 1 stoichiometry. Therefore, GOX activity was estimated as the maximum velocity (Vmax) of the rate of production of the red compound at an absorbance of 560 nm for 60 min. Vmax was calculated as the slope of the linear phase of the reactions using the software KCJunior v. 1.22 . Each reaction was repeated three times to estimate errors associated with sample preparation.We quantified the enzymatic activity of GOX from heads of 12 individuals per colony for each treatment and time period in our experiment. Head tissue was homogenized on ice in phosphate buffered saline (pH 7.4) using pestles. To quantify GOX activity, we used a peroxidase assay kit (ThermoFisher A22188) that produces the red compound resorufin with the presence of hydrogen peroxide  values normalized to expression of β-actin  was used as the calibrator, and so normalized expression values were made relative to this.We quantified gene expression of C CTG C; ) and Def AAA CG; ) from th ATC CA; ) and calrotocols . The ave2.4.GOX and Def1). Samples that showed standard deviations greater than 2 among replicates were inspected for outliers. We removed single values deviated more than ±2 from the other two replicates, or removed the whole sample if the two remaining replicates after outlier removal still deviated by more than 2. Generalized mixed linear models were built using ‘treatment’ and ‘time’ as fixed effects, and the variable ‘colony’ was included as a random effect. For the enzymatic GOX data, ‘plate’ was also included as a random effect to account for any variability from the specific groups of samples that was analysed in the same plate (31 samples and 1 negative control). Models for 7 and 14 day old bees were analysed separately. Linear models were analysed using the maximum-likelihood approach of the lme function of the R package nlme [We estimated the average GOX enzymatic activity of the three technical replicates for enzymatic activity (GOX) and the expression data  or 14 day old  bees  and no effect of AFB treatment  , with bees at T1 having lower GOX expression than bees at T0 or T2. A similar finding was determined for Def1, with no significant effect for the interaction between time and treatment  or AFB treatment . There was an effect of time , with significantly lower Def1 expression levels at T1 when compared with T0, and T2.The number of larval cells with overt AFB symptoms increased over time after initial infections in all experimental colonies, while control colonies showed no sign of diseased larvae . We did 52) bees . For gen= 0.936) . However4.GOX and Def1 were unaffected at the colony level. The fact that Def1 was not induced by exposure to AFB is not entirely unexpected. Even though Def1 is an antimicrobial peptide that responds to bacterial infection [GOX and Def1 expression levels during T1 for both control and treatment colonies. We are uncertain about the causes of this result and it is probably owing to various, wider environmental factors that were not measured in this experiment.GOX enzymatic activity and expression levels were not induced in 7 day old or 14 day old bees after honeybee colonies were challenged with a bacterial pathogen (AFB). Even though all infected colonies showed symptoms of disease, enzymatic production of GOX and transcription of nfection , AFB doenfection ,11, evennfection . Our resOur results indicate that even though GOX and Def1 are secreted into larval food for antiseptic properties, they remain constitutively expressed and are not increased owing to pathogen pressure. These findings are supported by previous work in GOX , and othDef1 expression have often been analysed as parameters of social immunity, as it has been hypothesized to prevent diseases through colony-food sterilization [GOX activity and lization . While tlization . GOX haslization , on graslization  and in olization , which slization , and clalization , wherebyTaken together, our results and the evidence of widespread use of GOX and Def1 suggest that some of the antimicrobial compounds produced by social insects can be co-opted to function as mechanisms of social immunity. Because social insects have stronger selective pressure of pathogen spread, they rely on a wide range of defences that include internal and external compounds, behavioural and physiological defences, constitutive or inducible mechanisms that benefit individuals and groups (


Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer.Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation.Hypomorphic Apc mice -induced HCC. uc.158− was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158− expression in human malignant hepatocytes. uc.158− expression was increased in We showed that uc.158− is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics. The Wnt/β-catenin pathway is frequently activated in liver cancer. The understanding of the molecular mechanisms of Wnt deregulation is necessary to develop more specific and effective therapeutics.Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs that harbour ultraconserved elements and are altered in human carcinomas.We demonstrate that T-UCRs are downstream mediators of the Wnt/β-catenin pathway in three different species, and we provide evidence of their role as competing endogeneous long non-coding RNAs.We have identified a mediator that is downstream of the Wnt/β-catenin pathway and specific for cancer development, improving the development of effective and selective novel therapeutics.We have studied several cohorts of human liver cancers and observed that deregulation of uc.158− is shared between hepatocarcinoma and cholangiocarcinoma, providing interesting insights into the molecular biology of these diseases and the implication for clinical trial design.Hepatocellular carcinoma (HCC) is the sixth most common malignancy in the world.CTNNB1) in 33% of human HCC. In addition, mutations of CTNNB1 are mutually exclusive with mutations in AXIN1.The Wnt pathway is frequently activated in HCC.Non-coding RNAs (ncRNA) are implicated in liver carcinogenesis.Apc580s/580s) Apcfl/fl mice were used and compared with Apc+/+ mice. Model generation and demonstration of the activation of Wnt/β-catenin pathway in this HCC model have already been reported.Apcfl/fl (tumour with activation of Wnt/β-catenin) and Apc+/+ . TAA rat tumour model was used as a model of rat cholangiocarcinoma (CCA). Generation of this model and activation of Wnt pathway in this tumour model have already been reported.Axin1fl/fl/Cre mouse model was used to investigate the effect of Axin1 disruption in the hepatocytes. This model was generated by crossing mice homozygous for the Axin1fl/fl allele with AhCre mice, as previously reported.Axin1fl/fl/Cre and Axin1+/+/Cre mice. Diethylnitrosamine-induced HCC: C57BL/6, 14 days old male mice were intraperitoneally injected with diethylnitrosamine (DEN) (40 μL of a solution containing 10 mg/mL DEN in PBS:80 mg/kg). Mice were sacrificed at 36 weeks after DEN injection; liver was assessed for macroscopic tumour number and size, and normal liver and tumour samples were placed into RNA later, snap-frozen in liquid nitrogen or fixed in neutral buffered formalin overnight at 4°C for the assessment of β-catenin.All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal works were approved by the appropriate committees. Experiments were carried out in compliance with UK Home Office Animal (Scientific Procedures) Act 1986 and the EU Directive 2010. Hypomorphic mutant , Apc+/+ (n:3) and DEN-treated mice (n:3) were used. mRNA was purified from total RNA after removal of rRNA . Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias, using a random priming method. The labelled cRNAs were hybridised onto the mouse LncRNA Array V.2.0 , which also included probes for protein-coding RNAs. The arrays were scanned by the Agilent Scanner G2505C . Agilent Feature Extraction software (V.11.0.1.1) was used to analyse the acquired array images. Quantile normalisation and subsequent data processing were performed using the GeneSpring GX V.11.5.1 software package (Agilent Technologies). After quantile normalisation of the raw data, differentially expressed LncRNAs with statistical significance between groups  were identified through volcano plot filtering, with a threshold fold change ≥2.0 and p≤0.05.RNA from the liver of The human CCA tissues were collected under approval of the Ethical Committee for Clinical Research of Padua and the University of Edinburgh ethics committee. Healthy liver was provided by the NHS Scotland Research (NRS) BioResource, NHS Lothian. All tissues were collected with informed consent. HCC tissues were provided by the University Hospital Basel and University of Ferrara after appropriate ethical approval. For the CCA tissue microarray (TMA), a retrospective series (1990–2011) of 102 surgically resected primary biliary cancers, including 85 CCA and 17 gall bladder carcinoma specimens, were retrieved from the formalin-fixed paraffin-embedded (FFPE) archives of the Department of Pathology and Diagnostics and the ARC-NET biobank of the University and Hospital Trust of Verona after appropriate ethical approval. CCAs were classified according to WHO 2010 as intrahepatic cholangiocarcinoma  and extrahepatic cholangiocarcinoma . Five 1 mm core TMAs were assembled. Additional 14 non-neoplastic controls were included in the TMAs. Three tissue cores per case were analysed.Statistical analyses were performed by GraphPad Prism 6 . Results are expressed as mean±SD, unless indicated otherwise. Groups that were normally distributed were compared with either a two-tailed Student's t-test  or using one-way analysis of variance to compare multiple groups. Non-parametric data were analysed using a Wilcoxon-Mann-Whitney U test when comparing two groups. Significance was accepted when p<0.05. *p:0.05–0.01; **p:0.009–0.001; ***p<0.001.Additional methods can be found in the online 10.1136/gutjnl-2016-312278.supp1supplementary dataApcfl/fl) were previously shown to develop HCC with activation of the Wnt/β-catenin pathway .Apcfl/fl and control Apc+/+ mice. Twenty-two T-UCRs were aberrantly expressed  tumours per liver with no nuclear activation of β-catenin (see online Apcfl/fl-HCC from DEN-induced HCC (Apcfl/fl and control Apc+/+ mice (RBPM39) gene. Given the fact that uc.158− does not overlap protein-coding genes, and that intergenic lncRNAs have been shown to be more stable,Hypomorphic Apc mice , while Huh-7 cells are known to be wild type (WT) for CTNNB1 and other genes of the Wnt pathway.AXIN1Axin1. These animals (Axin1fl/fl/Cre) were shown to develop microscopic foci of HCC in the absence of nuclear localisation of β-catenin.Axin1fl/fl/Cre in comparison with WT mice  than in WT controls (Apc deletion (AhCre+Apcfl/fl), which presents an increase in liver weight and proliferation, but not tumours. Indeed, we noticed that uc.158− expression was not changed following activation of the Wnt/β-catenin pathway in absence of cancer  specifically designed to interact with the conserved sequence of uc.158−. GAP1 and GAP3 provided the best inhibition .CTNNB1 mutations.It is becoming clear that a large number of lncRNAs have a determining role in regulating normal cell physiology as well as a variety of diseases including cancer.The mechanism of function of T-UCRs is still not clearly elucidated. Previous evidence suggests that they may act as decoy molecules for miRNAs.


