The present study demonstrated that the low molecular weight subfractions III exhibited the strongest inhibitory activity towards breast carcinoma cells MDA-MB-231, prostatic carcinoma cells PC3, and breast cancer cells MCF7 with the half-maximal inhibitory concentration (IC50) value of 52,25 μg/mL, 60,66 μg/mL, and 54,92 μg/mL, respectively. The highest percentage of inhibition was noted at a concentration of 300 μg/mL in all the examined tumor lines. A significant percentage (59.08%) of ex-LMSIII inhibition of the MDA-MB-231 tumor line was reached at a concentration of 15 μg/ml, while the concentration applied did not affect normal human fibroblast cells. The low molecular weight subfraction III was the most effective and additionally showed the highest free radical 1,1-diphenyl-2-picryl-hydrazyl scavenging activity (IC50 20.39 μg/mL) followed by the low molecular weight subfraction I (IC50 64.14 μg/mL) and II (IC50 49.22 μg/mL). The antibacterial activity of the tested preparations was evaluated against three microorganisms: Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The MIC minimal inhibitory concentration (MIC) values for the low molecular weight subfraction I, II, and III showed a stronger inhibition effect on S. aureus than on B. subtilis and E. coli cells. The MIC values for the low molecular weight subfraction II against S. aureus, B. subtilis, and E. coli were 6.25, 12.5, and 100 mg/mL, respectively.The aim of this study is to investigate  Grifola fron dosa, Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus, Tremella mesenterica, and Hericium erinaceus, are used for medicinal applications. There are also some non-edible mushroom species used solely for medicinal purposes, for instance Ganoderma lucidum, Schizophyllum commune, or Trametes versicolor. Bioactive substances from fungi can generally be divided into two groups of high molecular weight compounds, which include primarily polysaccharides and proteins and low molecular weight compounds, such as indoles, terpenoids, or phenols. The second group comprises low-molecular-weight secondary compounds that can penetrate the cell membrane and act as effectors of specific signal transduction pathways [Agrocybe cylindracea produce a laccase with HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity against HepG2 cells and MCF7 cells [Funalia trogii have been shown to have in vitro and in vivo anti-tumor efficacy [F. trogii and C. versicolor extracts on the HeLa cervical cancer cell line and human fibroblast cells [Ramaria flava exhibit a wide range of anticancer, antioxidant, and antibiotic activities [Xylaria hypoxylon [Pholiota adiposa and Ganoderma microsporum exhibited antiviral and antitumor activities as well [Inonotus xeranticus, Phellinus linteus [Ramaria flava [Medicinal mushrooms have long been used in Asian countries due to their bioactivities such as anticancer, antioxidant, antimicrobial, hepatoprotective, antineurodegenerative, antidiabetic, antiangiogenic, and hypoglycemic effects in animals and in humans –4. Of alpathways . Substanpathways –9. One opathways ,11. Bioapathways . Hu et aF7 cells . AnotherF7 cells –16. Natuefficacy . Other ast cells . It has ypoxylon . It has  as well –21. Mush as well –23. It h as well . Antioxi as well . Interes as well . Some au linteus , and RamC. unicolor was regarded as a nonedible fungus and was intensively studied as an efficient producer of extracellular laccase produced in noninduced conditions of growth [C. unicolor species possess cytotoxic and antiproliferative activity against cervical cancer cells (SiHa and CaSki) and melanomic cells [C. unicolor cultures are a very promising source of other bioactive substances, not only laccase, whose properties have been partially determined [For many years, f growth –29. The ic cells –31. The ic cells . C. unictermined .C. unicolor.The aim of the present report will be to evaluate the antitumor, antioxidant, and antibacterial activities of three subfractions separated from the total ex-LMS produced by Cerrena unicolor (Bull. ex Fr.) Murr. was obtained from the culture collection of the Regensburg University and deposited in the fungal collection of the Department of Biochemistry  under the strain number 139 (ITS sequence deposited in GenBank under accession number DQ056858) [Q056858) . The ferQ056858) . The medQ056858) . 10-day-Q056858) . The antQ056858) –33. In tProtein concentrations were determined using the Bradford reagent and bovine serum albumin as a standard . The tot−1.The analyses of ex-LMSI, ex-LMSII, and ex-LMSIII were performed using lyophilizates. FTIR spectroscopy was performed with a spectrometer (Thermo Scientific Nicolet 8700A with FT Ramana Nicolet NXR module) in the wavelength range 4000–400 cmc means the absorbance of the control sample andAt means the absorbance of the standard or tested compound. The antioxidant ability of the sample was expressed as IC50.The total antioxidant capacity of the three fractions was determined using the DPPH radical as a reagent, according to the procedure described by Paduch et al. . This mecontrol means the absorbance of the control and Asample is the absorbance at 734 nm of the tested compound/standard. A Trolox and ascorbic acid calibration curve was prepared for a concentration range from 6.25 to 800 μg/mL and IC50 values were obtained.The ABTS radical-scavenging activities of the fractions were determined using the method of van den Berg et al. , Duo-Chu4 · 7H2O (9 mM), 20 μL of a hydroxybenzoic acid solution with ethanol (9 mM) and 20 μL of H2O2 (8.8 mM) in a 37°C water bath for 30 minutes. The percent OH radical-scavenging effect of each sample was calculated using the following equation:control is the absorbance of the control reaction and the sample is replaced by 100 mL ethanol. The tests were performed in triplicate.The OH radical-scavenging activity assay was conducted according to the Fenton method –45 with Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 25922), and Bacillus subtilis (ATCC 6633) bacterial strains were used as indicator bacteria and inoculated into a commercially available Muller-Hinton Agar II medium (LabM (TM), IDG plc, UK), 38g/L, with the inoculum solution (100 μL) of ca. 1×105 CFU/mL of each kind of microorganisms smeared on the standard assay medium. 100 μL of the specimen (concentration 1 mg/mL) were added into the agar well in the center of Petri dishes and left to incubate for 2 hours at room temperature; afterwards, the plates were transferred to 37°C for 18 h. Sterilized physiological saline was used as a control. After the incubation of inoculated bacterial cultures treated with ex-LMSI, ex-LMSII, and ex-LMSIII, the inhibition zones were measured.5CFU/mL) was transferred to the wells of a 96-well tissue culture plate. Then, another 100 μL of diluted samples were added to each well and the inoculated 96-well tissue culture plates were incubated at 37°C for 24 h. The MIC was defined as the lowest concentration of samples inhibiting the visible growth of the tested microorganisms.The minimal inhibitory concentration (MIC) of ex-LMSI, ex-LMSII, and ex-LMSIII was evaluated using the two-fold serial dilution method . The samin vitro cell growth inhibitory activity against the human cancer cell lines  [2 and 100% relative humidity in a CO2 incubator. A 100-μL aliquot of cells (105 cells/mL) was transferred to the wells of a 96-well tissue culture plate. The cells were allowed to grow for 12 h and then they were treated with the sample. 100 μL of the test samples  were added to the wells, and the cells were further incubated for another 48 h at 37°C in an atmosphere of 5% CO2. 20 μL of MTT  were then added to each well and the cells were further cultured for 4 h. After removal of the medium, 100 μL of dimethyl sulfoxide (DMSO) was added to each well [control is the absorbance of the control reaction and Asample is the absorbance in the presence of the sample. Each test was done in triplicate and the concentration required for 50% inhibition of viability (IC50) was determined.The test sample was subjected to the MTT assay to determine the and PC3) . The celach well . SuitablAll results presented in the paper are expressed as mean and standard deviation (±SD) from three experiments (n = 3). Differences in mean values between the groups were analyzed by a one-way analysis of variance (ANOVA) with post-hoc Tukey HSD test and all tests were considered statistically significant at p ≤ 0.05.C. unicolor represents wood degrading fungi from the phylum Basidiomycota, which are exceptionally useful biotechnological tools in various industrial processes. In recent years, C. unicolor has been extensively studied as a very efficient source of extracellular laccase, which is an enzyme used on a wide scale in various industries. Recently, our results have indicated that laccase (LAC), crude endopolysaccharides (c-EPL), and the extracellular low molecular weight subfraction (ex-LMS) exhibit pro- and antioxidant properties as well as immunomodulatory, antibacterial, antiviral, and anticancer activities [C. unicolor ex-LMS preparation.tivities –33. In tC. unicolor is a source of natural active low molecular weight metabolites. Our analysis focused on three fractions of extracellular low molecular weight secondary metabolites from C. unicolor cultures . Our earlier study of the chemical composition of ex-LMS from C. unicolor showed the presence of sugars , proteins (189 μg/mL), and phenolic compounds (15 μM) [C. unicolor revealed distinct differences between the investigated preparations (Ramaria flava has been confirmed in other investigations [ (15 μM) . The anaarations . The ex-C. unicolor idiophasic cultures demonstrated an aminoglycoside substance pattern  showed higher absorption for proteins at ca. 1600 cm-1 and lower at ca. 3200 cm-1 compared to the ex-LMSI (a) and ex-LMSII (b) fractions. Similarly, the absorption at 1000 cm-1 is lower in the case of the ex-LMSIII fraction than for ex-LMSI and ex-LMSII.The FT-IR spectrum analysis of the ex-LMS subfractions of the  pattern . The chaen bonds . The absproteins . The banic chain . The abs50 values were 63.69 μg/mL for Trolox and 41.25 μg/mL for ascorbic acid in the DPPH method, 42.11 μg/mL and 28.23 μg/mL in the the ABTS method, and 54.61 μg/mL and 39.25 μg/mL in the the OH method, respectively. The DPPH radical-scavenging capacity of the analyzed samples is shown in 50 values, which correspond to the concentration of fungal samples that are able to scavenge 50% of free radicals present in the reaction mixture. High-IC50 values indicate low antioxidant activity. Ex-LMSIII shows the highest DPPH radical-scavenging activity, followed by ex-LMSI and ex-LMSII. The IC50 values of the samples were 20.39, 64.14, and 49.22 