Ctnnb1ΔE3) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1ΔE3 in keratocytes inhibited corneal epithelial stratification during postnatal development. Unlike the corneal epithelium of the littermate controls, which consisted of 5-6 cell layers at postnatal day 21 (P21), the mutant corneal epithelium contained 1-2 or 2-3 cell layers after Dox induction from embryonic day 0 (E0) to P21 and from E9 to P21, respectively. X-gal staining revealed that Wnt/β-catenin signaling activity was significantly elevated in the corneal keratocytes of the Dox-induced mutant mice, compared to the littermate controls. Furthermore, RT-qPCR and immunostaining data indicated that the expression of Bmp4 and ΔNp63 was downregulated in the mutant corneas, which was associated with reduced corneal epithelial proliferation in mutant epithelium, as revealed by immunofluorescent staining. However, the expression of Krt12, Krt14 and Pax6 in the mutant corneas was not altered after overexpression of Ctnnb1ΔE3 mutant protein in corneal keratocytes. Overall, mutant β-catenin accumulation in the corneal keratocytes inhibited corneal epithelial stratification probably through downregulation of Bmp4 and ΔNp63 in the corneal epithelium.We previously reported that genetic deletion of β-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/β-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized β-catenin mutant ( In mouse corneas, the outermost transparent layer of the eye serves as an ideal model for studying mesenchymal-epithelial interactions4. It is composed of a stratified squamous non-keratinized epithelium, a thick stroma scattered with keratocytes, and a single-layered endothelium5, all of which serve as a major refractive power to transmit light to the retina, as well as a protective barrier against dirt, germs and particles that can damage the eyes8.Bidirectional mesenchymal-epithelial interactions play essential roles in the development of organs with an epithelial parenchyma. Any disorder of these interactions may disrupt tissue formation and cell differentiation of both the epithelium and mesenchyme12. Both of these two signaling pathways play critical roles in ocular morphogenesis15. Gain and loss-of-function studies have revealed that Wnt/β-catenin signaling is involved in eye field formation, neural retina specification, and lens induction during early embryonic stages19. Loss of DKK2, an antagonist of the Wnt/β-catenin signaling pathway, suppresses corneal differentiation during mouse development21. Ectopic expression of Ctnnb1ΔE3 in corneal epithelial cells leads to corneal intraepithelial neoplasia22, which implies that Wnt/β-catenin signaling in the corneal epithelium needs to be repressed during embryonic development and adult homeostasis. BMP4 signaling is involved in cell differentiation and lens induction23. Crosstalk between Wnt/β-catenin and BMP4 signaling has been observed in multiple developmental events28. However, the roles of Wnt/β-catenin and BMP4 signaling pathways and signal crosstalk between them during corneal development are largely unknown, and the mechanism by which corneal keratocyte-derived signals contribute to these processes in the cornea has yet to be fully elucidated.To establish a functional cornea, complex developmental processes must be precisely coordinated by intrinsic regulators and reciprocal signal communication between the epithelium and stroma through signaling transduction, such as Wnt/β-catenin and BMP signaling pathwaysCtnnb1cKO) or co-receptor Lrp5 and Lrp6 (Lrp5cKO & Lrp6cKO), in mouse corneal keratocytes results in precocious corneal epithelial stratification9. In this study, taking advantage of the gain-of-function strategy, we found that expression of a stabilized β-catenin mutant, Ctnnb1ΔE3, in corneal keratocytes inhibited corneal epithelial stratification, an exactly opposite phenotype caused by β-catenin deletion in stromal cells during development. Interestingly, the protein levels of Bmp4 and ΔNp63 were downregulated in the cornea after expression of Ctnnb1ΔE3, which may be responsible for the inhibition of corneal epithelial stratification. Collectively, our data indicated that corneal keratocyte-derived Wnt/β-catenin signaling plays indispensable roles in corneal epithelial maturation during mouse ocular surface development.Recently, we reported that conditional disruption of Wnt/β-catenin signaling by deletion of its key mediator, β-catenin, resulted in precocious corneal epithelial stratification during morphogenesis and postnatal development9. This interesting phenotype prompted us to investigate whether ectopic overexpression of a stabilized β-catenin mutant, Ctnnb1ΔE3, in mouse keratocytes has any effect on corneal epithelial stratification during development. To this end, a new triple transgenic mouse strain  was generated to express Ctnnb1ΔE3 mutant protein (ΔE3β-catenin) in corneal keratocytes upon Dox administration. We administered Dox from E0 to P21 and from P9 to P21, respectively. Like the littermate controls, the Ctnnb1ΔE3 mutant mice were able to develop clear and transparent eyes (data not shown). However, hematoxylin and eosin (H&E) stain showed that, instead of forming 5-6 stratified corneal epithelial cell layers in the littermate controls at P21, expression of Ctnnb1ΔE3 in keratocytes resulted in forming significant thinner corneal epithelia ranging from 1 to 3 cell layers, depending on the time of Dox administration. . To detect any changes in corneal epithelial cell proliferation between littermate controls and mutant mice, the expression of PCNA was examined by immunofluorescent staining. The data showed that the percentage of PCNA-positive cells in the basal corneal epithelium of Ctnnb1ΔE3 mice was dramatically reduced to 53.85%, compared to 78.85% in littermate controls  caused corneal epithelium hyperplastic transformation, which also displayed loss of Krt12 and Pax6 expression in the corneal epithelium22. In this study, we asked whether expression of Ctnnb1ΔE3 in corneal keratocytes could influence corneal epithelial differentiation. Our data show that Krt12 expression in both mutant and littermate