The CNT–Al2O3 hybrid was synthesised by growing CNTs on Al2O3 particles via the chemical vapour deposition method. The CNTs were strongly attached onto the Al2O3 particles, which served to transport and disperse the CNTs homogenously, and to prevent agglomeration in the CNTs. The experimental results demonstrated that the CNT–Al2O3 hybrid-filled epoxy nanocomposites showed improvement in terms of the fracture toughness, as indicated by an increase of up to 26% in the critical stress intensity factor, KC1, compared to neat epoxy.The current study investigated the effect of adding a carbon nanotube–alumina (CNT–Al These hybrid compounds provide intriguing challenges and opportunities for chemists, physicists, biologists, engineers, and material scientists ,2. The h2) ,6, enhan2) [2) , improve2) [2) , and inc [2) [2) .2O3) particles and used as filler in polymer composites [2O3—such as its high level of hardness, refractoriness, excellent dielectric and good thermal properties—make it a suitable choice for hybridization with CNTs [2O3 particles, leading to agglomeration of the CNTs during the fabrication of the polymer composites. To overcome this problem, the CNTs and Al2O3 were hybridized via chemical vapour deposition (CVD) to improve the dispersion of the CNTs without damaging their structure and to maintain the properties of the CNTs. Furthermore, other advantages of CVD are its simplicity and ability to produce massive quantities of CNTs [Recently, CNTs have been hybridized with alumina  and a high-resolution transmission electron microscope (HRTEM) to observe the morphologies of the CNTs and Al2O3 particles. The aim of this study was to investigate the fracture toughness of epoxy nanocomposites. The dispersion of the CNTs in the epoxy nanocomposites were examined using FESEM and HRTEM. Possible explanations for the difference between the CNT–Al2O3 hybrid compound-filled epoxy and the physically-mixed CNT–Al2O3-filled epoxy will be discussed.In this paper, a CNT–Al2O3 hybrid compound was synthesized using the chemical vapour deposition (CVD). The catalyst was prepared by precipitating nickel nitrate hexahydrate (Ni(NO3)2·6H2O) on aluminium (Al) powder in a solution of sodium hydroxide (NaOH). The catalyst was then dried at 110 °C for 2 h and calcined at 900 °C to form a NiO–Al2O3 complex. The NiO–Al2O3 complex was subjected to a reduction process under hydrogen gas at 400 °C for 2 h, followed by the growth of the CNTs onto the Al2O3 particles under a methane and nitrogen gas atmosphere at a ratio of 1:7 at 800 °C for 30 min in a horizontal tube furnace. The reagent grade Ni(NO3)2·6H2O, Al powder, and NaOH were obtained from Merck & Company. CNTs and Al2O3 were physically mixed using a ball milling machine for 48 h at 20 rpm for comparison with the CNT–Al2O3 hybrid compound. Pure CNT  and Al2O3  were mixed at a ratio of 12:100 based on the energy dispersive X-ray  analysis reported in a previous paper [A CNT–Alus paper .2O3 hybrid compound and the physically-mixed CNT–Al2O3 were analysed using a field emission scanning electron microscope  and a high-resolution transmission electron microscope .The morphologies of the CNT–Al2O3 hybrid compound and physically mixed CNT–Al2O3 were dispersed in epoxy resin diglycidyl ether of bisphenol A (DGEBA) using a sonicator  at a frequency of 25 kHz for 30 min. The curing agent trimethylhexamethylenediamine (TMD) was then added to the mixture with a mass ratio to epoxy resin of 6:10. The mixture was placed in a vacuum at 76 cm Hg pressure for 30 min to remove any trapped air, and was then poured into a silicon mould. Finally, the epoxy composites were cured at 120 °C for 1 h. 1.0, 3.0, and 5.0 wt % of the CNT–AlKC1, for the epoxy nanocomposites according to the ASTM D5045 standard [KC1 was calculated using Equation (1):P is the maximum load; B is the specimen thickness; W is the specimen width; and Single-edge notch three-point bending (SEN-3PB) tests were conducted to obtain the fracture toughness in terms of the critical stress intensity factor, standard . The tesThe fracture surface of the epoxy nanocomposites was analysed using FESEM after coating with a 5–10 nm layer of Au-Pd by sputtering. The nano-scale morphologies of the Epoxy/HYB and Epoxy/MIX were analysed using HRTEM. The HRTEM samples with a thickness of 50 nm were prepared by cryo-ultramicrotomy using a microtome by Leica .2O3 particles. In addition, the CNTs were attached and distributed around the Al2O3 particles. The diameter of the grown CNT was approximately 10–30 nm. Meanwhile, the SEM image of the MIX illustrated poor distribution of the CNTs and Al2O3 particles , and the results are presented in KC1 following the addition of the HYB, but it was not significant for MIX. The fracture toughness of the Epoxy/HYB approached a maximum at 5 wt % HYB with 2.06 MPa·m1/2, representing an increase of over 26% in comparison to that of the neat epoxy. Meanwhile, the fracture toughness of the Epoxy/MIX approached a maximum at 5 wt % MIX with 1.79 MPa·m1/2, representing only a slight increase which was not significant compared to that of the neat epoxy. A few factors contributed to the enhancement of the fracture toughness, such as the hybrid filler architecture and morphology of the epoxy nanocomposites. The hybrid filler architecture played a crucial role in improving the fracture toughness of the epoxy nanocomposites. In this epoxy nanocomposite system, the hybrid filler combination consisted of rigid Al2O3 particles and CNTs which possessed extraordinary strength and modulus, leading to an improvement in the fracture toughness. As reported previously in many studies, CNTs possess a strength and modulus of about 63 GPa and 1000 GPa, respectively, which are far higher than the strength and modulus of neat epoxy [2O3 particles helped the CNTs to form a network by preventing the CNTs from agglomerating and simultaneously working to transport the dispersed CNTs. Thus, the CNT network formed by the HYB provided a more effective load transfer between the epoxy matrix and the filler, thereby improving the fracture toughness of the epoxy nanocomposites. In addition, the homogenous dispersion of CNTs also contributed to a wider surface for bonding with the epoxy matrix. A similar trend of the increasing fracture toughness by using CNTs as the filler was also reported by Shokrieh et al. [2O3 particles separately—inclined the CNTs to agglomerate due to the van der Waals forces and the high surface area. This agglomeration of CNTs reduced the surface area of the CNTs and introduced stress concentrations, which consequently reduced the ability of the epoxy nanocomposites to withstand the load.The fracture toughness of neat epoxy, Epoxy/HYB, and Epoxy/MIX was determined by the stress intensity factor, at epoxy ,21. Hench et al. . As disc2O3 particles were evenly surrounded by the CNTs, and the number of unattached CNTs in the epoxy matrix was minimal. The fracture surface of the Epoxy/MIX demonstrated that the MIX was not homogenously dispersed in the epoxy matrix, as shown in 2O3 particles were separated and some CNTs were detached from the Al2O3 particles. This was due to the weak interaction bonding between the CNTs and Al2O3 particles in the MIX as a result of the sonication process at high frequency vibration. The agglomeration of the Al2O3 particles and the CNTs in the epoxy matrix could be seen clearly from the close-up observation in The fracture surfaces of the Epoxy/HYB and Epoxy/MIX were observed using FESEM in order to understand the improvement in the fracture toughness. It was observed that the fracture surface of the neat epoxy appeared to be smooth, as shown in 2O3 particles. The formation of the cleavage planes was due to the change in the direction of the crack propagation as it crossed the CNTs or Al2O3 particles. Thus, this bridge effect—which prevented the cracks from opening—improved the fracture toughness of the epoxy nanocomposites. The decreased size of the cleavage plane made it more difficult for the cracks to propagate, as the CNTs and Al2O3 particles distorted the path of the crack tip. It could be seen that the size of the cleavage plane of the Epoxy/HYB was much smaller than that of the Epoxy/MIX. This means that more energy was absorbed by the generation of numerous micro cracks