μg/mL, respectively. The measurements of the ABTS test showed slightly lower scavenging capacity of the ex-LMS fractions (in particular ex-LMSIII) in relation to the DPPH method , 39.78 μg/mL (ex-LMSII), and 31.49 μg/mL (ex-LMSIII). The hydroxyl radical and its subsequent radicals are the most harmful reactive oxygen species, as they are mainly responsible for the oxidative injury in many biomolecules [50 values of the samples ranged from 49.13 to 69.12 μg/mL. Based on the comparison of the IC50 values, the order of the hydroxyl radical-scavenging activity was found to be as follows: ex-LMSIII fraction > ex-LMSII fraction > ex-LMSII fraction . Our results indicate that the analyzed ex-LMS fractions with lower phenol content exert a stronger radical scavenging effect, suggesting that phenols are not the main factor in their antioxidant activity. In our previous study, we demonstrated the antioxidant activity of the ex-LMS starting fraction of ectively . To our E. coli and S. aureus bacterial cells than the extracellular fraction of laccase or the polysaccharide fraction from C. unicolor [E. coli (Gram-negative bacteria) as well as S. aureus and B. subtilis (Gram-positive bacteria). In our experiments, the low-molecular subfractions ex-LMSI, ex-LMSII, and ex-LMSIII showed relatively strong antimicrobial activity against all the Gram-positive and Gram-negative bacterial species used  were incubated with various concentrations of ex-LMSI, ex-LMSII, and ex-LMSIII  [50 = 66.54 ± 4.27 μg/ml), and the inhibition rate was 71.66% at a concentration of 200 μg/ml. The in vitro anticancer activity of ethanol extracts was also confirmed by Wu et al. investigating the effects of Fomitopsis pinicola, Ganoderma sinense, Fomitopsis officinalis, and Polyporus melanopus extracts on cancer cell lines HepG2 and S-180 [Literature data indicating the antitumor activity of fungi are primarily focused on the use of mycelium extracts. For example, Liu et al. demonstrated the cytotoxic activity of a AMB-231) . They dend S-180 . In our C. unicolor culture fluid possessed anticancer, antioxidant, and antibacterial properties. Our results show that post-culture medium containing secondary metabolites of less than 10 kDa can be a rich source of natural anticancer, antioxidant, and antibiotic substances and can be important in the control of various human and animal diseases. These results suggest that C. unicolor can be an effective source of pharmaceuticals.It was found in the present study that the three bioactive subfractions of low molecular weight secondary metabolites: ex-LMSI, ex-LMSII, and ex-LMSIII isolated from


To develop a matched sealing material for socket rehabilitation of grey cast iron pipes, an epoxy resin is modified by the addition of different components to improve the flexibility. Three different modifications are made by adding ethylene-propylene diene monomer (EPDM) rubber powder, reactive liquid polymer (ATBN) and epoxidized modifier. In this paper the effect of the modification method as well as the influence of absorption of water on the mechanical and physical properties are analyzed in terms of: tensile strength, modulus of elasticity, adhesion performance, pressure resistance, glass transition temperature and water content. A comparison with neat epoxy shows for all materials that the modulus of elasticity and strength decrease. Unlike other tested modification methods, the modification with rubber powder did not enhance the flexibility. All materials absorb water and a plasticization effect arises with further changes of mechanical and physical properties. The application of the sealant on the grey cast iron leads to a reduction of the strain at break  and has to be evaluated. The main requirement of pressure resistance up to 1 MPa was tested on two chosen materials. Both materials fulfill this requirement. Developing Water Loss Prevention (DeWaLoP) is a project, led by Vienna Water, which includes a comprehensive concept for repairing leaky lead joint sockets. The sockets of the 150 year old grey cast iron pipes of Vienna’s water supplying system were caulked up to the 1920s with a hemp pack and a lead ring  and are to be replaced. The sealing was secured by a swollen hemp pack and a lead ring, stabilizing the hemp in the socket. A possible displacement of the lead ring and a decomposing of the hemp pack can cause leakage . HoweverFor this project, an in-pipe robot was designed, that crawls into water pipes (diameter 800 to 1000 mm), inspects, cleans and applies the sealing material . The tasBased on the previous investigations , epoxy mTg) of the modifier and the epoxy. Almost constant mechanical properties in the application temperature range and a decrease in the modulus of elasticity—in comparison to neat epoxy—can be observed. The material modification with epoxidized modifier (EP-EM 1) belongs to the group of internal plasticized epoxies. Epoxidized modifiers are added and dissolved as a second component in the resin. In the recent study, trifunctional modifiers are used which react with the curing agent and the modifying component will be incorporated in the epoxy matrix. The slope of the modulus of elasticity in the application temperature range of this modification is quite flat. The customized proprietary epoxy (C-EP 1) for sewer socket rehabilitation seems to be a copolymer of epoxy and flexibiliser . These formulations show a strong slope of the modulus of elasticity in the application temperature range. Strong differences in the mechanical properties between minimum and maximum application temperature arise. Tg of the materials should not be in the same range as the application temperature. Modification of epoxy leads to a decrease of the Tg which is also reported in [Tg.Changes of the mechanical properties in the application temperature range are not desired. In order to avoid this, the orted in ,10,11. TTg to lower temperatures. Using ATBN or rubber powder for modification, the materials show in addition to the Tg of the epoxy a second Tg of the rubber phase  resin mixed with the diluent 1,6-hexandiglycidylether. The epoxy equivalent is 200 (g/eq)A customized proprietary amine curing agentThe used base epoxy system is a proprietary but commercial available material. It is commonly used for pipe rehabilitation with an inliner, which means the in-pipe application of a needled felt liner impregnated with a thermoset material. The liquid resin and hardener used in this work for all modifications were:The acronym for the virgin, cured epoxy is EP.EPDM rubber powder, grain size of 63–100 µm and a hardness of 40 Shore AReactive liquid polymer; system of an amine-terminated butadiene-acrylonitril (ATBN) copolymer, with 16% acrylonitril and an amine equivalent weight of 900 g/eqepoxidized modifier 1, (EM 1): glycidyl ether of castor oil (three epoxy end-groups), epoxy equivalent 656 is g/eqepoxidized modifier 2, (EM 2): triglycidyl ether of propoxylated glycerol (three epoxy end-groups), epoxy equivalent is 644 g/eqThe modifiers used are:For reason of comparability two customized proprietary epoxies (primarily applied in the field for sewer rehabilitation) were also investigated .For preparing the formulations with reactive liquid rubber (EP/ATBN) the resin and ATBN were mixed together for 15 min in a bleaker and stirred by hand. The stoichiometric calculated amount of curing agent was added followed by further mixing for 5 min. The formulations with the epoxidized modifiers (EP/EM) were prepared as followed: the resin and the epoxidized modifier were stirred by hand in a bleaker for five minutes. The calculated amount of curing agent was added to the resultant mixture followed by further mixing for 5 min. Silica as a thixotropic agent for better processing was added to the formulations before adding the curing agent. For the epoxy/EPDM rubber powder (EP/EPDM) blend the resin and EPDM rubber powder were stirred by hand to a homogenous mixture. Finally the curing agent was added. The acronyms of the tested samples and their compositions are shown in Producing the characteristic samples for the mentioned methods, silicon molds were used. If needed, the surface was grinded (polished) to remove irregularities. M) and strain at break were determined according to ISO 527-1 [Tensile strength  with a TGA 2050 . Cured material  was investigated first, then the material after exposure to water for 28 days at 40 °C was tested. The mass loss up to 200 °C was considered for determining the water content of the sample. The TGA experiments were carried out using a heating rate of 20 K/min, air atmosphere as purge gas with a flow of 100 mL/min, a sample mass of app. 50 mg and an alumina pan. These tests were performed from ambient temperature to 600 °C according the standard ISO 11358 [Tg) and the storage modulus (Eʹ). All samples were measured with an oscillatory frequency of 1 Hz, a heating rate of 3 K/min and an amplitude of 70 µm in 3 point bending mode (50 mm support span). The glass transition temperature was determined at the maximum of the loss modulus Eʹʹ. According to the standard ISO 6721-1 [Dynamic mechanical analyses (DMA) were performed to assess the glass transition temperature , EPDM rubber powder and epoxidized modifiers as well as two customized epoxies were analyzed. The results show a comparison of the different methods to enhance the flexibility: Concerning the DMA results, nearly constant mechanical properties in the application temperature range (7–17 °C) could be observed at the formulations with reactive liquid polymer (ATBN) or EPDM. The customized proprietary epoxy and formulations with epoxidized modifier showed a slope in this temperature range. At all formulations a reduction of the mechanical properties like tensile strength and modulus of elasticity was observed. No enhancement of strain at break could be achieved by modifying with EPDM rubber powder. The glass transition temperature decreased and thus a change of the materials operating temperature range occurred. TGA measurements showed that all epoxy absorb water after immersion. Due to the water absorption, a plasticization effect was observed. Further changes in the materials operating temperature ranges have to be considered.Mechanical properties evaluated by tensile tests could not be reached in the adhesion tensile test. Inhomogeneous strain distribution in these adhesive compounds and a maximum of stress near the interphase were leading to crack initiation at lower loads. The modified epoxy EP/ATBN 2 as well as the customized epoxy C-EP 1 fulfilled the requirement of pressure resistance up to 1 MPa. The measured major strain was negligible. Modification with ATBN is the most effective method of epoxy flexibility increase for this application.