controls was restricted to the corneal epithelium, but was not found in the stroma  and regular reverse-transcription PCR (RT-PCR) showed that, at P10 and P21, Bmp4 expression was much stronger in the stroma than in the corneal epithelium  caused the inhibition of corneal epithelial stratification during ocular surface postnatal morphogenesis, which may be attributed to decreased proliferation of the basal corneal epithelial cells, caused by down-regulation of the expression of Bmp4 and ΔNp63 in the mutant mice. This phenotype is the exact opposite of the precocious corneal epithelial stratification observed in the β-catenin loss of function mutant mice (Ctnnb1cKO), as we reported previously9. Notably, we found that both Bmp4 and ΔNp63 were involved in these opposite phenotypes in corneal epithelium. Based on the data from the gain-of-function model, in this study, and loss-of-function data previously reported, we propose that during mouse corneal postnatal morphogenesis, stromal cell-derived canonical Wnt signaling negatively modulates the expression of Bmp4, which, in turn, functions as a paracrine growth factor to control corneal epithelial stratification via the transcriptional factor, p63. In this case, the expression of a stabilized mutant of β-catenin in stromal cells inhibited the expression of Bmp4, which would lead to decreased corneal epithelial cell proliferation and inhibition of epithelial stratification due to decreased ΔNp63 expression in the basal epithelial cells , keratocyte growth factor (KGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-a) have been characterized as the growth factors produced in corneal stroma and have intense impacts on epithelium morphogenesis by regulating its proliferation, motility, and differentiation4. Other growth factors and cytokines from the stroma may also contribute to these interactions during development, homeostasis, and wound healing of the cornea. In the current study, we discovered that corneal stromal cells were the main source of Bmp4 produced in corneas during development. Furthermore, we took advantage of two Dox-inducible compound transgenic mouse strains  to genetically manipulate β-catenin expression, specifically in corneal keratocytes, and successfully identified Bmp4 as a new mediator, derived from stromal cells to modulate corneal epithelial stratification during embryonic development and postnatal morphogenesis. Notably, our study also provided genetic evidence showing that Bmp4 expression in stromal cells is negatively regulated by canonical Wnt signaling during corneal postnatal morphogenesis9. Therefore, the interplay between canonical Wnt signaling and Bmp4 signaling may form the switch to coordinately control corneal epithelial stratification after mouse birth to establish multiple, well organized cell layers with proper function before eyelid opening around P12 to P14.Soluble growth factors and cytokines are generally considered to be the central mediators, which transmit signals between the stroma and epithelium in bidirectional communication39. However, to the best of our knowledge, most of the genetic studies examining the role of canonical Wnt signaling in mouse corneas were performed with corneal epithelium42. For example, it was documented that repression of canonical Wnt signaling activity in corneal epithelium is an essential prerequisite for the differentiation of corneal epithelial cells from their progenitors, as reported in the Dkk2 knockout mice21 and in Pitx2-deficient mice20. Similarly, canonical Wnt signaling should be stringently controlled in corneal epithelium for the proper maintenance of normal corneal structure and function at the mouse adult stage because expression of a stable mutant form of β-catenin (Ctnnb1ΔE3) in adult corneal epithelial cells led to corneal intraepithelial neoplasia22. We made use of the keratocan (Kera) gene promoter/enhancer region to drive a reverse tetracycline transactivator (rtTA) and generated the Dox-inducible mouse lines, KR and KeraRT by transgenic and knock-in strategies, respectively. These two strains can be used to manipulate gene expression in corneal keratocytes since kera gene is exclusively expressed in the neural crest derived cells, including corneal keratocytes43. Benefitting from these two special driver mouse lines, we knocked out β-catenin (Ctnnb1cKO) previously9 and ectopically expressed a stable mutant form of β-catenin (Ctnnb1ΔE3) in the present study, specifically in corneal keratocytes. Excitingly, we observed opposite corneal epithelium phenotypes from these two mutant mice after modifying β-catenin genetically in corneal keratocytes. Additionally, the same or similar molecular mechanism, by which canonical Wnt signaling impacts corneal epithelial maturation, may be implicated in these phenomena because the same molecules, such as Bmp4 and ΔNp63, were involved. In addition to the inhibition of corneal epithelial stratification, we noticed that the expression of corneal stromal specific markers, Lum and Kera, in mutant stroma was reduced after Ctnnb1ΔE3 expression (data not shown), which may imply that the differentiation status of corneal keratocytes had been altered after expression of Ctnnb1ΔE3. In view of the changes in the components of the TGFβ signaling pathway in the Ctnnb1cKO mice9, we propose that reduced expression of Lum and Kera might be attributed to the changes in TGFβ signaling activity. The exact molecular mechanism is being investigated in our laboratory.Canonical Wnt signaling plays vital roles in ocular morphogenesis, homeostasis, wound healing, and pathogenesis of many ocular diseasesCtnnb1cKO mice9. Therefore, we are currently investigating the possible roles of the FGF signaling pathway in the Ctnnb1ΔE3 mutant mice, besides Bmp4 as the first priority candidate, which may both be regulated by canonical Wnt signaling in corneal keratocytes and in turn may mediate corneal epithelial stratification in a paracrine manner.Bmp4 was previously ascertained as the key mediator linking the corneal epithelial phenotype, instigated by loss of β-catenin. However, we did not exclude other growth factors and