dissipated in the Epoxy/HYB during the fracture process.Based on the surface roughness, it could be seen that the neat epoxy had river stripe marks with minimal deformation. The addition of the HYB and MIX into the epoxy matrix resulted in improved energy dissipation and toughening through the formation of many micro-cracks with cleavage planes on their fracture surfaces. The network of cleavage steps formed the cleavage planes, and each plane consisted of at least CNTs or Al2O3 particles were dispersed together in the epoxy matrix. The CNTs were well-dispersed around the Al2O3 particles, and there was less agglomeration of CNTs. The Al2O3 particles functioned as transportation for the CNTs to disperse homogenously and to prevent the CNTs from agglomerating. Meanwhile, the Epoxy/MIX demonstrated poor dispersion and agglomeration of the CNTs and Al2O3 particles, as shown in 2O3 particles appeared to disperse alone instead of helping the CNTs to disperse in the epoxy matrix. Therefore, it could be concluded that the dispersion of the CNTs in the Epoxy/HYB composite was dependent on the Al2O3 particles, while the dispersion of the CNTs in the Epoxy/MIX was not dependent on the Al2O3 particles.HRTEM was carried out to further evaluate the dispersion of the HYB and MIX in the epoxy nanocomposites. The morphologies of the Epoxy/HYB and Epoxy/MIX were examined at magnifications of 19,500× and 29,000×. KC1 of the neat epoxy (0.9 MPa·m1/2), which resulted in fracturing of the epoxy matrix. The propagation of cracks in the Epoxy/HYB was delayed by the better dispersion of fillers compared to the Epoxy/MIX. As shown in The failure modes of the epoxy composites were the result of various factors and mechanisms, such as stress transfer between the matrix and the reinforcement, interface debonding, stress concentration points, and crack propagation . The strThis study investigated the effect of HYB and MIX on the fracture toughness of the epoxy nanocomposites at various filler loadings. Based on the experimental findings, it can be concluded that the incorporation of HYB and MIX in the epoxy composites resulted in an improvement in the fracture toughness of the epoxy nanocomposites. The Epoxy/HYB showed a higher fracture toughness compared to the Epoxy/MIX. The fracture toughness of the Epoxy/HYB was enhanced by about 26%, whereas the fracture toughness of the Epoxy/MIX was enhanced by about 9%. When the fracture surface was observed under FESEM and HRTEM, it could be seen that the HYB was homogenously dispersed and there was some agglomeration of MIX in the epoxy matrix. It can be concluded that the better dispersion of HYB in the epoxy matrix led to higher fracture toughness.


EMT), an indispensable programme for driving renal fibrosis. As an adapter protein, four and a half LIM domain protein 2 (FHL2) acts as a coregulator of β‐catenin in several other cell types. To determine whether FHL2 affects β‐catenin signalling and thus is involved in tubular EMT, we examined its expression and function in the process of TGF‐β1‐induced EMT. FHL2 mRNA and protein were induced by TGF‐β1 in rat tubular epithelial cells (NRK‐52E), an effect that intracellular Smad signalling was required. Ectopic expression of FHL2 inhibited E‐cadherin and enhanced α‐smooth muscle actin (α‐SMA) and fibronectin expression, whereas knockdown of FHL2 partially restored E‐cadherin and reduced α‐SMA and fibronectin induction stimulated by TGF‐β1. Overexpression of FHL2 increased β‐catenin dephosphorylation (Ser37/Thr41), nuclear translocation and β‐catenin‐mediated transcription and up‐regulated expression of β‐catenin target, EMT‐related genes, such as Snail, Twist, vimentin, plasminogen activator inhibitor‐1 and matrix metalloproteinase‐7. Conversely, knockdown of FHL2 increased β‐catenin phosphorylation (Ser33/37/Thr41), decreased its nuclear translocation and inhibited β‐catenin‐mediated transcription and target genes expression. TGF‐β1 induced a FHL2/β‐catenin interaction in NRK‐52E cells, especially in the nuclei. In a mouse model of obstructive nephropathy, FHL2 mRNA and protein were induced in a time‐dependent fashion, and the extent and pattern of renal β‐catenin activation were positively correlated with FHL2 induction. Collectively, this study suggests that FHL2, via modulating β‐catenin signalling, may implicate in regulation of TGF‐β1‐mediated tubular EMT and could be a potential therapeutic target for fibrotic kidney disease.β‐Catenin signalling plays an important role in regulating tubular epithelial‐to‐mesenchymal transition ( More than 10% of the population worldwide suffers from chronic kidney disease (CKD), and end‐stage renal failure is an ineludible outcome in a considerable proportion of patients with CKD As myofibroblast is well known as the main type of ECM‐producing cells in the interstitium of fibrotic kidney, the origin of myofibroblasts during renal fibrosis has been intensely investigated and debated for several years Previous studies present that activation of β‐catenin signalling induces tubular EMT and enhances the magnitude of EMT induced by TGF‐β1 It has been proposed that four and a half LIM domain‐only protein 2 (FHL2) is implicated in Wnt/β‐catenin signalling pathway The data in this study indicate that expression of FHL2 is up‐regulated in the tubular epithelial cells treated with TGF‐β1 and in the fibrotic kidney after obstructive injury. And the further investigations reveal that FHL2 functionally implicates in regulating tubular EMT, and it functions, at least partially, through modulating the activity of β‐catenin signalling.Rat proximal tubular epithelial cell (NRK‐52E) was provided by the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . The cells were cultured in Dulbecco's modified Eagle's medium–Ham's F12 medium supplemented with 10% foetal bovine serum . Serum‐starved cells were incubated with recombinant TGF‐β1  for different periods of time at the concentration of 2 ng/ml except as otherwise indicated. The cells were then harvested for real‐time RT‐PCR and Western blot analyses. In some experiments, cells were pre‐treated with several chemical inhibitors at the concentrations specified for 30 min., followed by incubating with or without 2 ng/ml TGF‐β1. PD98059 (MEK1 inhibitor) and Wortmannin (PI3K inhibitor) were provided by Sigma‐Aldrich , SP600125 (JNK inhibitor) was provided by Merck (Germany), and SB202190 (p38 MAPK inhibitor) and SB431542 (TGF‐β1 type I receptor inhibitor) were provided by R&D Systems.Plasmid transient transfection was performed as described previously NRK‐52E cells were transiently transfected with negative control small interfering RNA (siRNA) or FHL2 siRNA  for 48 hrs using Lipofectamine 2000 reagent according to the manufacturer's instructions as described previously Total RNA was extracted using TRIzol RNA isolation system (Invitrogen). The first strand of cDNA was synthesized using 2 μg of RNA in 20 μl of reaction buffer by reverse transcription using ReverTra Ace  and oligo(dT)12–18 primers at 42°C for 30 min. Cell lysates and kidney tissue homogenate were prepared, and Western blot analysis of protein expression was performed using routine procedures as described previously For preparation of nuclear protein, NRK‐52E cells were collected and washed twice with cold PBS. Nuclear protein was extracted using NE‐PER Nuclear and Cytoplasmic Extraction Reagents, on the basis of the protocols provided by the manufacturer , as described previously Immunoprecipitation was performed using an established method Immunofluorescence staining of cultured NRK‐52E cells was carried out using a routine protocol Immunohistochemical staining of paraffin‐embedded mouse kidney sections (3 μm thickness) was carried out by an established procedure as described previously Renilla reniformis luciferase driven under TK promoter  was also cotransfected. Luciferase assay was carried out by utilizing a dual luciferase assay system kit according to the protocol specified by manufacturer (Promega). Relative luciferase activity (arbitrary units) was shown by the fold induction over the controls.The effect of FHL2 on β‐catenin‐mediated transcription was determined using the TOPflash TCF reporter plasmid containing two sets of three copies of the TCF binding site upstream of the thymidine kinase (TK) minimal