The canonical Wnt/β-Catenin signaling pathway is widely involved in regulating diverse biological processes. Dysregulation of the pathway results in severe consequences, such as developmental defects and malignant cancers. Here, we identified Ube2s as a novel activator of the Wnt/β-Catenin signaling pathway. It modified β-Catenin at K19 via K11-linked polyubiquitin chain. This modification resulted in an antagonistic effect against the destruction complex/β-TrCP cascade-orchestrated β-Catenin degradation. As a result, the stability of β-Catenin was enhanced, thus promoting its cellular accumulation. Importantly, Ube2s-promoted β-Catenin accumulation partially released the dependence on exogenous molecules for the process of embryonic stem (ES) cell differentiation into mesoendoderm lineages. Moreover, we demonstrated that UBE2S plays a critical role in determining the malignancy properties of human colorectal cancer (CRC) cells in vitro and in vivo. The findings in this study extend our mechanistic understanding of the mesoendodermal cell fate commitment, and provide UBE2S as a putative target for human CRC therapy. K11-linked polyubiquitin chain is another common modification in eukaryotic cells5. Extensive studies have provided insights into its biochemical mechanisms and cellular functions in cell cycle progression, pluripotency, and differentiation10. In general, the process of ubiquitination is achieved by three types of enzymes, namely Ub-activating enzyme , Ub-conjugating enzyme , and Ub ligase (E3)3. Ub-conjugating enzyme E2S (Ube2s) is a K11 linkage-specific E212. It selectively cooperates with E1, another priming E2 (Ube2c/d), and the E3 complex anaphase-promoting complex/cyclosome (APC/C) to elongate K11-linked polyubiquitin chain on substrates for 26S proteasome-mediated degradation10. The critical role of Ube2s in regulating cell cycle and differentiation inevitably implicates it into tumorigenesis. To date, aberrant expression of Ube2s has been detected in multiple human primary cancers15. Strikingly, Ube2s overexpression alone is sufficient for the onset of some types of cancers15.In the process of ubiquitination, ubiquitin (Ub) protein is covalently attached to substrates either as a monomer or a polymer chain linked via its N-terminus or any of its seven lysine (K) residues, K6, K11, K27, K29, K33, K48, and K63. Among them, the cellular function of K48- and K63-linked polyubiquitin chains is well understood. It is generally believed that K48 linkage marks substrates for degradation, whereas K63-linked polyubiquitination results in non-degradative outcomes, such as signal transduction17. As the core component of this pathway, β-Catenin is tightly regulated by post-translational modifications that fine-tune its protein level and optimal activity. At the molecular level, when Wnt ligands bind to the Frizzled receptor and its co-receptor, low-density-lipoprotein-related protein 5/6 (LRP5/6), β-Catenin is dissociated from the Axin destructive complex and subsequently translocates from the cytoplasm into nucleus for transcription regulation18. The Axin destructive complex is composed of several proteins, including Axin, glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli (APC), and casein kinase 1 (CK1). In the absence of activation stimuli, β-Catenin is recruited to the destructive complex for sequential phosphorylation at serine 45 (S45) by CK1 followed by S33, S37, and threonine 41 (T41) by GSK321. Consequently, the phosphorylated S33 and S37 of β-Catenin act as the signals recognized by an E3 complex Skp1/Cul1/F-boxβ-TrCP which promotes K48-linked polyubiqutination and proteasomal degradation 26.The canonical Wnt/β-Catenin signaling pathway pivotally regulates diverse cellular processes, including embryonic development, stem cell maintenance, and differentiation27. Meanwhile, Sox2 is associated with Ube2s via direct physical interaction10. Sharing a common interacting partner suggests that Ube2s and β-Catenin may be functionally connected in the same pathway. In addition, Ube2s-overexpressed mES cells exhibit specific induction of Esrrb which is a downstream target of the Wnt/β-Catenin signaling28, indicating that Ube2s could serve as an activator of the pathway. Importantly, β-Catenin has been found to be modified by K11-linked polyubiquitin chain29. Since Ube2s is one of the most established E2 mediating K11 linkage, it could potentially be involved in monitoring the cellular activity of β-Catenin. In this study, we explored the role of Ube2s in regulating β-Catenin and uncovered that Ube2s directly interacted with β-Catenin to ubiquitinate its K19 residue via K11 linkage. This modification promoted β-Catenin stablization through antigonizing its proteasomal degradation mediated by the destruction complex/β-TrCP signaling. Consequently, Ube2s promoted mesoendoderm lineage specification from mES cells. Meanwhile, it enhanced the malignancy properties of colorectal cancer (CRC) both in vitro and in vivo, which can be markedly reduced upon UBE2S deletion alone. Our study presents UBE2S as a potential novel target for enhanced CRC treatments and production of specific mesoendodermal lineages from mES cells.Interestingly, several lines of evidence suggest the potential association between Ube2s and β-Catenin. Previous studies reported that transcription factor SRY (sex-determining region Y)-box 2 (Sox2) is an interaction partner of β-Catenin in breast cancer and mouse embryonic stem (mES) cells 6-tagged β-Catenin fusion proteins to interrogate their association using an in vitro GST pull-down assay. Our result showed that they were capable of forming a protein complex through direct physical interaction  assay. Results confirmed their endogenous interaction Fig. . Moreoveion Fig. .Fig. 1Ub6 failed to retard the degradation of β-Catenin  treatment to block de novo protein biosynthesis, we found that Ube2s elevation failed to promote the degradation of β-Catenin. Instead, it enhanced the β-Catenin stability Fig. . Furthernin Fig. . This ob36. Co-IP assay revealed that these two proteins indeed interacted in the mES cells  of β-Catenin to prevent its degradation. To this end, we expressed β-Catenin mutants in which the K19 and K49 residues were substituted with an arginine (R), respectively. Using in vitro ubiquitination assay, we found that the K19R mutation compromised the Ube2s-mediated polyubiquitin signal on β-Catenin, while the K49R mutation did not display any effect  stably overexpressing Ube2s. Realtime PCR and ICC results showed that with 3 μM CHIR99021 treatment in the defined culture medium, the control cells  displayed a dramatic increase in mesoendoderm marker T, implicating a successful induction of differentiation -inducible UBE2S. The downstream targets of β-CATENIN, MYC and CCND1, were activated upon dox induction  using the CRISPR/Cas9-mediated gene knockout system. Two individual colonies (UBE2S∆-1 and UBE2S∆-2) were prepared for further characterization  rather than K19 or 49 with K11- or K63-linked polyubiquitin chain to increase its accumulation and activity in hematopoietic stem cells58. Although all these factors stabilize β-Catenin, their activity exhibits cell-type specificity. Accordingly, repression of these factors in CRC cells may not realize an effective removal of the redundant β-Catenin. For instance, EDD depletion fails in decreasing β-Catenin in CRCs, which rules out the possibility of its involvement in regulating CRC pathogenesis29. In this study, we identify Ube2s as a novel post-translational modifier of β-Catenin. It ubiquitinates β-Catenin via K11 linkage at its residue K19 and thus increase the stability of β-Catenin. Strikingly, manipulating the expression level of Ube2s alone appears to be sufficient for controlling the proliferation and metastasis of CRC cells. As such, our study strongly suggests Ube2s as a putative target for a future therapy of CRCs.Dysregulation of the Wnt/β-Catenin pathway frequently results in severe consequences, such as malignant CRCs32. For example, CHIR99021 coordinates with LIF and/or PD0325901 to maintain the self-renewal and pluripotency of mES cells in serum-free culture medium59. At the molecular level, CHIR99021 enhances cytosolic β-Catenin accumulation and subsequent nuclear translocation to activate Esrrb which helps to maintain the stemness of ES cells28. However, without LIF or PD0325901, CHIR99021 alone fails to maintain the undifferentiated state of mES cells, instead it promotes differentiation59. Furthermore, with defined culture medium in vitro, CHIR99021 treatment-induced β-Catenin accumulation can specifically direct mES cell differentiation into mesoendoderm lineages43. Two other studies also provide evidences showing that sustained activation of Wnt/β-Catenin pathway stimulates mesoendoderm lineage formation from ES cells61. By virtue of its control of β-Catenin stability, Ube2s asserts an important role in the differentiation process. Of note, our previous study shows that Ube2s ubiquitinates Sox2 and promotes its proteasomal degradation. Interestingly, the study by Thomson et al. shows that Sox2 inhibits mesoendoderm lineage formation43. Therefore, Ube2s exhibits twofold effect in promoting mesoendoderm differentiation from mES cells through enhancing β-Catenin accumulation and simultaneously removing the inhibitory Sox2. As such, engineering the level of Ube2s could serve as a key step toward the optimization of the protocols for in vitro mesendoderm lineage differentiation. It is noteworthy that β-Catenin can function independent of the Wnt signaling pathway. It associates with multiple Cadherin proteins, such as E-Cadherin, to regulate cell–cell adherence63. Interestingly, E-Cadherin-related intercellular adherence is required by ES cell maintenance and differentiation64. Thus, we could not rule out the possibility that Ube2s regulates mesoendodermal lineage commitment through its ubiquitination of E-Cadherin-linked β-Catenin.Besides tumorigenesis, the Wnt/β-Catenin pathway is critically involved in regulating pluripotency and cell differentiation2. The cells were maintained on gelatin-coated dishes in Dulbecco’s modified Eagle medium , supplemented with 15% heat-inactivated fetal bovine serum , 0.1 mM β-mercaptoethanol (GIBCO), 2 mM L-glutamine, 0.1 mM MEM non-essential amino acid, 5000 units/ml penicillin/streptomycin, and 1000 units/ml of LIF . Mouse F9 teratoma cells, human embryonic kidney 293 cells and human CRC HCT116 and HT29 cells were obtained from ATCC . The cells were cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) at 37 °C with 5% CO2.Mouse E14 ES cells (ATCC) were cultured under a feeder-free condition at 37 °C with 5% COUbe2s, β-Catenin-K19R, and β-Catenin-K49R ORFs were inserted into vector pCAG-puro, respectively, for