cytokines which may contribute to the phenotype, since changes in the expression of several growth factors, including FGF family members, were detected in the Ctnnb1ΔE3 in mouse corneal keratocytes inhibits corneal epithelial stratification. Bmp4 and ΔNp63 may be responsible for the inhibition of epithelial stratification caused by Ctnnb1ΔE3. Our data suggest that corneal keratocyte-derived Wnt/β-catenin signaling plays indispensable roles in corneal epithelium maturation during mouse ocular surface development.In summary, aberrant expression of the stabilized β-catenin mutant, KeraRT 32, TetO-Cre (TC)30, Ctnnb1fE3 33 and Axin2LacZ 34 have been previously described. Compound transgenic mice were generated via natural mating of each individual mouse line. All the mice were bred at the Animal Facility of the School of Optometry, Indiana University. Experimental procedures for handling the mice were approved by the Institutional Animal Care and Use Committee, Indiana University. Animal care and use are conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The identification of each transgenic allele was performed by polymerase chain reaction (PCR) using tail genomic DNA as templates. Primer pairs used in genotyping are summarized in Supplementary Table All of the genetically modified mouse lines, ad libitum). Control animals were littermates with either single or double transgene(s).Mice were subjected to systemic induction by Dox chow . To induce mice from the embryonic stage, pregnant dams were given an intraperitoneal (IP) injection of doxycycline , pH7.4; Clontech Laboratories), then fed Dox chow  overnight at room temperature followed by post-fixation in 4% PFA/PBS at 4 °C overnight. Whole mount and paraffin sections were subjected to examination via a stereomicroscope .Excised eyes were fixed in 4% PFA/PBS  for 30 min at 4 °C, then incubated in X-gal staining solution  were stained with Hematoxylin and Eosin and examined under a stereomicroscope . For immunofluorescent staining, tissue sections were de-paraffinized, rehydrated and subjected to antigen retrieval in sodium citrate buffer  at boiling temperature for 30 minutes. Corneal sections were then blocked with 3% bovine serum albumin (BSA) in PBS containing 0.05% NP-40 for 1 hour at room temperature, then incubated overnight at 4 °C with the primary antibodies diluted in the same buffer. After three washes in PBST (PBS/0.1% Tween-20), slides were incubated at room temperature for 1 hour with Alexa Fluor 488- or Alexa 555-conjugated secondary antibodies (Invitrogen) and 1 μg/ml DAPI  as a nuclear counterstain, washed with PBST again, and mounted with Mowiol (Sanofi-Aventis U.S.). Sections were photographed using a Zeiss microscope equipped with a camera (Axiocam Mrm). For data acquisition, we used the Axiovision 4.6 software (Carl Zeiss). The specifications for the antibodies used in this study are listed in supplementary Table KeraRT; TC; Ctnnb1fE3; TH2-GFP44 as experimental mice) and two double transgenic mice , which were Dox-induced from P0 to P10. The left stromal cells with GFP green fluorescence were collected under stereo microscope and stored at −80 °C until used. Total RNA (10 μg) was isolated from the mouse corneal stroma using Trizol reagent (Invitrogen), then annealed to random primers and reverse transcribed with avian reverse transcriptase (RT) kits (Promega), according to the manufacturer’s instructions. RT-PCR was performed using C1000Touch Thermal cycler (Bio-Rad Laboratories Inc.). 30–35 PCR cyclers were carried out to detect the expression of Bmp4 and housekeeping gene Gapdh. RT-qPCR was performed using the CFX96 real-time system equipped with a C1000™ Thermal Cycler (Bio-Rad Laboratories Inc.). After the initial 3 minute denaturing step at 95 °C, 40 subsequent cycles at 95 °C lasting 15 seconds, 62 °C for 15 seconds, and 72 °C for 20 seconds were performed. The cycle threshold values were used to calculate the normalized expression of genes of interest against Gapdh using Q-Gene software. Primer pairs are listed in Supplementary Table Corneal epithelial cells were scrapped and removed under Zeiss stereo microscope by using a rotating burr (Algerbrush II) from two ketamine/xylazine-anesthetized compound transgenic mice  containing 1x protease inhibitor cocktail (Sigma P8340). Cell lysates (20 µg) from each sample were separated on a 4–20% linear gradient Tris-HCl denaturing polyacrylamide Ready Gel (Bio-Rad) and transferred to PVDF membrane (Whatman). Membranes were blocked with 5% nonfat milk in TBST  and probed with primary antibody in the same buffer overnight at 4 °C. After three washes in TBST, membranes were probed with HRP-conjugated secondary antibody for an hour at room temperature and bond second antibody was further detected using an enhanced chemiluminescence assay  and examined and photographed using a VersaDoc 4000MP imaging system (Bio-Rad). Antibodies are listed in Supplementary Material Table t-test  was used to analyze the significance of difference; P < 0.05* was considered statistically significant and P < 0.01** was considered highly statistically significant.A two-tailed Student’s supplementary information


Salmonella enterica sv Typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium difficile, Bacteroides fragilis, Escherichia coli, Haemophilus parasuis, Lawsonia intracellularis, Shigella dysenteriae, and Staphylococcus epidermidis employ a variety of molecular strategies to alter the appropriate functioning of diverse signaling pathways. Among these, Wnt/β-catenin has recently emerged as an important target of several virulence factors produced by bacteria. The mechanisms used by these factors to interfere with the activity of Wnt/β-catenin is diverse and include the repression of Wnt inhibitors' expression by the epigenetic modification of histones, blocking Wnt–Fzd ligand binding, activation or inhibition of β-catenin nuclear translocation, down- or up-regulation of Wnt family members, and inhibition of Axin-1 expression that promotes β-catenin activity. Such a variety of mechanisms illustrate an evolutionary co-adaptation of eukaryotic molecular signaling