promoter and luciferase open reading frame (Millipore). NRK‐52E cells were cotransfected with TOPflash plasmid (1 μg) and either FHL2 expression vector or empty vector pcDNA3. For normalizing the transfection efficiency, an internal control reporter plasmid (0.1 μg) Male CD‐1 mice weighing 20–22 g were provided by the Shanghai Experimental Animal Center . Unilateral ureteral occlusion (UUO) was performed using an established protocol, as described previously P value ˂ 0.05 was considered statistically significant.All data examined were presented as mean ± S.E.M. Statistical analysis of the data was processed by SigmaStat software . Comparisons between groups were made using one‐way analysis of variance, followed by the Student–Newman–Keul's test. A We investigated FHL2 regulation by TGF‐β1 in NRK‐52E cells. Real‐time RT‐PCR analyses demonstrated that TGF‐β1 markedly induced FHL2 mRNA levels  and partially restore E‐cadherin expression suppressed by TGF‐β1 . In addition, down‐regulation of FHL2 partially prevented fibronectin and α‐SMA overproduction in response to TGF‐β1 stimulation . Hence, it seems that knockdown of FHL2 expression partially inhibits tubular EMT induced by TGF‐β1.To further determine the potential role of FHL2 in tubular EMT, FHL2 expression was down‐regulated by transfection with specific FHL2 siRNA in NRK‐52E cells. Transfection with FHL2 siRNA not only led to a distinct decrease in endogenous FHL2 mRNA and protein levels Fig. A and B, via affecting its phosphorylation. Given that the nuclear translocation of β‐catenin is an indispensable procedure for it to control its target genes transcription in nucleus, FHL2 could be an endogenous mediator for modulating the activity of β‐catenin signalling during the process of tubular EMT.Previous studies demonstrate that activation of β‐catenin signalling can induce tubular EMT For assessing the functional consequence of FHL2‐regulated β‐catenin signalling activation in tubular epithelial cells, we examined the β‐catenin‐mediated transcriptional activity in a TOPflash luciferase reporter system. We found that either exogenous FHL2 or TGF‐β1 markedly induced β‐catenin‐mediated transcriptional activity in NRK‐52E cells Fig. A. A combversus lane 1) and hinder their induction stimulated by TGF‐β1 to varying degrees . Therefore, it is suggested that through regulating β‐catenin‐mediated transcriptional activity and β‐catenin‐targeted gene expression, FHL2 plays an essential role in governing tubular EMT.Given that the activation of β‐catenin signalling plays a crucial role in mediating tubular EMT, we further examined the effect of FHL2 on the expression of β‐catenin target, EMT‐related genes, including Snail, Twist, vimentin, PAI‐1 and MMP‐7. We found that forced expression of FHL2 was able to notably induce these genes expression in NRK‐52E cells Fig. C. ConverFor exploring the potential reason that FHL2 affects β‐catenin signalling in tubular epithelial cells, we examined the interaction of FHL2 with β‐catenin under TGF‐β1‐stimulated condition using a coimmunoprecipitation approach. Incubation of NRK‐52E cells with TGF‐β1 induced FHL2 to interact with β‐catenin, as depicted by increased FHL2/β‐catenin complex formation after TGF‐β1 treatment Fig. A and B. in vivo, we investigated FHL2 expression in obstructive nephropathy, a mouse model of renal interstitial fibrosis. Immunohistochemical staining exhibited that compared with the sham controls , active β‐catenin and β‐catenin. Expectedly, Wnt/β‐catenin signalling was dramatically activated, as illustrated by a time‐dependent increase in active β‐catenin level in the obstructed kidneys (Fig. via regulating β‐catenin activity in this model.To further uncover the correlation between FHL2 induction and Wnt/β‐catenin signalling activation eys Fig. D and E. eys Fig. D, as preeys Fig. F, suggesOur findings presented in current study suggest that FHL2, an adaptor protein containing LIM domains only, through modulating β‐catenin signalling, implicates in the regulation of TGF‐β1‐induced tubular EMT. Our data demonstrate that FHL2 expression is induced by TGF‐β1, which largely relies on intracellular Smad signalling, a crucial pathway for mediating tubular EMT. Furthermore, ectopic expression of FHL2 leads to repression of E‐cadherin and induction of fibronectin and α‐SMA, an effect that is similar to that by TGF‐β1. Conversely, knockdown of FHL2 partially restores E‐cadherin and ameliorates the induction of fibronectin and α‐SMA stimulated by TGF‐β1. In addition, TGF‐β1 causes increased FHL2 abundance both in cytosol and nuclei and an increase in the interaction between FHL2 and β‐catenin in tubular epithelial cells, especially in the nuclei. It seems that the increased FHL2 abundance by TGF‐β1 brings a change of β‐catenin phosphorylation and subcellular localization, which results in activation of β‐catenin signalling, exhibiting the enhancement of β‐catenin transcriptional activity and its target genes expression. In a mouse model of obstructive nephropathy, FHL2 expression is induced and the activation of β‐catenin signalling is closely correlated with FHL2 induction. These results have established that FHL2, acting as an endogenous regulator for β‐catenin signalling activity, implicates in the regulation of tubular EMT, thereby participating in the pathogenesis of renal fibrosis.via interacting with β‐catenin, FHL2 could affect β‐catenin‐mediated gene transcription in a cell‐specific manner Belonging to LIM domain‐only protein family, FHL protein possesses four complete and one half LIM homeodomains. FHL2 is the most studied one among five members of FHL protein subfamily, that is FHL1, FHL2, FHL3, FHL4 and ACT. As LIM domain serves as a specific scaffold, protein containing LIM domains exerts its functions through interacting with various proteinaceous binding partners FHL2 is predominantly expressed in muscle and cardiovascular systems, and it can also be found in tissues of different origin One of the significative findings in the present study is that the role of FHL2 involved in TGF‐β1‐induced tubular EMT is dependent on, at least partially, its modulation for the activity of β‐catenin signalling. Observations suggesting this notion are as follows. First, after TGF‐β1 treatment, the abundance of FHL2 is increased both in cytosol and nucleus, and FHL2 physically interacts with β‐catenin, especially in the nuclei. Second, ectopic expression of FHL2 causes activation of β‐catenin signalling, whereas knockdown of endogenous FHL2 leads to inhibition of β‐catenin signalling activity. Third, after short‐term TGF‐β1 stimulation, the abundance of β‐catenin translocated into nucleus, β‐catenin transcriptional activity and its target genes expression are all increased, while the total β‐catenin abundance is not changed. However, down‐regulation of FHL2 attenuates β‐catenin transcriptional activity and its target genes expression evoked by TGF‐β1. Therefore, it is conceivable to postulate that TGF‐β1‐induced up‐regulation of FHL2 facilitates tubular EMT substantially through binding to β‐catenin, which results in the enhancement of β‐catenin transcriptional activity in the nuclei.The authentic role of tubular EMT contributing to the pathogenesis of renal interstitial fibrosis has been debated for several years. Studies using genetic cell lineage tracing indicate that the major portion of myofibroblasts having the ability to produce ECM in renal interstitium does not originate from tubular EMT in vivo.For all we know, this is the first report displaying that FHL2 is up‐regulated in fibrotic kidney. The concomitant of β‐catenin activation with FHL2 induction in obstructive nephropathy Fig.  suggestsin vivo will be helpful to bring new insights into the mechanism of tubular EMT.In summary, we have presented herein that the induction of FHL2 by TGF‐β1 plays a crucial role for β‐catenin signalling activation and tubular EMT progression. In addition, our studies indicate that through interaction with β‐catenin, FHL2 acts as a pivotal mediator for TGF‐β1 regulating β‐catenin signalling in tubular epithelial cells. To be sure, further investigations on the regulation and function of FHL2–β‐catenin interaction in the circumstance of renal fibrosis The authors confirm that there is no conflict of interests.