overexpression in mES cells. Human UBE2S ORF was inserted into vector pcDNA4.0/TO(+) for overexpression in HCT116 cells. The Ube2s shRNAs were cloned into pSuperpuro  between BglII and HindIII sites, and 19-bp shRNAs with a 9-bp loop were expressed by the pSuperpuro plasmids. The two target sequences for Ube2s knockdown in mES cells are 5′-GCTACTTCCTGACTAAAAT-3′ and 5′-GGAGGTCTGTTCCGTATGA-3′. The two target sequences for Ube2s knockdown in HCT116 cells are 5′-GCATCAAGGTCTTTCCCAA-3′ and 5′-GGCGGTTATAAAGAGGCAG-3′. Transfection of plasmids was performed using Lipofectamine 2000 .Mouse The recombinant GST-tagged proteins were expressed in BL21 after induction with 0.2 mM IPTG at 16 °C and conjugated to glutathione–sepharose beads (GE) in lysis buffer . After a 1.5-h incubation at 4 °C, the supernatant was removed with a 5-min spin at 1000 rpm, and the beads were washed twice with the lysis buffer at 4 °C. The recombinant GST-tagged proteins were eluted from the beads with the elution buffer .The recombinant 6×His-tagged proteins were expressed in BL21 after induction with 0.2 mM IPTG at 16 °C and conjugated to Ni superflow beads in wash buffer . The beads were washed five times at 4 °C, and the recombinant 6×His-tagged proteins were then eluted from the beads with the elution buffer .t-test; ***p < 0.001; **p < 0.01; *p < 0.05).Total RNAs were extracted using Trizol . cDNA synthesis was performed with 500 ng of total RNA using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix  according to the manufacturer’s instructions. mRNA levels were measured by qRT-PCR analysis based on SYBR® Premix Ex Taq™  with the BioRad real-time PCR machine. Results were normalized to β-actin. All the primers used in the study give rise to single product with the right size in agarose gel analysis. The data are presented as the mean ± SD . Twenty micrograms of the total proteins were separated by SDS–PAGE and transferred to PVDF membrane. The membrane was blocked with 5% milk and probed with specific primary antibodies and secondary antibodies. The blots were developed with ECL Advance Western Blotting Detection Kit . The antibodies used in this study include anti-β-Catenin antibody , anti-Ube2s antibody , anti-β-TrCP antibody , anti-Cdc27 antibody , anti-HA antibody , anti-GST antibody , anti-Ub , anti-P-β-Catenin T41/S45 , anti-P-β-Catenin S33/S37/T41, anti-β-Tubulin , and anti-LaminB antibody .Five-hundred micrograms of protein samples in a total volume of 500 μl reaction solution were immunoprecipitated with 2 μg of antibody and 20 μl of Protein-G beads . The samples were rotated at 4 °C overnight. The beads were washed four times with 1 ml of cold NP40 lysis buffer containing protease inhibitors . The beads were then boiled for 10 min in the presence of 20 μl 2× sample buffer and the eluted proteins were fractionated by SDS-PAGE in 10% gels. Proteins were detected by immunoblotting as described above.Purified GST-tagged protein were precleared with Glutathione Sepharose 4B  for 1.5 h and incubated with His-tagged fusion proteins at 4 °C overnight. Protein-bound sepharose beads were washed four times with lysis buffer and eluted in SDS-PAGE sample buffer. Eluted proteins were analyzed by immunoblotting.Cells were fixed with 4% formaldehyde for 30 min and washed four times with PBST buffer (0.25 g Tween-20 dissolved in PBS solution). Subsequently, the cells were blocked with 5% horse serum diluted in PBST buffer. Primary antibodies were applied in blocking solution at 4 °C overnight. The primary antibodies used in this study include anti-T antibody  and anti-HA antibody . After washing with PBST buffer, the coverslips were incubated with Alexa546-conjugated anti-mouse  and Alexa488-conjugated anti-goat  secondary antibodies for 1 h. The secondary antibody solution also contained 2 μg/ml DAPI. After washing with PBST buffer three times, the cell images were captured. All red and green images were adjusted identically in order to generate the merged images.Cdc27 complex solution, and 200 nM β-Catenin in an ATP cocktail  was incubated at 30 °C for 90 min. Reactions were terminated by addition of trichloroacetic acid (TCA) , and subsequently analyzed by SDS-PAGE and western blotting with the antibody against β-Catenin .A 0.5-ml conjugation reaction containing 300 nM E1 (Uba1), 600 µM Ub, 500 nM Ube2s, 500 nM Ube2c, 5 µl Apc43. Briefly, mES cell medium was changed to fresh N2B27 medium. After 48 h of culturing, 3 μM CHIR99021  was added to treat the cells for 2 days. The resulting cells were analyzed by immunocytochemistry staining or real-time RT-PCR assays.Mesoendoderm differentiation assay was performed as described previously by Thomson and colleagues5 cells were seeded in the upper chamber. The cells were allowed to invade for 48 h. Cells that invaded the lower surface of the filter were counted in five random fields under a light-microscope at high magnification.The transwell assay was conducted by using Transwells  with 8 μm pore-sized polycarbonate membrane filters in 24-well culture plates. The upper surface of the filter was coated with 12.5 μl Matrigel per filter . The Matrigel was dried and reconstituted at 37 °C into a solid gel on the filter surface. After starving in FBS-free DMEM overnight, 3 × 105 cells onto 96-well plates. Confluent monolayers were wounded using a pipette tip. The cell migration distance was measured under a light-microscope at high magnification in at least three random fields.The scratch assay was performed by seeding 2 × 106 cells from the UBE2S∆ cell line and the control line were injected subcutaneously into the bilateral flanks of 6-week-old nude mice, respectively. For the in vivo metastasis assay, the dox-inducible UBE2S overexpression HCT116 stable cell line was injected into tail veins of 6-week-old BALB/c female nude mice. For the group with UBE2S overexpression, dox was added in the feed after cell injection. The group without dox feed was used as control. Every group has seven mice. The mice were sacrificed 4 or 5 weeks after cell injection. Formalin-fixed lungs isolated were paraffin-embedded, and tissue sections were stained with H&E to evaluate the cellular morphology.The experimental mouse work followed the animal care protocol LA2013-1 approved by Peking University Animal Research Ethics Board and was conducted at the Peking University Health Science Center, China. For the in vivo tumorigenesis assay, 5 × 10Supplementary figure legendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5


Tg parts with mechanical properties similar to the amine-epoxy systems, which are the mainstream two-component epoxy resin systems for automobiles.Imidazole cured epoxy resin systems were evaluated for one-component, fast-curing resins for liquid molding of automotive composite parts according to industry requirements. It was demonstrated that an epoxy resin-1-(cyanoethyl)-2-ethyl-4-methylimidazol(EP-1C2E4MIM) system would cure in a few minutes at 120 °C, while exhibiting acceptable pot life, viscosity profiles, and low water absorption. Moreover, this system yielded high  Owing to the high strength-to-weight ratio and specific stiffness, carbon fiber-reinforced plastics (CFRPs) are increasingly used in a variety of applications that require high-performance and light weight ,3,4,5,6.Many strategies have been executed to develop one-component epoxy resins with workable pot life and viscosity profiles . One of Tg of the cured epoxy resin, while also maintaining the low cost of the total system. In general, imidazoles have intrinsic high curing reactivity towards epoxy resins, and the curing behaviors can be facilely tailored by introducing various substituent groups on the imidazole rings [Imidazoles are a class of epoxy hardener ,25,26. Ble rings ,28. So fle rings ,30,31. IDGEBA (E-51) with an epoxy equivalent weight of 182~192 was supplied by Nan Ya Plastic Co. . 2-ethyl-4-methylimidazole  and 1C2E4MIM (99%) were purchased from HWRK Chem. Co., Ltd. . A commercial grade amine-epoxy system 10-min fast-cure epoxy resin 3585/5003-1 was obtained from Wells Advanced Materials (WAM 3585/5003-1) Co., Ltd.  as a benchmark. T700 3K carbon fiber plain weave fabric was supplied by Zhongfu Shenying Carbon Fiber Co., Ltd. .The rheological properties and curing behaviors of the uncured epoxy resins were investigated using a dynamic shear rheometer (DSR) and a differential scanning calorimeter (DSC). The thermo-mechanical properties of the cured epoxy resins were studied by dynamic mechanical analysis (DMA). The mechanical properties of the cured epoxy resins, including fracture toughness, tensile, and flexural properties, were tested according to ASTM standards. For carbon fiber composite laminates, the tensile, flexural, compressive, and interlaminar shear properties were tested according to corresponding ASTM standards.The viscosity of the epoxy resin was measured by DSR (Haak MARS60) with parallel plates, Φ = 40 mm, a gap of 1 mm, and 1.592 Hz.The pot life for the epoxy resin at ambient temperature and the gelation times were determined by DSR. The pot life was determined as the time at which the viscosity reached 1000 Pa·s during ambient storage at 23 ± 2 °C.The cure time of the epoxy resin was determined by DSC (NETZSCH DSC214). Isothermal DSC was performed by rapidly ramping the sample temperature from 0 to 120 °C, with the rate of 40 °C/min, and holding for various periods of time. Dynamic scans at 10 °C/min over a temperature range from 25 to 220 °C were used to determine the total heat of the reaction.The dynamic thermal mechanical properties were tested on a DMA  with a dual cantilever at a heating rate of 2 °C/min from 30 to 250 °C, and a frequency of 1 Hz.The mechanical tests for neat resins and composites were carried out using a universal testing machine (Instron Model 2360) with a 30-KN load cell. The tensile and flexural tests for neat resins were performed according to ASTM D 638 and D 790, respectively ,33. The The mechanical properties of the carbon fiber composite were also tested according to ASTM standards. The tensile test was performed according to ASTM D 3039  with a sTg), and hot-wet flexural properties. Water absorption was measured by putting the neat resin sample into a deionized water heat bath of 70 °C and weighing the sample every few days until a constant weight was reached. The moisture absorption was calculated by Equation (1), as follows:G0 is the initial weight of the coupon, and Gi is the coupon’s weight on the i-th day.The hot-wet properties were characterized by water absorption, hot-wet glass transition temperature  to reduce the defects on sample’s surfaces. The benchmark epoxy resin 3585/5003-1 and the hardener were mixed at room temperature and de-gassed for 20 min at room temperature, then