to a battery of soluble or structural components synthesized by pathogenic bacteria. This review gathers the recent efforts to elucidate the mechanistic details through which bacterial virulence factors modulate Wnt/β-catenin signaling and its physiological consequences concerning the inflammatory response and cancer.The Wnt/β-catenin signaling pathway is crucial to regulate cell proliferation and polarity, cell determination, and tissue homeostasis. The activation of Wnt/β-catenin signaling is based on the interaction between Wnt glycoproteins and seven transmembrane receptors—Frizzled (Fzd). This binding promotes recruitment of the scaffolding protein Disheveled (Dvl), which results in the phosphorylation of the co-receptor LRP5/6. The resultant molecular complex Wnt–Fzd–LRP5/6-Dvl forms a structural region for Axin interaction that disrupts Axin-mediated phosphorylation/degradation of the transcriptional co-activator β-catenin, thereby allowing it to stabilize and accumulate in the nucleus where it activates the expression of Wnt-dependent genes. Due to the prominent physiological function, the Wnt/β-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli, may lead to alterations in cell proliferation, apoptosis, and inflammation-associated cancer. The virulence factors from pathogenic bacteria such as  Such phosphorylation adds a chemical label to β-catenin that is recognized and polybuquitinated by the Skp1-Cdc53-F-Box E3 ubiquitin ligase complex (SCFβ−TRCP) and degraded by the proteasome 26S. The activation of the canonical pathway is dependent on the binding of Wnt glycoprotein ligands to Frizzled (Fzd) receptors and its co-receptor, low-density lipoprotein receptor-related protein 5/6 (LRP5/6) on the plasma membrane. Several molecular events take place after the formation of the ternary complex Wnt–Fzd–LRP5/6. One of them is the translocation of the scaffolding protein disheveled (Dvl) to the complex, and the phosphorylation of LRP5/6 followed by the recruitment of the destruction complex to the receptor. The consequence of this series of protein–protein interactions is the inhibition of Axin-mediated β-catenin phosphorylation, which leads to β-catenin stabilization, cytoplasmic accumulation, and nuclear translocation. Once in the nucleus, β-catenin displaces the corepressor Groucho and binds to the DNA-bound transcription factors T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) to activate Wnt-dependent gene expression  is one of the main defense mechanisms of the innate immune system that protects us against physical, chemical, or biological aggressions . The actThis review highlights new findings that strongly support the regulatory role of Wnt/β-catenin signaling components in the bacterial-induced IR and cancer. Each section contains concise but detailed descriptions on how pathogenic bacteria activate or inhibit the Wnt/β-catenin signaling and what physiological alterations they may cause. We have chosen to present pathogenic bacteria according to their impact on human health and the number of reports cited. Salmonella enterica is one of the major public health issues in third- and first-world countries. According to statistical data in the United States, the number of illnesses caused by this enteric bacterium every year is about 1.2 million, with 23,000 hospitalizations and 450 deaths   , which ipathways .S. enterica effector is the anti-virulence factor A (AvrA), a protein related to Yersinia acetyltransferase effector YopJ and the Xanthomonas campestris pv vesicatoria protein AvrBsT  and SOCS3  in a colon cancer mouse model (The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) regulates many fundamental biological processes including cell proliferation and differentiation, inflammation, and apoptosis . In homese model . It is tArabidopsis thaliana) from Pseudomonas syringae  from Ralstonia solanacearum  contain the genomic cytotoxin-associated gene A pathogenicity island (cagAPAI) that mediates the assembly of a syringe-like structure type IV secretion system (T4SS) , Crk (a proto-oncogene adaptor protein), Csk , Shp2 (a protein tyrosine phosphatase encoded by the gene PTPN11), and others that contain an SH-2 domain  . Injecti2 domain . These C2 domain , triggerH. pylori causes colorectal and gastric cancer because it can activate the Wnt/β-catenin signaling pathway mainly by translocating the virulence factor CagA to the cytoplasm of epithelial cells. Once inside the host cell, CagA interacts and phosphorylates the oncoprotein c-Met receptor that, in turn, activates NF-κB and the expression of numerous pro-inflammatory cytokines and chemokines, enzymes, and angiogenic factors (H. pylori is TFF1 (trefoil factor 1)  . Akt inhactor 1) . In epitpression . Promotenfection . As a re on CagA . It was . pylori .Helicobacter pylori can induce gastric cancer by activating cell proliferation, cell invasion, and angiogenesis  . The suiewed in ]. It is M. tuberculosis, Wnt3a and Wnt5a downregulation and Wnt6 upregulation promote an anti-IR, via canonical (Wnt3a) and non-canonical (Wnt5a and Wnt6) signaling in mouse and human models . This feature constitutes a useful model to investigate the characteristic attaching/effacing (A/E) injuries of the gut epithelium induced by E. coli in humans  expression in two different ways. The first mechanism involves epigenetic modification of the enhancer region of the methyltransferase zeste homolog-2 (EZH2) gene that, in turn, promotes the H3 lysine-27-trimethylation (H3K27me3) in the promoter region of the WIF1 gene  in HeLa cells and inhibit small GTPases by glycosylation resulting in cell rounding and death , 64; how toxin B . When to toxin B . The inhc-Myc even in the presence of the GSK3 inhibitor LiCl, indicating the existence of additional steps in its inhibitory mechanism , which is a heat-labile metalloprotease. Rabbits challenged with high concentrations of BFT show tissue damage and hemorrhage, an increase in IL-8 expression, NF-κB activation, and disruption of the epithelial barrier  but not their proliferation, viability, or cell cycle regulation. They showed that this stimulation was driven by the