We identified Src-homology 2 domain-containing phosphatase 2 (SHP-2) as a critical binding partner of CEACAM1 that could modulate β-catenin Y86 phosphorylation. Hence, CEACAM1 serves as a scaffold that controls membrane proximal β-catenin signaling. In vivo, mammary tumors of WAP-T/CEACAM1null mice displayed increased nuclear translocation of β-catenin and a dramatically enhanced metastasis rate compared to WAP-T mice. Hence, CEACAM1 controls EMT in vitro and in vivo by site-specific regulation of β-catenin phosphorylation. Survival analyses of human mammary carcinoma patients corroborated these data, indicating that CEACAM1 is a prognostic marker for breast cancer survival.We analyzed the molecular basis for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-controlled inhibition of epithelial-mesenchymal transition (EMT) in a mouse model for mammary adenocarcinoma (WAP-T mice). We demonstrate that silencing of CEACAM1 in WAP-T tumor-derived G-2 cells induces epithelial-mesenchymal plasticity (EMP), as evidenced by typical changes of gene expression, morphology and increased invasion. In contrast, reintroduction of CEACAM1 into G-2 cells reversed up-regulation of genes imposing mesenchymal transition, as well as cellular invasion. We identified the Wnt-pathway as target for CEACAM1-mediated repression of EMT. Importantly, β-catenin phosphorylation status and transcriptional activity strongly depend on CEACAM1 expression: CEACAM1 Malignancies of the breast, along with colon cancer, cause the most frequent cancer-associated deaths in women. Although long-term survival has improved, 16.7% of breast cancer patients develop deadly metastatic disease . This epCEACAM1 is a member of the carcinoembryonic antigen (CEA) family in the superfamily of immunoglobulins. It is expressed on epithelial, endothelial or hematopoietic cells and its transcript undergoes extensive splicing, producing variants with or without a transmembrane domain, and long (CEACAM1-L) or short cytoplasmic tails (CEACAM1-S) . CEACAM1in vitro . p values: *p ≥ 0.05; **p ≥ 0,01; ***p ≥ 0.001. Figures were generated with Inkscape (www.inkscape.org) and Photoshop (Adobe). Fluorescence and phase contrast pictures were processed with ImageJ. Confocal pictures were processed in Imaris 5.0 (Bitplane).Numeric data were first entered in Excel  and processed with GraphPad PRISM 4.0 (GraphPad Software) for generation of the graphs as well as for statistical analyses. For the results of the qPCR, invasion- and luciferase-assays, we performed Student's


Our findings suggest that PGCP negatively associates with Wnt/β-catenin signaling during metastasis. Targeting this regulation may represent a novel and effective therapeutic option for liver cancer by preventing metastatic activity of primary tumor cells.Tumor metastasis is the leading cause of cancer death. In the metastatic process, EMT is a unique phenotypic change that plays an important role in cell invasion and changes in cell morphology. Despite the clinical significance, the mechanism underlying tumor metastasis is still poorly understood. Here we report a novel mechanism by which secreted plasma glutamate carboxypeptidase(PGCP) negatively involves Wnt/β-catenin signaling by DKK4 regulation in liver cancer metastasis. Pathway analysis of the RNA sequencing data showed that PGCP knockdown in liver cancer cell lines enriched the functions of cell migration, motility and mesenchymal cell differentiation. Depletion of PGCP promoted cell migration and invasion via activation of Wnt/β-catenin signaling pathway components such as phospho-LRP6 and β-catenin. Also, addition of DKK4 antagonized the Wnt/β-catenin signaling cascade in a thyroxine (T4)-dependent manner. In an  Liver cancer ranks as the third leading cause of cancer death worldwide . Tumor mEpithelial-mesenchymal transition (EMT) is a unique phenotypic change that occurs during the process of tumor metastasis. EMT is characterized by a loss of epithelial features, including the reduction of E-cadherin, cytokeratin and claudin, while gaining the properties of mesenchymal cells, such as upregulation of N-cadherin, fibronectin and vimentin. EMT has been shown to contribute to cell invasion and therapeutic resistance through regulation of several transcription factors , which are commonly induced in invasive tumors –6. LiverVarious signaling pathways regulate the expression of the metastasis-related genes in liver cancer. Among these is Wnt/β-catenin signaling pathway, shown to promote cell migration and invasion in several types of cancer –11. A kePlasma glutamate carboxypeptidase (PGCP) is a secreted protein hydrolyzing the circulating peptides in the extracellular microenvironment. In particular, PGCP liberates I-thyroxine (T4) from thyroglobulin (Tg), converted to 3,5,3′-triiodothyronine (T3) by type I 5′-deiodinase (DIO1) , 19. Thein vitro and in vivo evidence that PGCP depletion promoted cell migration and invasion via activation of Wnt/β-catenin signaling cascade. DKK4 antagonized the effect in a T4-dependent manner. In addition, the expression pattern of PGCP in liver cancer tissues was inversely correlated with β-catenin expression. Thus, our findings provide a novel mechanism of PGCP-mediated negative regulation of liver cancer metastasis potentially representing an effective target for cancer therapy.Given this information, we hypothesized that PGCP may be a negative regulator of liver cancer metastasis by inducing DKK4 expression and thereby blocking Wnt/β-catenin signaling. Our results provide CDH1, DSP, OCLN and CLDN1, and an up-regulation of mesenchymal cell markers CDH2, FN1, COL1A1, SNAI2 and FOXC2 (data not shown).To address the biological functions of PGCP in liver cancer progression, SK-Hep1 cells were transfected with PGCP siRNA (siPGCP), and then RNA sequencing was employed to compare the global gene expression profiles of PGCP-deficient cells versus cells transfected with a negative control siRNA (siCont). Pathway analysis of the molecular signature revealed that PGCP knockdown enriched the functions involved in mesenchymal cell differentiation, cell motility, and cell migration Figure . AccordisiRNA silencing of PGCP significantly increased the rate of