casted into a preheated aluminum mold coated with release agents and cured for 30 min at 120 °C.A carbon fabric-reinforced epoxy resin laminate was chosen as the composite material becaus of its widespread use in automotive and aeronautical industries. The carbon fabric-reinforced composite coupons were prepared via the vacuum-assisted resin infusion process (VARI). Eight plies of plain weave carbon fabric and one layer of peel ply (30 cm × 30 cm) were laid on a glass plate mold. The de-gassed resin was preheated to 70 °C and infused into the carbon fiber preform, followed by curing for 30 min at 120 °C.It is well established that the reactivity of imidazole hardeners depends heavily on their chemical structures. Herein, we selected imidazole compounds with and without an electron-withdrawing group; 1C2E4MIM and 2E4MIM, respectively. The latency of EP-1C2E4MIM and EP-2E4MIM was investigated by measuring the viscosity change with time at 25 °C. As shown in Ti), which was determined by the activation energy of the imidazole. However, when the 1C2E4MIM level was increased, the temperature of the maximum exothermic temperature (Tmax) showed a slight reduction, while the total enthalpy (ΔH) showed a significant increase. This indicated that the cross-linking reaction was driven to a higher level of completion when the level of 1C2E4MIM was increased from 2 wt % to 6 wt %. From the practical point of view the 1C2E4MIM level of 6 wt % was already too high, because its heat of reaction (ΔH) reached 441 J/g which could induce unexpected intense exothermic reactions and be considered risky in industry. As a result, 1C2E4MIM levels higher than 6 wt % were not further tested.The curing behaviors of the EP-1C2E4MIM system were investigated by DSC using isothermal and non-isothermal modes . As showTg of the neat resin measured by DMA, along with different levels of 1C2E4MIM, which served as a confirmation of the isothermal DSC results. The data showed that resins with 4 wt % and 6 wt % 1C2E4MIM had their Tg plateaued, indicating that the resins had reached a complete degree of cure within 15 min. However, that with 2 wt % had a much lower value of Tg, indicating that the cross-linking level was far from adequate.The results of isothermal DSC at 120 °C also showed that the cross-linking reaction rates increased with increasing 1C2E4MIM level. Tg of the neat resin can be determined. It is well understood that the Tg of a thermoset resin is mainly affected by intra-molecular stiffness  and cross-linking density [Tg would indicate a higher level of cross-linking. In this case it shows that 2 wt % 1C2E4MIM does not yield adequate cross-linking density and 6 wt % does not provide additional benefit in raising Tg further compared to that of 4 wt %. The thermal mechanical properties of the cured neat resin were characterized by DMA.  density ,30,31. TTg increased as the 1C2E4MIM level increased, indicating an increased cross-linking density. Similarly, their Tan δ curves have only one peak and no relaxations of storage modulus at low temperature, pointing to a homogeneously cured resin. However, when the 1C2E4MIM concentration was further increased beyond 4 wt %, the excessive amount of 1C2E4MIM would induce side reactions, causing phase separation and resulting in two separate glass transition temperatures; one of them was the same as that at 4 wt % (165.6 °C), and the other was lower (137.1 °C).Perhaps the more significant insight taken from Tg at that 1C2E4MIM level, it was clear that 6 wt % was not a favorable level based on the intense heat release as shown by DSC results as well as the lowering of the overall Tg as shown by the DMA. This was the primary reason for selecting 4 wt % as the 1C2E4MIM level for further composite evaluation.Although it was unclear what the mechanisms were that caused the side reactions to start to produce molecules or networks exhibiting lower KIC), is shown in KIC values of the neat resins with 2 and 4 w t% 1C2E4MIM were 0.51 and 0.56 MPa·m1/2, similar to that of the benchmark resin. It was obvious that the neat resin with 6 wt % 1C2E4MIM showed the highest KIC, which was about 42% higher than that of the epoxy resin with 4 wt % 1C2E4MIM and about 40% higher than that of the benchmark resin. This distinctive high toughness of the 6 wt % 1C2E4MIM sample might be due to the two-Tg/two-phase structure discussed above, for the portion with a lower Tg could potentially provide some additional toughening. However, the high toughness achieved by 6 wt % 1C2E4MIM could not overweigh its problems with excessive exothermal heat. Additionally, in considering the cost of 1C2E4MIM at high levels, 4 wt % 1C2E4MIM remained the level of choice for subsequent tests.To investigate the mechanical properties of the cured epoxy neat resins with different levels of 1C2E4MIM, tensile and fracture toughness tests were performed according to ASTM standards. Meanwhile, a commercial fast-curing epoxy resin, 3585/5003-1, was used as the benchmark and tested side by side. As shown in Because the EP-1C2E4MIM resin system evaluated in this work was a fast cure one, it was therefore highly reactive with a potentially limited shelf life. If the shelf life is too short, it would not be suitable for industrial use. For this reason the resin was stored under industry standard storage conditions, at −18 °C, for 12 months and its cure profile was measured by a viscosity-temperature scan using DSR. As can be seen from the results in Tg and deterioration of mechanical properties. The water absorption measured by weight gain at 70 °C as a function of time using the neat resin with 4 wt % 1C2E4MIM is shown in Water resistance is important for composite resins because the absorbed water may cause plasticizing effects, leading to the reduction of Tg change when placed under the “hot” condition, which subjected the resin to heat aging for 48 h at 100 °C. Moreover, the Tg did not drop, as would occur in most epoxy resin systems, after being subjected to “hot-wet” conditions, although there was a slight drop in its storage modulus, as shown in The excellent water resistance of this resin was also reflected by the zero Tg of epoxy resins is mainly determined by the crosslinking density of epoxy networks instead of defects, it is reasonable that this resin exhibited an almost unchanged Tg value due to its low water absorption.Likewise, the flexural strength of the 1C2E4MIM cured epoxy resin remained almost unchanged after heat aging, as shown in The epoxy resin with 4 wt % EP-1C2E4MIM was used as a matrix to fabricate carbon fiber composite test panels. Because the viscosity of the resin at infusing temperature is an important parameter for the VARI process, for it affects the processing time and wettability, the resin viscosity as a function of temperature is shown in Vf) of the carbon fiber-epoxy composite panels was 68.2%. The data showed that the tensile strength of the composite made with the EP-1C2E4MIM system was 631 MPa, much higher than that made from 6808 epoxy resin (374 MPa) [The mechanical test results of carbon fiber-epoxy composite panels are shown in 374 MPa) . Employe374 MPa) . The fleTg and flexural strength when cured at the same conditions, and therefore was deemed more favorable for the intended applications. It was demonstrated that the resins made with 4 wt % 1C2E4MIM loading had a combination of fast cure, reasonable pot life, high Tg, long shelf life at −18 °C, and low moisture uptake, and thus was suitable for liquid molding processes such as VARI. The mechanical properties of the composite made with this resin and a T 700 grade carbon fabric were also tested and shown to be superior to that of a current commercial product.Imidazoles 2E4MIM and 1C2E4MIM were investigated as hardeners for fast cure one-component epoxy resins, which could be used in high-throughput automotive composite part production. Compared to 2E4MIM-cured epoxy resins, 1C2E4MIM-cured epoxies had longer pot life as well as higher


Eighty-nine patients  were enrolled. The median age was 65 years . Thirty-seven patients (42%) were classified as high-intermediate or high risk according to the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI). Patients were categorized into either high or low PB-/BM-CD11b+CX3CR1+ monocyte group according to the cutoffs identified by the receiver-operating-characteristics analysis . The high PB-CD11b+CX3CR1+ monocyte group was significantly associated with high-intermediate and high risk NCCN-IPI group (P = 0.004). With a median follow-up of 27.7 months , the low PB-CD11b+CX3CR1+ monocyte group showed significantly better overall survival (OS) than the high PB-CD11b+CX3CR1+ monocyte group . In contrast, no significant difference was observed between the high and low BM-CD11b+CX3CR1+ monocyte groups. Among patients with high-intermediate to high risk NCCN-IPI, the high PB-CD11b+CX3CR1+ monocyte group showed significantly worse OS than the low PB-CD11b+CX3CR1+ monocyte group . Taken together, PB-CD11b+CX3CR1+ monocyte percentage correlates with the NCCN-IPI risk stratification, which enables identification of subgroups with extremely poor clinical outcomes.Despite their critical roles in angiogenesis and host immunosuppression within the tumor microenvironment, the prognostic significance of myeloid-lineage cells expressing CD11b and CX3CR1 in diffuse large B-cell lymphoma (DLBCL) has not been well studied. We prospectively enrolled newly-diagnosed DLBCL patients at two Korean institutions between May 2011 and Aug 2015. CD11b Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of aggressive non-Hodgkin's lymphomas in Korea . AlthougMyeloid-lineage cells, including monocytes and their precursors, play central roles in cancer growth and progression by directly promoting angiogenesis , 4 and s+CX3CR1+ monocytes are a subpopulation of CD11b+Gr1+ cells  vs. 5/52 [9.6%]) than the low PB-CD11b+CX3CR1+ monocyte group. Thus, high PB-CD11b+CX3CR1+ monocyte group was significantly associated with worse PFS  and OS  of the 89 patients . Among ts Figure . In cont+CX3CR1+ cell groups were significantly associated with PFS and OS  performance status, extranodal involvement, risk stratification by NCCN-IPI , and PB-+CX3CR1+ monocyte percentages in PB or BM with other clinical variables. The high PB-CD11b+CX3CR1+ monocyte group was significantly associated with unfavorable clinical variables, including ECOG performance status ≥2 (P = 0.031), elevated serum LDH levels (P = 0.036), and extranodal involvement (P = 0.003), and also showed a trend towards older age (P = 0.099) (Table +CX3CR1+ monocyte percentages were significantly associated with risk stratification by NCCN-IPI (P = 0.004). However, BM-CD11b+CX3CR1+ monocyte percentages were not significantly associated