activation of Wnt/β-catenin pathway. Osteogenesis and osteogenesis stimulated by LPS were confirmed by RT-PCR of osteogenesis-related genes ALP , RUNX2 (Runt-related transcription factor 2), OCN , and OSX (osterix)  . TAZ , H. parasuis may turn pathogenic, causing Gd or an acute respiratory disease, depending on the site of infection. Sometimes, an acute inflammatory systemic response causes massive fibrin exudates in the pleuroperitoneal cavity leading to sudden death (Glässer's disease (Gd) is an acute infection in pigs characterized by a combination of meningoencephalitis, polyserositis, pericarditis, polyarthritis, and, in some cases, primary pneumonia. It is caused by specific serovars of en death .H. parasuis was reported (H. parasuis strain SH0165 was observed to increase β-catenin and reduce phospho-β-catenin at Ser33/37/Thr41 as compared with the non-virulent strain HN0001 in pig kidney (PK-15) cell line and newborn pig tracheal (NPTr) epithelial cell lines. Relative luciferase expression assays showed that the Wnt/β-catenin pathway was activated by increasing doses of the virulent strain and by LiCl, a known GSK3β inhibitor that stimulates the Wnt signaling pathway. This activation correlated well with an increase in β-catenin and a decrease in phospho-β-catenin. β-Catenin was also translocated from the membrane to the cytoplasm and to the nucleus by inoculation with the virulent strain and treatment with LiCl , PAI-1 (plasminogen activated inhibitor 1), and COX2 (cyclooxygenase 2), which significantly produced a recovery of the expression levels of the EMT-related genes E-cadherin, collagen IV, cytokeratin, N-cadherin, Snail, Vimentin, and S100A4  is an infectious disease caused by  control .L. intracellularis infection was investigated by Huan et al.  and a drastic decrease by 28 dpc. Fluorescence and mRNA levels of MUC2  reached the lowest levels by 14 dpc, consistent with the maximum of L. intracellularis fluorescent signal in the ileum. A cell proliferation marker Ki67 and the apoptosis marker caspase-3 also reached their highest fluorescent signals by 14 dpc. These assays demonstrated the correlation between L. intracellularis ileum crypt colonization and the increase in cell proliferation and apoptosis, which result in PE tissue damage. β-Catenin also increased in the upper and lower halves of the crypt by 14 dpc, which was in agreement with the repression of the Wnt/β-catenin-dependent genes ASCL2 (achaete-scute family BHLH transcription factor 2), LGR5 (leucine-rich repeat containing G-protein receptor 5), SOX9 (SRY box 9), and Cyclin D1 , HES1 , and OLFM4   proliferation and differentiation. These pathways transit amplifying (TA) progenitor cells from which secretory cells and intestinal enterocytes differentiate, regulating in that way the intestinal homeostasis. The evaluation of Wnt/β-catenin and Notch signaling during the disruption of intestinal homeostasis caused by n et al. . They deyclin D1 . The int 28 dpc) . ChangesShigella dysenteriae is one of the four species of the genus. It is integrated by 15 serotypes, which are one of the main causative agents of dysentery in developing countries. It destroys the colonic epithelium without causing systemic infection. The typical symptoms produced are diarrhea with blood and mucus accompanied by abdominal cramps and fever  after infection with S. dysenteriae  and specific transcription factors , as well as cytoskeletal targets activated during the pro-IR, have been characterized in a number of bacterial infections. More recently, signaling pathways like Wnt/β-catenin and Notch, whose main function is not related to inflammation, were added to the arsenal of molecular mechanisms touched by pathogenic bacteria. The complexity is even greater because the classical NF-κB pro-inflammatory signaling molecules cross-talk with other signaling pathways such as the Wnt/β-catenin, which are not apparently related to inflammation. This interrelationship frequently gives unexpected phenotypes that need to be taken into account to understand the temporal stages of infection. An integration of signaling at each stage and its proper interpretation may help researchers identify the important clues to fight lethal bacteria. Once signaling molecules are identified, the next step would be to design specific drugs to deactivate the virulence factors. In order to accomplish this ambitious goal, the structural details of virulence factors need to be elucidated. Such biochemical studies combined with bioinformatics, molecular dynamics approaches, and traditional methods  will undoubtedly pave the way to win the battle against infectious diseases.OS-G and JV-A helped with the writing of the manuscript. VB-A conceived, wrote, edited, and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, downregulation of TNKS suppressed the glucose uptake, lactate excretion, and cellular ATP levels and increased cellular O2 consumption rates. Molecular mechanism studies revealed that TNKS promoted aerobic glycolysis at least in part due to upregulation of pyruvate carboxylase (PC) via activation of Wnt/β-catenin/snail signaling. In agreement with these findings, expression of TNKS is positively associated with snail and PC in clinical ovarian cancer samples. Our findings identified TNKS as an oncogenic regulator of ovarian cancer cells proliferation that promotes aerobic glycolysis via activation of Wnt/β-catenin signaling, indicating that the TNKS might serve as a potential molecular target for clinical therapy of Wnt/β-catenin dependent ovarian cancer.Tankyrase (TNKS) plays important roles in the malignancy of several cancers such as human lung tumor, breast cancer, and hepatocellular cancer. However, its exact functions and molecular mechanisms in ovarian cancer remain unclear. In this study, we found that TNKS was aberrantly overexpressed in human ovarian cancer tissues and associated with poor patient prognosis. TNKS inhibition or knockdown not only reduced ovarian cancer cell proliferation, colony formation, migration, invasion, and tumorigenic potential in nude mice but also enhanced the drug susceptibility of ovarian cancer cells