cell migration and invasion in both examined tumor cell lines (SK-Hep1 and SNU-387) as compared to the cells treated with siCont . As PGCPSince cancer cells gain mesenchymal characteristics to initiate metastasis , we nextP=0.02) between the PGCP and β-catenin expression as shown by the depletion of PGCP based on Spearman's rank correlation coefficient analysis  contribute to EMT and cell invasion by activating transcription factors SNAIL, SLUG and ZEB1 , 23. To s Figure . These rTo identify upstream signals of GSK3β regulated by PGCP, we used a candidate analysis approach. Various upstream proteins, such as ERK, mTOR and Wnt, are involved in the inactivation of GSK3β and tumor metastasis . In the DKK4 is positively regulated by T3 and TR in liver cancer , 15, andBecause DKK4 is also a secretory protein, we added the supernatant from 293T cells overexpressing DKK4 to the SK-Hep1 cells treated with siCont or siPGCP, and then compared the levels of p-LRP6, p-GSK3β and β-catenin. The presence of DKK4 protein in culture medium abolished the PGCP knockdown-mediated induction of phospho-LRP6 and cell migrations, inactivation of GSK3β, and increase in β-catenin levels causing inactivation of Wnt/β-catenin signaling Figure . GSK3β pTo analyze whether the effects of PGCP knockdown on cell migration and invasion were dependent on the concentration of T4, and to exclude the contribution of free Tg present in serum, the siPGCP- and siCont-treated SK-Hep1 and SNU-387 cells were cultured with and without serum. In the absence of serum, the phosphorylation of LRP6 was either unaffected (SK-Hep1) or slightly increased (SNU-387) by PGCP knockdown. In contrast, serum supplementation caused a significant induction of LRP6 phosphorylation in both PGCP-depleted tumor cell lines Figure . Becausein vivo, we established stable shCont and shPGCP SK-Hep1 cell lines. As expected, PGCP knockdown caused induction of mesenchymal markers   Figure , up-regu) Figure  and incr) Figure . In expe) Figure . Also, i) Figure . These rCanonical Wnt/β-catenin signaling is strongly associated with tumor growth and metastasis. When elevated, β-catenin binds to TCF/LEF transcription factors and regulates a number of genes involved in tumorigenic and metastatic processes , 10, 28.Here, we provide evidence that PGCP associates with Wnt/β-catenin signaling which mediates metastasis. The evidence was supported by the finding of a reverse correlation between the PGCP and β-catenin expression in human liver cancer tissues. In addition, the down-regulation of PGCP not only induced cell migration/invasion in SK-hep1 and SNU-387 cells but also promoted lung metastasis in a mouse model. Thus, we established that the PGCP function is strongly associated with tumor metastasis in liver cancer. As PGCP is a secretory protein , 21, we in vivo via transmembrane transduction of Wnt signals. In addition, in LRP6-mice model, LRP6 promoted Wnt signaling and accelerated tumorigenesis in breast cancer [The expression level of LRP6 is up-regulated in liver cancer . LRP6 ist cancer , 33. OurHypothyroidism is a common disorder of the endocrine system resulting from a deficiency of thyroid hormone. It has been reported in cancer patients as a possible risk factor for liver cancer . ThyroidThe DKK family is strongly associated with tumor metastasis. DKK4 suppresses cell invasion, whereas DKK1 promotes invasion and metastasis in serous ovarian cancer and liver cancer –17. ManyIn conclusion, we report for the first time that PGCP blocks liver cancer metastasis through the inactivation of Wnt/β-catenin signaling by regulating DKK4 expression and thus may represent an effective target for control of liver metastasis Figure . We also2 at 37°C.The human liver cancer cell line SNU-387 and SK-Hep1 were purchased from the Korean Cell Line Bank  and was cultured in RPMI and MEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified atmosphere of 5% COA lentiviral pool encoding shPGCP (TRCN0000073949) and shRNA control (SHC005) was purchased from Sigma-Aldrich . Viral transduction was performed according to the manufacturer's instructions. The sense sequence of PGCP shRNA is 5′-CCGGCCAGTCTAC TTGATGACTTATCTCGAGA TAAGTCATCAAGTAGACTGGT TTTTG-3′. siRNA duplexes to PGCP, DKK4, and β-catenin were purchased from ST Pharm , Genosys  and Genolution , respectively. siCont (negative control siRNA) was used for control treatment. siRNA sequences are described in the The anti-CPQ (HPA023235) antibody was purchased from Sigma-Aldrich; anti-N-cadherin (#4061), vimentin (#5741), phospho-GSK-3β (Ser9) (#9323), non-phospho (active) β-catenin (#8814) and phospho-LRP6 (#2568) antibodies were purchased from Cell Signaling Technology ; anti-β-actin (sc-47778), β-catenin (sc-7963), and HRP-conjugated secondary  antibodies were obtained from Santa Cruz Biotechnology . L-thyroxine (T1775) was obtained from Sigma-Aldrich.6/mouse). Cells were injected into the tail veins of mice (n = 5). Animals were sacrificed after 4 weeks, the lungs were fixed in Bouin's solution, and the metastatic nodules were counted.Cells were harvested at approximately 70% confluence. Female BALB/c nude mice (4 weeks of age) were injected with SK-Hep1 cells  and invasion chambers  were rehydrated in serum-free medium. Complete medium with 20% FBS (700 μl) served as a chemoattractant in the bottom chamber, and 5 × 103VO4, 1 mM NaF, and 1 × protease inhibitor cocktail). Cell lysates were centrifuged at 14,000 × g for 15 min at 4°C and then boiled in 5 × sample buffer. The protein samples were subjected to Western blotting with the indicated antibodies at a 1:500 dilution ratio.Western blotting was performed according to the manufacturer's instructions –39. The Immunofluorescence was performed according to the manufacturer's instructions . Cells gTotal RNA was isolated from the indicated cell lines using a Qiagen RNeasy Mini Kit according to the manufacturer's instructions , 42. RNAThe T4 assay was performed according to the manufacturer's instructions. The concentration of T4 was measured by the Human Free Thyroxine (FT4) ELISA kit . After treatment of SK-Hep1 cell lines with siCont or siPGCP for 48 h, the supernatant was collected, and the thyroxine assay was performed.