with any other clinical variables  were classified as higher risk (high-intermediate to high) subgroup and 52 patients (58.4%) were classified as lower risk (low to low-intermediate) subgroups based on the NCCN-IPI. In the higher risk NCCN-IPI subgroup, patients with a high percentage of PB-CD11b+CX3CR1+ monocytes had significantly worse PFS  and OS  than those with a low percentage of PB-CD11b+CX3CR1+ monocytes  [+CX3CR1+ monocyte level might be used to further discriminate the risk of disease progression and death in higher risk NCCN-IPI subgroup. Thus, our results will be useful for discriminating patients at high risk for disease progression and death. Furthermore, these findings have therapeutic implications. For lower risk NCCN-IPI subgroup, traditional R-CHOP chemotherapy shows excellent outcomes regardless of PB-CD11b+CX3CR1+ monocyte status. In contrast, patients in the higher risk NCCN-IPI subgroup with high PB-CD11b+CX3CR1+ monocyte percentages showed very poor OS . Interestingly, the lower survival rate observed in this study may be improved by adding novel drugs that inhibit fractalkine/CX3CR1 signaling. A novel small-molecule CX3CR1 antagonist has shown preclinical evidence of activity by interfering with progression and metastasis in a breast cancer model [+CX3CR1+ monocytes.Our study also demonstrated that PB-CD11bor lung) . With reor lung) . In addier model . Thus, c+CX3CR1+ monocyte groups. Furthermore, although our subgroup analysis provided some useful information, the results of the subgroup analysis might be underpowered. Future studies with larger populations are required to bolster our results.A potential limitation of our study is the relatively small number of patients with short follow-up period (approximately 2.3 years). Indeed, only approximately 20% of patients died, reflecting the limitation of an arbitrary cutoff rather than the biological cutoff that reflects the different clinical outcomes between the high and low CD11b+CX3CR1+ monocytes with risk stratification with the NCCN-IPI, and also identifies a subgroup with extremely poor clinical outcomes. This study also suggests the possibility of PB-CD11b+CX3CR1+ monocytes as a potential therapeutic target in higher risk subgroup of DLBCL.Taken together, our study establishes the significant association of PB-CD11bFrom May 2011 to August 2015, this multicenter, prospective, pilot study included patients with newly diagnosed, biopsy-proven, CD20-positive DLBCL. Other eligibility criteria included no prior history of chemotherapy or radiotherapy and adequate organ function to receive chemotherapy with curative intent. Patients were excluded if they had secondary transformed DLBCL, symptomatic central nervous system involvement, or other concurrent cancers requiring treatments. Patients suffering from severe infection, liver cirrhosis, or psychiatric disorder were also excluded. This study was approved by the Institutional Review Board at each institution. All patients provided written informed consent.2, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1.4 mg/m2 intravenously one day 1, and prednisone 100mg orally for 5 days) every 21 days. Prophylactic granulocyte-colony stimulating factor was recommended but was not mandated. Dose modification with respect to adverse effects in the preceding cycle was performed at the discretion of treating physician. The NCCN-IPI was determined for prognosis [Patients were staged according to the Ann Arbor staging system. Baseline staging evaluation included a physical examination, complete blood count, serum biochemistry with LDH, computed tomographic (CT) scanning of the chest and abdomen-pelvis, CT scanning or magnetic resonance imaging of other involved lesions, fluorodeoxyglucose positron emission tomography, and bilateral BM aspiration and trephine biopsy. Predefined treatment consisted of six to eight cycles of standard R-CHOP immunochemotherapy  was measured by flow cytometric analysis in fresh PB and BM aspirate samples before administration of R-CHOP therapy. PB- and BM-CD11b+CX3CR1+ cells were measured by the same procedure. Briefly, 2 mL of PB or BM aspirates was added to 20 mL of RBC lysis buffer , and then the cells were stained with APC-conjugated anti-human CX3CR1 antibody , and FITC-conjugated anti-human CD11b Ab  for 30 min at 4°C. At least 50,000 MNCs were gated based on forward- and side-scatter profiles, and further subdivided into CD11b+ and CX3CR1+ subpopulations. The percentage of CD11b+CX3CR1+ cells was analyzed using a Navios flow cytometer  curve analysis was used. ROC curve analysis demonstrated that the cutoffs of 3.68% (PB), 3.45% (BM), and 1.77 (PB/BM) had the best sensitivity and specificity to predict disease progression or death. Then, patients were categorized into high or low PB- or BM-CD11b+CX3CR1+ monocyte groups according to the cutoffs. Spearman's correlation coefficient was used to assess the association between PB-CD11b+CX3CR1+ and BM-CD11b+CX3CR1+ cells. PFS and OS were defined as the time from the date of diagnosis to the date of disease progression, death, or last follow-up, as appropriate, and were estimated using the Kaplan-Meier method. Differences in survival outcomes between two groups were tested using the log-rank test. A Cox proportional hazards regression model was used for univariate and multivariate analysis of PFS and OS. Variables with P <0.05 in the univariate analysis were included in the multivariate analysis; a forward conditional method was used, and the results were reported as a hazard ratio and 95% CI. A two-sided P <0.05 was considered statistically significant. All data analyses were carried out using SPSS software, version 19.0 .Pearson's χfication , and thefication . To dete


For both the fabricated sensors, the experimental detection limit was 0.5 μM, while the linear range was 0.5 μM–5.0 mM. The observed results revealed that the simply grown IZO nanomaterials could efficiently be used to fabricate highly sensitive chemical sensors.Herein, we report the growth of In-doped ZnO (IZO) nanomaterials, i.e., stepped hexagonal nanorods and nanodisks by the thermal evaporation process using metallic zinc and indium powders in the presence of oxygen. The as-grown IZO nanomaterials were investigated by several techniques in order to examine their morphological, structural, compositional and optical properties. The detailed investigations confirmed that the grown nanomaterials, i.e., nanorods and nanodisks possess well-crystallinity with wurtzite hexagonal phase and grown in high density. The room-temperature PL spectra exhibited a suppressed UV emissions with strong green emissions for both In-doped ZnO nanomaterials, i.e., nanorods and nanodisks. From an application point of view, the grown IZO nanomaterials were used as a potential scaffold to fabricate sensitive phenyl hydrazine chemical sensors based on the I–V technique. The observed sensitivities of the fabricated sensors based on IZO nanorods and nanodisks were 70.43 μA·mM Phenyl hydrazine is used for the synthesis of agricultural pesticides and insecticides, in pharmaceutical industries, rocket propellant, and explosives. Even a very low concentration of it may prove very fatal and produces drastic environmental and health hazards. The exposure of phenyl hydrazine may lead to dermatitis, skin irritation, liver and kidney damage, and hemolytic anemia . AccordiModifications of the structural, morphological, optical, chemical, electronic, and physicochemical properties of the ZnO semiconductor nanomaterials either through doping or through the formation of composites have always attracted the researcher to synthesize such materials ,6,7,8,9.2·V−1·s−1 at room temperature [3+, further enhances the conductivity [3+ (0.094 nm) is greater than that of Zn2+ ions (0.074 nm), the incorporation of the In3+ ions into the ZnO crystal lattice further produces tensile strains leading to higher surface defects [ZnO is an II–VI, n-type semiconductor with a wide direct band gap of ~3.27 eV, large exciton binding energy (~60 meV), and high electron mobility of 115–155 cmperature ,35,36. Ductivity ,38. It huctivity . Additio defects . These p2 and a power conversion efficiency of 2.7% as compared to the current density of 8.02 mA/cm2 with 1.8% efficiency for the bare ZnO nanoparticles. Badadhe et al. [2S at 250 °C through 3 at. %. In-doped ZnO thin films deposited onto pre-cleaned glass substrates through a conventional spray pyrolysis technique. However, for 50 ppm CO gas, 1 at. % and 2 at. % In-doped ZnO nanostructures prepared by Dhahri et al. [2+ ions from the crystal lattice by In3+ ions, along with the higher rate of O2 adsorption. Ge et al. [Chava et al.  synthesie et al.  observedi et al.  showed ai et al.  explorede et al.  observede et al. .In this paper, a facile thermal evaporation method is reported for the growth of In-doped ZnO (IZO) with two distinct morphologies as a function of reaction conditions on a silicon substrate. The grown nanomaterials were characterized for their morphological, structural, optical, and crystal properties through relevant characterization techniques. Further, the grown IZO nanomaterials were utilized as efficient electron mediators for the fabrication of phenyl hydrazine chemical sensor. Finally, a comparison was made between the In-doped ZnO nanorods and nanodisks for the electrochemical sensing properties towards phenyl hydrazine.The stepped hexagonal nanorods and disk-shaped IZO nanomaterials were grown on silicon substrate by thermal evaporation of metallic zinc (Zn) and indium (In) powders in the presence of oxygen. For the growth of stepped hexagonal nanorods, metallic Zn and In powders were mixed well in the ratio of 5:2, while for the disk-shaped structures, the Zn and In powders were mixed in the ratio of 10:3. Both of the mixtures were separately transferred to the ceramic boats and were placed at the center of the high-temperature ceramic tube furnace. Two different experiments were performed for the growth of stepped hexagonal nanorods and disk-shaped structures. Prior to the experiments, the Si(100) substrates were cleaned with DI water, ethanol, and acetone, sequentially and were dried by nitrogen gas. For both of the experiments, several pieces of Si(100) were placed adjacent to the source boat and ceramic tube furnace chamber was down to 1 torr using a rotary vacuum pump. For the nanorods growth, the reaction was done at 700 °C, while disks were grown at 850 °C under the continuous flow of high purity nitrogen and oxygen gases with the flow-rates of 40 and 100 sccm, respectively. The reaction was terminated in 1.5–2.5 h. After completing the reaction for the desired time, the furnace was allowed to cool to room temperature and the deposited materials were characterized