through arresting cell cycle and inducing apoptosis. These phenotypic changes correlated with downregulation of targets  of Wnt/ Ovarian cancer is the fourth leading cause of gynaecological malignancy around the world, and estimated 239,000 new patients are diagnosed with ovarian cancer each year , 2. Currβ-catenin, which activates the canonical Wnt pathway [β-catenin pathway including Snail (SNAIL), Axin2 (AXIN2), and matrix metalloproteinases (MMPs). In addition, it is common that the related genes of Wnt/β-catenin pathway are mutated in ovarian cancer, suggesting that the Wnt/β-catenin pathway plays a significant role in the development of ovarian cancer [Wnt signaling is essential during multiple embryonic developmental progresses and its aberrant activation plays a crucial role in the proliferation, differentiation, metastasis, and multidrug resistance of many human cancers, including ovarian cancer , 6. Wnt  pathway . There an cancer .β-catenin degradation with consequent hyperactivation of Wnt/β-catenin signaling [Tankyrases (TNKS) are members of the poly(ADP-ribose) polymerase (PARP) family, including TNKS1/PARP5A and TNKS2/PARP5B . Both TNignaling . Since tignaling . Reflectignaling , hepatocignaling , gastricignaling , breast ignaling , and colignaling . As suchβ-catenin/snail signaling. Our study demonstrated that TNKS inhibition might give a reliable therapeutic benefit for ovarian cancer.Given the strong implication of in the molecular pathogenesis of ovarian cancer, we rationalized that TNKS may play important roles in the development of ovarian cancer. In the current study, we found elevated TNKS expression in a subset of human ovarian cancer. TNKS knockdown in ovarian cancer cells inhibited cell proliferation, cell metastatic abilities, and drug susceptibility. Moreover, TNKS could promote aerobic glycolysis by inducing pyruvate carboxylase (PC) expression through activation of Wnt/β-catenin (ab32572), and phosphorylated β-catenin (ab27798) were from Abcam Ltd. . The polyvinylidene difluoride (PVDF) membrane and the annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-staining apoptosis detection kit were obtained from Millipore . The BioCoat Matrigel invasion chamber was from BD Biosciences, Inc. . The Dual-Glo Luciferase Assay System kit was purchased from Promega  and the EliVision Plus kit was from Maixin Bio . Lactate assay kit (BioVision) and an ATP Bioluminescence assay kit were purchased from Promega Thermo Scientific .XAV939, Wnt3a, taxane, cisplatin (CDDP), MTS, and Glucose Oxidation assay kit were purchased from Sigma-Aldrich . Fetal calf serum and RPMI-1640 medium were from Gibco . Lipofectamine 2000, goat anti-mouse and anti-rabbit secondary antibodies conjugated to HRP were from Invitrogen . Monoclonal antibodies against GAPDH (sc-47724), GLUT1 (sc-377228), PDK1 (sc-293160), cyclin D (sc-70899), snail (sc-271977), G6PD (sc-373886), GCK (sc-17819), DLD (sc-271569), ALDOA (sc-377058), PGK1 (sc-130335), PC (sc-166649) and polyclonal antibody against MDR (sc-55510), and MMP-9 (sc-21733) were from Santa Cruz Biotechnology . Polyclonal antibodies against TNKS (ab227471), HK (ab150423), We followed the methods of our previous studies , 17. OvaThis method was performed as previously described , 17. ParThe three ovarian cell lines  and human normal ovarian epithelial (HNOE) cells were purchased from Cell Bank of the Chinese Academy of Sciences . These four cell lines were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in an atmosphere of 5% CO2. The OVCAR-3 cells were transfected with pll3.7 control vector (shCtrl) or pll3.7-TNKS vector (shTNKS) with Lipofectamine 2000 to obtain stably transfected cells.−ΔΔCt method.Total RNA was extracted using a Simple RNA Extraction kit  according to the manufacturer's instructions as previously described , 17. RevWestern blotting was carried out as previously described , 17. Ali3 into 96-well plates overnight or another time. After treatment with a variety of agents including taxane and CDDP, MTS (5 mg/mL) was added to each well. The plates were incubated at 37°C for 4 h and followed measured at 490 nm using a microplate reader .As previously described , 17, celμm in diameter were counted.As previously described , 17, fiv4 cells were seeded per upper chamber in serum-free RPMI 1640, whereas the lower chambers were loaded with RPMI 1640 containing 5% FBS. After 24 h, the nonmigrating cells in the upper chambers were removed with a cotton swab, and cells migrating through the membrane to the underside of the membrane were stained with 0.05% crystal violet and counted. Cell invasion assays were performed similarly, but with Matrigel.Cell migration was examined by wound-healing and transwell assays, and in vitro invasion was evaluated by using a Matrigel invasion chamber as previously described . For wou0/G1 phases by serum starvation and then cultured for 48 h. The cells were washed, harvested, fixed with ice-cold PBS in 70% ethanol, and stored at 4°C overnight. After centrifugation, the cells were resuspended in 100 g/ml RNase A at 37°C for 30 min and subsequently stained with 50 g/ml PI at 4°C for 30 min in the dark. The cells were analyzed with a FACScan flow cytometer  at 488 nm. Apoptosis was evaluated using an annexin V-FITC/PI double-staining assay. The annexin V-FITC/PI double-staining assay was performed according to the manufacturer's instructions.Cell cycle and apoptosis analysis were performed by flow cytometry as previously described . For celCells were seeded onto 35 mm culture dishes, and after 6 h the culture medium was replaced with fresh complete medium and incubated for additional 48 h. The media were then collected for measurement of glucose and lactate concentration and cells harvested for protein lysates. Glucose levels were determined using a glucose assay kit. Lactate levels were determined using a lactate assay kit according to the manufacturer's instruction. Activity of mitochondrial respiratory chain complex I in cellular lysates was assessed using Complex I Enzyme activity microplate assay kit according to the manufacturer's protocol. All values were normalized on the basis