In contrast, SAMs made from BPT, 2-Cl-BPT and 2-Br-BPT did not form stable CNMs after a significantly higher electron dose of 9 mC/cm2.The determination of the negative ion yield of 2′-chloro-1,1′-biphenyl (2-Cl-BP), 2′-bromo-1,1′-biphenyl (2-Br-BP) and 2′-iodo-1,1′-biphenyl (2-I-BP) upon dissociative electron attachment (DEA) at an electron energy of 0 eV revealed cross section values that were more than ten times higher for iodide loss from 2-I-BP than for the other halogenides from the respective biphenyls (BPs). Comparison with dissociative ionization mass spectra shows that the ratio of the efficiency of electron impact ionization induced fragmentation of 2-I-BP, 2-Br-BP, and 2-Cl-BP amounts to approximately 1:0.7:0.6. Inspired by these results, self-assembled monolayers (SAMs) of the respective biphenyl-4-thiols, 2-Cl-BPT, 2-Br-BPT, 2-I-BPT as well as BPT, were grown on a Au(111) substrate and exposed to 50 eV electrons. The effect of electron irradiation was investigated by X-ray photoelectron spectroscopy (XPS), to determine whether the high relative DEA cross section for iodide loss from 2-I-BPT as compared to 2-Br-BP and 2-Cl-BP is reflected in the cross-linking efficiency of SAMs made from these materials. Such sensitization could reduce the electron dose needed for the cross-linking process and may thus lead to a significantly faster conversion of the respective SAMs into carbon nanomembranes (CNMs) without the need for an increased current density. XPS data support the notation that DEA sensitization may be used to achieve more efficient electron-induced cross-linking of SAMs, revealing more than ten times faster cross-linking of 2-I-BPT SAMs compared to those made from the other halogenated biphenyls or from native BPT at the same current density. Furthermore, the transfer of a freestanding membrane onto a TEM grid and the subsequent investigation by helium ion microscopy (HIM) verified the existence of a mechanically stable CNM created from 2-I-BPT after exposure to an electron dose as low as 1.8 mC/cm Carbon nanomembranes (CNMs) are two-dimensional molecular sheets with a thickness of one to a few nanometers, high mechanical strength, and high thermal stability –3. DepenThe cross-linking is typically achieved through irradiation with electrons in the energy range of 50–100 eV , but has14In typical aromatic monomers of SAMs, such as biphenyls, the cross-linking is primarily attributed to electron-induced C–H bond cleavage in the monomers, but also C–C cleavage . In turnTypically, the energy distribution of such secondary electron peaks well below 10 eV is significant at threshold , and has a high energy tail, the extension of which depends on the energy of the primary irradiation and the nature of the substrate  or above their ionization energy (DI), DEA is a resonant and very bond selective process leading to the production of a negative ion fragment and a neutral, radical counterpart. The initial step is the formation of a transient negative ion (TNI) through vertical transition from the ground state neutral to the respective anionic state, strained within the initial neutral geometry. The TNI is thus bound to relax through re-emission of the electron (autodetachment) or through dissociation. The cross section for the formation of the TNI at very low incident energies follows an  to 0 eV –29. DissThe bond selectivity in DEA and the fact that the cross sections for this process may be significant at 0 eV electron incident energy opens up the attractive possibility to use this process to purposely enhance the cross-linking efficiency and explore the potential of site selectivity in the cross-linking step as a tool to control the properties, and ultimately, the functionality of the produced nanomembranes. However, for single bond rupture in DEA, the prerequisite at 0 eV incident electron energy (where the attachment cross sections are highest) is that the electron affinity (EA(X)) of the charge-retaining fragment (X) exceeds the respective bond dissociation energy (BDE(M−X));Hrxn is the reaction enthalpy for the bond rupture, which is approximately equal to the respective threshold energy (Eth). For the higher halogens Cl, Br and I, the BDE(C6H5−X) in the respective halo-benzenes decreases significantly from X = Cl (4.14 eV), to Br (3.49 eV) to I (2.82eV) [Here Δ(2.82eV) . The EA (2.82eV) . Corresp6H5−H) is about 4.5 eV [6H5–H bond endothermic by more than 3 eV.are 0.53, 0.13 and −0.24 eV for X = Cl, Br and I, respectively. For comparison, the electron affinity of hydrogen is about 0.75 eV and that of the phenyl radical is about 1.1 eV . The BDEt 4.5 eV , making For the corresponding halogenated biphenyls, we expect the DEA cross section for chloro-biphenyl to be low, as this channel is not accessible where the attachment cross section is highest, that is, near 0 eV. The DEA cross section for the bromo analogue should be somewhat higher as this channel is accessible at lower energies. Finally, for the iodo-biphenyls, where the DEA channel is exothermic, we expect significant cross sections at 0 eV. Similarly, from the DEA threshold energy, we expect C–H cleavage through DEA to be present but inefficient. In fact, Houplin et al.  recentlyThe comparison of the cross-linking efficiency of SAMs made from these halogenated monomers is thus well-suited to put the hypotheses to the test that predetermined, high cross section DEA sites may be purposely used to enhance the cross-linking efficiency and eventually may offer a viable route for site-selective cross-linking. The former is desirable to speed up the large-scale production of CNMs. The latter may open up new avenues in tailoring the properties and functionality of nanomembranes.−, Br− and I− ion yield from 2-chlorobiphenyl (2-Cl-BP), 2-bromobiphenyl (2-Br-BP) and 2-iodobiphenyl (2-I-BP), respectively, through DEA in the energy range from 0–10 eV. In DEA, the halogen loss is the only observed fragmentation channel and the formation of the halogen ions is largely confined to a fairly narrow contribution at about 0 eV. Importantly, the relative cross section for the I− formation is about ten times larger than the relative cross section for the Br− formation and more than 200 times larger than that for the Cl− formation. As mentioned above, the electron attachment cross section at very low electron energies is proportional to E−1/2 [− from 2-I-BP, while the formation of Cl− and Br− through DEA to 2-Cl-BP and 2-Br-BP, respectively, is endothermic. The fact that we still observe the formation of Cl− and Br− from the respective biphenyls at 0 eV , and we find that these agree well with those available in the NIST database . From thTo put the hypothesis to the test that targeted generation of a radical site by DEA may be used to increase the cross-linking efficiency, self-assembled monolayers (SAM) of biphenyl-thiols (BPTs) and halogenated biphenyl-thiols  were prepared on an Au(111) substrate from solution . Subsequently, these were irradiated by electrons at an electron energy of 50 eV. Cross-linking of the molecular layers and the formation of mechanically stable carbon nanomembranes (CNMs) was then monitored through X-ray photoelectron spectroscopy (XPS) during the irradiation process. This enables the transition of the SAMs into CNMs to be observed and allows the determination of the required dose for the respective CNM formation also by directly testing the mechanical strength and microscope images of the cross-linked SAMs.2. The chlorine 2p doublet for 2-Cl-BPT is seen at 200 eV with an energy separation of 1.6 eV [1/2 + Cl 2p3/2, while for bromine, only the Br 3p3/2 is used to quantify the signal of interest. It is clear from 5/2 peak (I–C) at 620 eV can be observed, even after 1 min (0.6 mC/cm2). For the two other halogenated SAMs, the decrease of the halogen intensity is, however, rather moderate. Interestingly, in case of the iodine spectra 2-I-BPT after the decrease of the I 3d5/2 peak, a new peak of an iodine species (InS) evolves at a binding energy of 619 eV. Presumably, this peak can be attributed to free iodine caused by the electron interaction forming an iodine–gold bond. A similar observation was made by Hirayama et al. while investigating dissociating iodoalkanes on a gold substrate [f 1.6 eV  and the f 1.6 eV . The chlubstrate .Ihalog/Ihalog0, is similar for Br 3p and Cl 2p, whereas the I 3d5/2 peak, exposed to the same current density, decays substantially faster. For a reduction in the normalized intensity by 50%, which corresponds to Ihalog/Ihalog0 = 0.5, in the case of bromine and chlorine, an irradiation time of 5 to 10 minutes (3–6 mC/cm2) is needed. For iodine, the same reduction is already reached after 1 to 3 minutes (0.6–1.8 mC/cm2). The intensity reduction vs irradiation dose for chlorine and bromine is rather similar, which is consistent with the fact that both of these processes are endothermic in DEA and the intensity at 0 eV, apparent in In + signals in their mass spectra [Comparing the relative electron dose dependence of the dehalogenation process in the SAMs  and the  spectra , the dif3/2 peak at a binding energy (BE) of 162 eV and a formation and increase of a new sulfur species with a S 2p3/2 peak at BE = 163.5 eV due to radiation-induced formation of new sulfur species, such