in terms of their morphological, structural, and optical properties.The necessary characterization techniques were utilized for the evaluation of the morphological, structural, optical, compositional, and crystal phases of the as-synthesized IZO nanostructures. Field emission scanning electron microscopy  attached with energy dispersive spectroscopy (EDS) explored the morphological, structural, and compositional characteristics. In-doped ZnO nanostructures were subjected to X-ray diffraction  analysis using Cu-Kα radiation (λ = 1.54 Å) in the diffraction angle range of 20–65° elaborated the crystal structure, purity, crystal sizes and crystallinity. The optical properties of the deposited materials were examined by room-temperature photoluminescence (PL), measured with the He-Cd (325 nm) laser lines as the exciton source.To fabricate the phenyl hydrazine chemical sensors, firstly, the grown IZO nanomaterials were transferred from the substrates to the glassy carbon electrodes (GCE) and wetted by phosphate buffer solution (0.1 M PBS) with pH = 7.4 and dried gently with the flow of high purity nitrogen gas.2HPO4) and 0.2 M monosodium phosphate (NaH2PO4) solution in 100 mL de-ionized water. Consequently, 5 weight % Nafion solution was dropped onto the modified electrode to form a net-like structure on the electrode, which can hold the functional materials during the sensing measurements. Prior to the experiments, the GCE was polished with alumina slurry and was then sonicated in de-ionized water. The modified electrode, i.e., nafion/In-doped ZnO/GCE was finally dried at 40 °C for 12 h. All of the electrochemical experiments were performed at room temperature by Keithley 6517A-USA  electrometer with computer interfacing with a simple current-voltage (I–V) technique. For the two electrode system, the modified electrodes were used as working electrode, in which the Pt wire was employed as a counter electrode. The sensitivity of the fabricated sensors was determined by plotting a calibration curve of current vs. concentration.The PBS  solution was prepared by mixing 0.2 M disodium phosphate , (222), (411), and (332) [3+ ions.The crystallinity and crystal properties of as-grown IZO nanomaterials were examined by X-ray diffraction and the observed results are shown in nd (332) ,49,50. HThe crystallite sizes (d) of the thermally deposited IZO nanostructures were evaluated by using Debye–Scherrer equation (Equation (1)) ,47.(1)dThe morphology of the IZO nanostructures thermally synthesized at 700 °C is shown in The morphology of the IZO nanostructures thermally synthesized at 850 °C is shown in +) with a negatively charged electron (e−) of the singly ionized oxygen (O) vacancies on ZnO surface lattice [In order to evaluate the structural and optical property of the thermally deposited In-doped ZnO nanostructures, Photoluminescence spectroscopic analysis was performed at room temperature using a He-Cd source having 325 nm excitation wavelength. The corresponding PL spectra are shown in  lattice ,58.IZO nanostructures based electrochemical sensors were tested for the detection 0.05 μM phenyl hydrazine in 0.1 M phosphate buffer solution (PBS) with pH = 7, as compared to blank PBS.The current responses were measured from 0.0 to +1.5 V. Significant increase in the current response for even very low concentration of 0.05 μM phenyl hydrazine as compared to blank PBS confirms that both the doped ZnO nanostructures could act as efficient electron mediators and electro-catalysts for the electrochemical detection of phenyl hydrazine at room temperature a,b. For For the estimation of important sensing parameters like sensitivity, linear dynamic range, detection limit, and correlation coefficient, a series electrochemical sensing experiments were conducted using both the IZO nanostructured modified electrodes using different concentrations. A series of phenyl hydrazine solutions with a concentration range of 0.5 µM–5.0 M in 0.1 M PBS were prepared. −1·cm−2, whereas for IZO nanodisks modified GCE it was 130.18 μA·mM−1·cm−2. For both of the modified electrodes the LDR and experimental detection limits were 0.5 μM–5.0 mM and 0.5 μM, respectively. The high sensitivity of IZO nanodisks based sensors are due to the high surface to volume ratio of as-grown nanodisks when compared to the IZO nanorods. The low-dimensionality and reduced dimensions of the IZO nanodisks can be well-understood by the observed SEM images, as shown in It has been reported that the greater the growth temperature the greater the density of defects on the surface of the nanomaterials due to the diffusion of the oxygen from the crystal lattices creating anion vacancies and enhancing the positive charge density ,61,62. AThe phenyl hydrazine responses for different sensors reported in the literature are summarized in 3+ ions induces the surface lattice defects along with an active surface area for the effective adsorption of the O2 molecules from the surrounding air, as well as the phenyl hydrazine molecules from the PBS [2 groups and π-electron density of the phenyl rings of phenyl hydrazine molecules further enhance the attractions between the analyte molecules and the active sites of the IZO nanostructures. The adsorbed O2 molecules are subsequently converted into oxygenated anionic species i.e., O2−, O2−, and O− etc. by extracting the conduction band electrons of the IZO nanostructures [Doping of the ZnO nanostructures with In the PBS ,72. The ructures   nanomaterials, i.e., stepped hexagonal nanorods and nanodisks were grown on silicon substrate by simple thermal evaporation process and characterized in detail using several techniques. The detailed morphological studies confirmed that both IZO nanomaterials possess well-crystallinity with wurtzite hexagonal phase and grown in high density. A suppressed UV emissions and strong green emissions for both IZO nanomaterials, i.e., nanorods and nanodisks were seen in the room-temperature PL spectra. The fabricated phenyl hydrazine chemical sensors based on as-grown IZO nanomaterials exhibited high sensitivities, i.e., 70.43 μA·mM


The vertebrate eye is a highly specialized sensory organ, which is derived from the anterior neural plate, head surface ectoderm, and neural crest-derived mesenchyme. The single central eye field, generated from the anterior neural plate, divides to give rise to the optic vesicle, which evaginates toward the head surface ectoderm. Subsequently, the surface ectoderm, in conjunction with the optic vesicle invaginates to form the lens vesicle and double-layered optic cup, respectively. This complex process is controlled by transcription factors and several intracellular and extracellular signaling pathways including WNT/β-catenin signaling. This signaling pathway plays an essential role in multiple developmental processes and has a profound effect on cell proliferation and cell fate determination. During eye development, the activity of WNT/β-catenin signaling is tightly controlled. Faulty regulation of WNT/β-catenin signaling results in multiple ocular malformations due to defects in the process of cell fate determination and differentiation. This mini-review summarizes recent findings on the role of WNT/β-catenin signaling in eye development. Whilst this mini-review focuses on loss-of-function and gain-of-function mutants of WNT/β-catenin signaling components, it also highlights some important aspects of β-catenin-independent WNT signaling in the eye development at later stages. During gastrulation, the eye field, a group of the retinal precursor cells, is specified within the anterior neural plate. At this stage, these cells are anteriorly and laterally surrounded by the telencephalic progenitor cells. Subsequently, the eye field is divided into two lateral parts, which extend toward the surface ectoderm and give rise to the optic vesicle Figure . The hea2+, and the canonical WNT/β-catenin signaling pathway, the focus of this review. WNT/β-catenin signaling is initiated by binding of the WNTs to the Frizzled/LRP5/6 receptor complex, which leads to the accumulation of β-catenin and nuclear translocation. In the nucleus, β-catenin interacts with the TCF/LEF family of transcription factors and regulates their target genes. In the absence of WNTs, β-catenin is phosphorylated by a “destruction complex” composed of multiple proteins, including AXIN2 and GSK3β, and targeted for degradation , WNT/Ca-catenin in the presumptive lens placode and surrounding head surface ectoderm results in abnormal lens morphogenesis due to cell-cell adhesion defects. Conversely, the lens induction in the β-catenin loss-of-function mutant is not affected as expression of lens-specific markers is maintained , and Lef1 are expressed in the ciliary margin , FZD4, or LRP5 genes. Norrin contains separate binding sites for FZD4 and for LRP5  in the postnatal brain vasculature, Lrp5 plays a major role and Lrp6 plays a minor role in the retinal vascularization .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gastric balloons for weight loss have historically been placed after a screening endoscopy. However, the utility and yield of these endoscopies has not been studied. Therefore, we wanted to evaluate the utility of screening endoscopy and to assess patients who had balloons placed without endoscopy.Data was collected on two cohorts. Cohort 1 consisted of patients who had a screening endoscopy prior to or upon balloon placement. Cohort 2 consisted of patients who were followed after having a balloon placed under fluoroscopic guidance without endoscopy. Balloon intolerance and findings on removal endoscopy were assessed in both cohorts.n = 253), two patients had severe symptoms on history; balloon placement was contraindicated based on screening endoscopy findings. Eleven patients with a history of hiatal hernia and the presence of severe belching demonstrated an insignificant hiatal hernia on endoscopy. In cohort 2 (n = 50), all patients had an unremarkable history. Three previously asymptomatic patients had balloon intolerance and one was found to have a 4-cm hiatal hernia and oesophagitis upon balloon removal. Out of 194 patients, 25 were either intolerant to the balloon or had relevant findings on removal endoscopy. Findings on screening endoscopy did not correlate with balloon intolerance or findings on removal endoscopy.In cohort 1 (These results demonstrate that a careful history can identify patients who may have contraindications for balloon therapy and that balloons can be placed safely after taking a careful history without screening endoscopy. Screening endoscopy may not be useful in predicting balloon intolerance or potential complications. The global increase in obesity warrants adequate prevention programmes and effective treatment