of the Bradford protein assay.Intracellular ATP level was assayed by a bioluminescence method with the ENLITEN ATP Assay System Bioluminescence Detection Kit according to the kit instructions. Cells were seeded in six-well plates for 24 h and then collected and homogenized with 500 ml of ice-cold homogenization buffer (0.25 M sucrose and 10 mM HEPES-NaOH (pH 7.4)). Equal volume of ice-cold 10% TCA (trichloroacetic acid) was added to homogenate and shaken for 20 s and then centrifuged at 10,000g for 10 min at 4°C. Supernatant (400 ml) was collected and added to 200 ml of Tris-acetate buffer  for neutralization. The supernatant was diluted 50-fold with deionized water, and 20 ml of the diluted extract was used for ATP assay by a luminometer.4 cells/well) were seeded in 96-well plates for 24 h before transfection as previously described [OVCAR-3 cells  in accordance with institutional guidelines. This study was approved by the local Ethical Committee of the First Affiliated Hospital of Xiamen University .Four-week-old female BALB/c nude mice were used as previously described . A totalP). Multigroup comparisons of the means were carried out by one-way analysis of variance (ANOVA) test with post hoc contrasts by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.Data were analyzed using GraphPad Prism software . Data are obtained from at least three independent experiments and presented as the means ± SD. Student's t-test (two-tailed), one-way ANOVA, Fisher's exact test, and Pearson's r were used to compare data and to calculate their probability value (P < 0.05), whereas no association was found with age (P > 0.05). These results demonstrated the clinical significance of TNKS serving as a potential molecular target for ovarian cancer patients.Gene expression data from Oncomine demonstrates that TNKS1 gene expression levels substantially higher in ovarian cancer tissues than in normal tissues . ConsistTo assess the functional role of TNKS in ovarian cancer cells, we knocked down expression of TNKS in OVCAR-3 cells (high TNKS expression) using TNKS-specific short hairpin RNA lentiviruses (shTNKS). Expression of TNKS in shTNKS cells was significantly lower than that of shCtrl cells . In resuTo further investigate the oncogenic potential of TNKS, flow cytometry was performed to assess the cell cycle progress and cell apoptosis. Results from cell cycle analysis showed that TNKS inhibition or knockdown increased the number of cell in G1 phase but decreased the number of cells in S and G2/M phases . In addiNext the effect of TNKS knockdown on ovarian cancer cells migration and invasion was evaluated by using wound-healing and transwell assays. As shown in 2 consumption rates were also enhanced , TNKS and PC , and snail and PC   . Those aβ-catenin signaling [β-catenin signaling.TNKS shows various biological functions through regulating Wnt/ignaling , 18. Abeignaling , 20. In β-catenin signaling contributed to fast proliferation, distal metastasis, and multidrug resistance of ovarian cancer [β-catenin signaling, both of TNKS mRNA and protein levels were overexpressed in human ovarian cancer tissues, compared with the normal fallopian tube epithelium tissues. Epithelial ovarian cancer is a distinct histotype of ovarian cancer that accounts for more than 80% of ovarian cancer patients. In our study, ovarian cancer and its adjacent noncancerous tissues collected from 75 patients who underwent surgery were all fallopian tube epithelium tissues. Furthermore, Inhibition of TNKS by XAV939 resulted in inhibition of cell proliferation, migration and invasion, and the resistance to common chemotherapeutic drugs. Effect of TNKS knockdown by RNA interference achieved similar effects as pharmacological inhibition of TNKS. Mechanistically, those antitumor effects caused by TNKS inhibition or knockdown were associated with increased AXIN protein levels, decreased β-catenin protein levels, and decreased β-catenin/TCF4 transcriptional activity, which resulted in inactivating Wnt/β-catenin signaling and then downregulating the expression of Cyclin D, MMP-9, Snail, and MDR, which were closely associated with the development of ovarian cancer [β-catenin signaling. Our findings were in line with previous studies that repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt/β-catenin signaling and inhibited the progression of lung cancer [Previous reports have demonstrated that the frequently hyeractivated Wnt/n cancer , 22. As n cancer –26. Takeg cancer , hepatocg cancer , colon cg cancer , gastricg cancer , and breg cancer , indicatβ-catenin/snail signaling. Many cancers including ovarian cancer need glycolysis to provide primary source of energy even under aerobic condition. This phenomenon is commonly known as the Warburg Effect and is marked by an increased glucose uptake and lactate production [2 consumption rates. Activation of Wnt/β-catenin/snail signaling is known to promote the activity of aerobic glycolysis in tumor cells [β-catenin signaling participating in the development of tumor cells are complicated and changeable [β-catenin signaling and regulate aerobic glycolysis of colon cancer [β-catenin signaling, suggesting PC may become a new target to involve in TNKS-regulated the development of ovarian cancer.Importantly, in order to investigate the mechanisms underlying the tumorigenic function of TNKS, we found that TNKS inhibition or knockdown suppressed the aerobic glycolysis of ovarian cancer cells by reducing PC through inhibition of Wnt/oduction . Our stuor cells . The mecangeable . Evidencn cancer , 32–34. β-catenin signaling. The modulation of this molecular process may be a method of inhibiting ovarian cancer cells growth by downregulation of TNK to interfere with cell metabolism. In particular, the PC may become a new target for further inhibitor design to interfere with Wnt/β-catenin dependent ovarian cancer progression.Taken together, our findings identified TNKS as an oncogenic regulator of ovarian cancer cells proliferation that promote aerobic glycolysis via activation of Wnt/