as disulfides and/or thioethers [The decrease of the intensity maximum of the thiol sulfur S 2pioethers . A straiioethers .However, since the latter procedure has to be conducted for each individual exposure time it is significantly more labor intensive. Nevertheless, for reasons of reliability, here we have applied both these approaches to follow the cross-linking of the respective halogenated biphenyls as a function of the irradiation time and thus the electron dose.2). The raw data of four characteristic irradiation stages is plotted in 3/2/S 2p1/2 doublets for the S2p spectrum. The doublets were fitted presuming an energy separation of 1.2 eV [3/2 peak at BE = 162 eV is related to the thiolate anchor group at the end of the molecule attached to the Au(111) substrate in the native SAM. The other is related to the formation of new sulfur species, for example, disulfide, which appears with a S 2p3/2 peak at 163.5 eV and reflects the loss of thiol groups. A reduction of the 162 eV S 2p doublet and increase of the 163.5 eV, that is, a transformation of the relative ratio of the different S 2p doublet peaks, is visible for all four SAMs when comparing the first (0 min) and last (120 min) XP spectra of the molecules. This reduction can be taken as an indicator of the cross-linking process, as already mentioned above. Moreover, if the intensity ratio of the newly formed sulfur species (InSs) and to the thiolate–Au peak (IRS-Au) is plotted vs the electron irradiation dose (or time), a saturation of the thiol group reconfiguration into the other sulfur species can be identified, implying completion of the cross-linking of the SAMs and the formation of CNMs [InSs/IRS-Au for the sulfur species of BPT, 2-Cl-BPT, 2Br-BPT and 2-I-BPT along with an exponential fit applied to the data. Turchanin et al. observed a saturating electron dose for BPT between 40 to 60 mC/cm2, which is in accordance with the data (black curve) presented in 2 (20–40 min). In contrast, the data for 2-I-BPT reveals a very effective cross-linking, compared to the other halogenated and non-halogenated SAMs. A saturation of the intensity ratio plot already occurs between 1.8 and 3 mC/cm2 (1–3 min). This is more than 10 times faster than that observed for BPT and the other halogenated BPTs. Such finding is remarkable, considering that the 2-I-BPT precursor molecule is only halogenated at a single position in of the upper phenyl ring. However, it should be taken into account that these compounds are also subjects to different steric restrictions determining their relative orientation. For instance, the twist angle between the phenyl rings is a result of a compensation of the repulsion of the ortho hydrogens, or halogen atoms respectively, and the delocalized π-electrons of the neighboring phenyl ring on the other hand. This interaction leads to a twist angle of 45° for BPT 60° for 2-Cl-BPT, 63.8° for 2-Br-BPT, and 67° for 2-I-BPT [2. Some ruptures within the membrane are visible, which were probably caused during the transfer process onto the TEM grid or which could also be induced by the PMMA cleaning process within the critical point dryer. 2 is an unambiguous proof of the successful cross-linking of a SAM and respective CNM formation. Comparative attempts were also made to transfer an irradiated SAM made from BPT, 2-Cl-BPT and 2-Br-BPT to other substrates or TEM grids after a lowered electron exposure of up to 15 min (9 mC/cm2). These experiments suggested that these layers were not stable enough to survive the transfer process, thus it was not possible to image an intact carbon nanomembrane derived from these precursors neither by optical microscopy nor by HIM.The interpretation of the spectra of the halogenated as well as the conventional non-halogenated BPTs is based on the characteristic S 2pf 1.2 eV  and the  2-I-BPT –39, resp 2-I-BPT  as well The high DEA cross sections observed at 0 eV for 2-I-BP as compared to 2-Br-BP and 2-Cl-BP, led to the hypothesis that radical formation through effective DEA channels may be purposely used to enhance the cross-linking efficiency for the production of CNMs. This hypothesis was put to the test through comparison of the cross-linking efficiency upon electron irradiation of SAMs produced from 2-I-BPT as compared to 2-Br-BPT, 2-Cl-BPT and BPT. XPS was used to follow the effect of electron irradiation on the bonding in the halogenated biphenyl-thiols, while a direct test of the mechanical strength and optical microscopy was used for more direct confirmation of the cross-linking efficiency.The normalized intensity in the XPS data was shown to decrease for iodine, bromine and chlorine, confirming that the dehalogenation of 2-I-BPT takes place much faster than for 2-Br-BPT and 2-Cl-BPT at the same current density.2, to a TEM grid. This clearly shows the mechanical stability achieved through cross-linking of 2-I-BPT even at an electron radiation dose as low as 1.8 mC/cm2. This is to be compared to 40–60 mC/cm2 [Furthermore, the extent and rate at which new electron-induced sulphur species are formed is considerably faster for 2-I-BPT as compared to the other halogenated BPTs and the native BPT. Based on this analysis, the cross-linking was more than ten times faster for 2-I-BPT as compared to the other halogenated and conventional BPT at the same current density. The efficiency of cross-linking in the 2-I-BPT SAMs was confirmed by successful transfer of a 2-I-BPT-based CNM, generated by an electron radiation dose of only 1.8 mC/cm0 mC/cm2  which waThe superior performance of 2-I-BPT compared to 2-Br-BPT, 2-Cl-BPT and the native BPT strongly indicates that this enhanced cross-linking efficiency is rooted in the significantly higher DEA cross section of 2-I-BPT. This in turn opens up the possibility to use high cross-sectional DEA processes to purposely enhance the cross-linking efficiency of SAMs for the production of CNMs. Moreover, the site selectivity of the DEA process also offers the potential to direct the cross-linking to specific molecular sites for production of functional CNMs.The 2-halobiphenylthiols were synthesized by a palladium-catalyzed Kumada reaction of the respective 1,2-dihalobenzenes (1-bromo-2-chlorobenzene in case of 2-Cl-BPT) with the Grignard reagent of (4-bromophenyl)triisopropylsilylsulfide . The rem−8 mbar and the acquisition pressure was maintained at about 5 × 10−7 mbar. The electron energy resolution was around ≈110 meV, determined by the FWHM of the well-documented SF5− formation from SF6. The energy scale was calibrated with respect to the SF6− formation from SF6 at 0 eV. In the current experiment, the inlet system was maintained at room temperature, but to avoid condensation on the electrical lens, the components and resulting charging the monochromator is maintained at 120 °C by means of halogen lamps. Consequently, there is a temperature gradient along the inlet capillary and we expect the target gas to be above room temperature. From previous experiments we expect the gas temperature to be between 40 and 60 °C when entering the reaction zone.Dissociative electron attachment and dissociative ionization experiments were conducted in a crossed beam apparatus that has been described in detail elsewhere . We thus−10 mbar. The electron energy was set to 50 eV and the electron bombardment was performed in nine incremental steps of irradiation time between 1 and 120 minutes. The electron dose was calibrated by means of a mobile Faraday cup built for the sample stage of the analysis chamber. The electron current between the flood gun and the cup was measured for an array of lateral positions. As a result, one minute of electron irradiation at a beam energy of 50 eV corresponds to an electron dose of 0.6 mC/cm2 on the sample. SAMs were grown by immersing a commercially produced Au substrate with (a 300 nm Au(111) layer sputtered on mica) into a solution of the relevant biphenyl diluted in dimethylformamide (DMF). After 72 h at 300 K, a homogenous molecular layer was formed. The Au(111)/mica substrate was precleaned by ozone cleaning and ethanol rinsing. The XPS experiments were also performed under UHV conditions using the monochromatic X-ray source XM1000 and the SPHERA hemispheric analyzer from Omicron Nanotechnologies. The analyzer was operated in the constant analyzer energy (CAE) mode at varying energies of 20–50 eV, depending on the element under detection. Since 2-I-BPT is highly sensitive to light exposure, its preparation and the experiments were performed either in the dark or using a yellow-light environment. The HIM images were recorded using a Zeiss Orion Plus HIM at 34.7 kV and with a blanker current of 0.3 pA at secondary-electron-detection mode. For a better resolution in the HIM, the samples were transferred to a Quantifoil R0.6/1 TEM grid with a mean hole diameter of 0.6 µm and an average hole–hole distance of 1 µm. For the exfoliation of the CNM from the gold, PMMA was used as a coating for the nanomembrane. With the help of aqueous iodine, the PMMA–CNM stack was detached from the gold substrate. Subsequently, the PMMA was dissolved by acetone within a critical-point dryer.The CNMs were prepared by electron irradiation of self-assembled monolayers of halogenated BPT at UHV conditions, meaning a chamber pressure of approximately 1 × 10