strategies . Evidenc2 or a BMI ≥35 kg/m2 with at least one obesity-related comorbidity [Bariatric surgery is only considered for individuals with a BMI ≥40 kg/morbidity , 7.Historically, intragastric balloons have required endoscopy and sedation. The oesophagus and stomach were typically assessed for contraindications either with a screening endoscopy or concomitant to device placement. However, endoscopy is not without risks in obese patients.We first correlated endoscopic findings upon balloon placement to the patient history and to endoscopic findings upon balloon removal and then evaluated what aspects of the patient history were most critical to assess. Using these results, we studied the outcome of patients who had an unremarkable history in whom the balloon was placed only with fluoroscopy.In order to accomplish these aims, we first evaluated patients who had their balloons inserted after a screening endoscopy or under direct endoscopic visualisation (cohort 1). Subsequently, in cohort 2, we followed patients who had balloons inserted under fluoroscopy without endoscopy after a detailed medical history.All patients referred for intragastric balloon therapy were screened using a detailed medical history. A detailed history was taken focused on upper gastrointestinal symptoms including heartburn, belching, nausea, vomiting, epigastric pain/dyspepsia and the relationship of these symptoms to diet and posture, a history of peptic ulcer disease, the presence of a hiatal hernia, medication use, surgery and type of surgery and a family history of gastrointestinal neoplasia. Those who reported to have a hiatal hernia first underwent an X-ray examination with contrast. To guarantee a consistent reporting of the history, only patients who participated in previous clinical trials conducted by the author were included.Endoscopy for positioning and removal of the balloon was performed under conscious sedation with midazolam while monitoring vital signs. In both cohorts, the balloons were removed with endoscopy.Three different balloons were placed in this series Table . Both thIn this series, no endoscopy was performed but the diaphragm was visualised under fluoroscopy and the Orbera balloon positioned 10 cm below the diaphragm –16.t test or Mann-Whitney U test in case of a non-Gaussian distribution. Subgroups were compared using ANOVA with Bonferroni’s correction for multiple testing or Kruskal-Wallis in not normally distributed values. Chi square statistics with Fisher’s exact test when indicated were used to compare categorical values and risk differences and the odds ratio (OR) with 95% confidence intervals (CI) were calculated. The Newcombe-Wilson method was used to calculate the 95% confidence interval for the prevalence differences in baseline characteristics. A p value <0.05 was considered significant.Descriptives are given as means and standard deviation (SD). Group characteristics were compared using Student’s 2. A total of 840 patients were excluded, of which 815 requested balloon placement in a private practice setting  were referred for intragastric balloon therapy of whom 303  with a mean age 36.7 ± 9.8 years were considered eligible with a mean weight of 138.4 ± 27.4 kg and a mean BMI of 46.4 ± 7.8 kg/mOf these 303 patients, 253 were part of cohort 1 and 50 were part of cohort 2. There were no differences between cohort 1 and cohort 2 in age, body weight and BMI  had epigastric pain or dyspepsia and 13 (5.2%) had a known hiatal hernia  of patients had diet-related heartburn with no position dependence and only 16 (6.4%) needed antacids, H4% of patOnly some aspects of the history correlated to endoscopic or biopsy findings Table . Severe In 57 patients who opted for surgery and not for balloon treatment, both an endoscopy and X-ray with contrast were performed. Six (11%) patients reported to have a hiatal hernia. In one patient, this was confirmed by both endoscopy and X-ray. In the other five, a small, insignificant hiatal hernia was seen only on X-ray and not on endoscopy.Seven patients did not receive a balloon at all and were excluded from this dataset Table . Anothern = 2), grade C (n = 1) or grade B (n = 1) oesophagitis which rapidly healed after removal of the balloon. Eleven (7.3%) patients had ulcers: seven were asymptomatic, two had symptoms of balloon intolerance not necessitating balloon removal and two presented with bleeding ulcers. Neither the patient history nor the findings on screening or placement endoscopy correlated to these adverse events.Four (2.6%) patients did not tolerate the balloon and upon endoscopy, no abnormalities were found, and two (1.3%) patients reacted favourably to adjustment in balloon volume Table . AnotherOn three occasions, balloon removal itself resulted in lacerations at the gastro-oesophageal junction caused by vomiting, one of which was a Mallory-Weiss tear.In cohort 2, the same detailed history as described previously was taken for each patient. Special attention was given to the details on history that came forward from cohort 1 . No patient reported any gastrointestinal symptoms. The Orbera balloon was then placed without endoscopy, under fluoroscopic guidance.All but three patients (94%) had an unremarkable balloon therapy period. Full endoscopic reports were available in 43 patients. Upon removal of the Orbera balloon, there were minimal findings on endoscopy Table . Thirty-Upon removal, one major complication occurred. The grasper caught the oesophageal wall and upon removal of the balloon, an oesophageal tear occurred, subsequently causing an oesophageal perforation which had to be closed surgically.Helicobacter pylori status and findings on biopsies, oesophageal manometry, 24-h pH monitoring and gastric emptying time (data not shown). None of the findings was associated with the findings observed upon removal of the balloon.Twenty-five patients in cohorts 1 and 2 had findings upon balloon removal or were intolerant of the balloon. We attempted to correlate intolerance and findings upon balloon removal to history, screening endoscopy, A search of the literature shows that the majority of studies on intragastric balloon treatment report results of weight loss and changes in co-morbidities, but data on complaints, related endoscopic findings and their utility in screening out patients with contraindications are lacking . The goaIn this study, both patients in cohort 1 who had contraindications had severe symptoms on history that correlated with findings on endoscopy of a hiatal hernia with grade D oesophagitis and a peptic ulcer, respectively. Indeed, in the 253 patients assessed in cohort 1, a history alone would have been adequate as a screening method, questioning the use of routine endoscopy for screening balloon patients. Moreover, in 57 patients in our study assessed with both screening endoscopy and X-ray with contrast, X-ray was actually more effective in identifying small anatomical abnormalities such as hiatal hernia. This result further questions whether endoscopy should be the first test performed if further work-up is required after taking a careful history.H. pylori screening is optional (recommendation grade D) [2 blockers or PPIs should have an upper GI endoscopic evaluation. Both guidelines preceded two recent meta-analyses which attempted to put the (routine) use of preoperative endoscopy into perspective [As data on screening endoscopy before endoscopic bariatric therapy were lacking, we searched the literature on the use of endoscopy as a screening procedure in the field of bariatric surgery. In 2013, the American Society for Metabolic and Bariatric Surgery (ASMBS), The Obesity Society and the American Association of Clinical Endocrinologists published guidelines for the preoperative evaluation of a patient undergoing bariatric surgery that indicate a screening endoscopy is not part of a standard work-up, and gallbladder ultrasound and grade D) . In 2015grade D) . Patientspective , 20. BotGiven these findings, we investigated whether balloons could be placed without endoscopy, using only a careful history as a screening tool, with emphasis on symptoms of severe nausea and vomiting, belching, heartburn and a history of hiatal hernia that were brought to the foreground by retrospective findings from cohort 1. We placed balloons in 50 patients under fluoroscopic guidance without a screening endoscopy. In 47 (94%) patients, there were no adverse events during balloon therapy. Three patients did not tolerate the balloon and in one, a large hiatal hernia and grade B oesophagitis was noted upon removal.Could findings on a screening endoscopy predict balloon intolerance and complications? In our 151 patients who received a balloon after a screening endoscopy or under direct endoscopic visualisation and who had a follow-up endoscopy for balloon removal, this was not the case. None of the findings on endoscopy correlated to complications during balloon therapy. Even more sophisticated examinations such as manometry, 24-h pH measurements and gastric emptying studies were not helpful in predicting the outcome. Given our experience and the available literature, other than a careful history, the only intervention that appears to protect patients prior to balloon therapy is to begin acid suppression therapy. Indeed, Rossi et al. have shown that intragastric balloon therapy increases the risk of erosive oesophagitis and indicates that acid suppression therapy should be instituted prior to and during the therapy period . While tNot only is the yield of endoscopy as a screening procedure for balloon therapy very low, it is expensive and not without risk. Obesity has previously been linked to an increased frequency of sedation-related adverse events, including cardiac and respiratory events, in patients undergoing advanced endoscopic procedures with propofol , 23. MorH. pylori (19% in the present series) and a low incidence of upper gastrointestinal cancer.The limitations of our study should be acknowledged. Although it was useful for standardisation across cohorts, these data come from one institution experienced in balloon therapy and from patients involved in previous clinical studies. The conclusions made in this study are limited by the small size of cohort 2, particularly in the setting of a rather low frequency of adverse events, as demonstrated in our large study on 815 patients treated in a private practice setting . Also, oOur experience demonstrates that intragastric balloons may safely be placed without endoscopy using fluoroscopic guidance and a careful screening history prior to placement. Moreover, endoscopy prior to balloon placement does not predict complications during balloon therapy, and endoscopy is not without risk, particularly during intragastric balloon removal.