As a result of the research, the possibility of directional control of the operational properties of epoxy composites by the use of small additives of thermally expanded graphite-graphene structures has been proved. The rational content of the structuring additive in the composition of the epoxy composite (0.05 parts by mass.) was selected, which ensured an increase in the studied complex of physico-mechanical properties. The influence of thermally expanded graphite on the process of structure formation of an epoxy composite has been established. The addition of thermally expanded graphite increases thermal, fire and heat resistance as well as the coefficient of heat-conducting epoxy composite. The most common type of a binder in industry is various epoxy oligomers. The most important problem is the fragility of this type of material, so there are various types of system modifications3. The use of plasticizers and fillers provides necessary performance characteristics of composite materials. The addition of plasticizers provides the elasticity of polymeric materials and changes their glass transition temperature4. The addition of fillers allows to increase the mechanical and physicochemical characteristics of composites7. To provide polymer composite materials and their products with better operational characteristics, various modifying additives and fillers are used5. Mineral fine-dispersed fillers such as talc, basalt, and chromite are wide spread, however, the expected useful effect often exceeds the actual one observed in practice, which is due to the low adhesion of mineral materials to polymer matrices and their tendency to agglomerate. As nanofillers, various types of carbon nanotubes, fullerenes, graphenes, astralenes, technical carbons, as well as titanium and silicon dioxide, diamond batch, white carbon black, etc., are widely used14. Due to the high specific surface, thermal stability and excellent mechanical characteristics, carbon materials  are promising for widespread use in lightweight polymer composites, providing them with enhanced performance characteristics21. Despite the enormous number of works devoted to the study of the effects of various modifying systems (including carbon), there are still insufficiently studied issues related to the influence of modifiers on the structure formation processes, the structure and performance characteristics of polymer composite materials, which predetermines the direction of the research of this work.Currently, polymer composite materials which combine unique properties, high deformation and strength characteristics, and at the same time low specific weight are widely applied in industryThe main goal of this research is to enhance physicochemical and mechanical properties of epoxy composites using thermally expanded graphite obtained by thermal exfoliation of electrochemically oxidized graphite powder.-20 (GOST 10587-93) because it has low viscosity, a narrow limit of the epoxy groups content, the stability of physico-chemical properties. As a hardener of epoxy oligomer, an amine type hardener was used - polyethylene polyamine (PEPA) (TS 6-02-594-85), capable of forming a three-dimensional network structure without heating.The compositions were developed on the basis of epoxy resin EDFor the plasticization of epoxy composites, oligo(resorcinophenyl phosphate) with terminal phenyl groups (ORPP) purity: 99% manufactured by ICL Industrial Products America Inc. (USA), was used in the work. Its chemical formula is shown in Supplementary Fig. 22.ORPP – oligomeric halogen-free plasticizer with flame retardant properties. The choice of ORPP is due to the presence of combustion inhibitor – phosphorus . During thermal decomposition of the composite, the presence of phosphorus provides an increase in the yield of carbonized structures, which are the physical barrier for the inter-diffusion of the oxidant and combustible gases to the combustion zone, which, in general, reduces the flammability of epoxy composite22.The ratio of epoxy oligomer, plasticizer and hardener was previously determined experimentally: 100 parts by mass of ED-20, 40 parts by mass of ORPP and 15 parts by mass of PEPA3 in the galvanostatic mode at the current I = 100 mA/g of graphite, with the capacity Q = 400 mA h/g23. At the next stage, the hydrolysis of the synthesized graphite nitrate was carried out: the reaction mixture was quickly diluted with cold distilled water (t = 15–18 °C) by stirring. Samples were kept in the water for 15 minutes. After dilution, the solid phase was filtered with the help of a Buchner funnel and washed with cold distilled water to pH = 5–7 for wash water. The specific consumption of water for the hydrolysis of graphite nitrate was 100 ml, and for the washing of the obtained oxidized graphite we used 500 ml of H2O per 1 g of the product. The drying of hydrolyzed graphite nitrate to constant weight was carried out in the drying oven at 105 ± 2 °C. The particle size of the solid phase in the aqueous suspension is in the range of 102–106 nm, the fraction of exhaust gas with a dimension of 102–103 nm in the suspension is 5–10%. The weighted fraction of the OG is separated from the main OG fraction by decanting. Thermal reduction and exfoliation of the oxidized graphite was carried out under static conditions for 5 seconds at 250 °C in the SNOL – 1.6.2.5.1/9 – I4 muffle furnace. The schematic of overall synthesis strategy of TEG was shown in Supplementary Fig. Thermally expanded graphite obtained by thermal exfoliation of electrochemically oxidized and hydrolyzed graphite was used as a structuring additive. Electrochemically oxidized graphite (OG) was obtained by electrochemical oxidation of natural dispersed graphite with a particle size of <160 μm in 58% HNO4. The mixture was degassed at 25 ± 5 °C for 30 min under vacuum before curing. The preparation process of TEG/epoxy composites was shown in Supplementary Fig. In plasticized epoxy composition TEG was added as a modifying agent (0.01–1.0 parts by weight). To increase the uniformity of distribution and hinder the aggregation of TEG particles, as well as the activation of its surface and binder, ultrasonic treatment of the composition was used. The parameters of the ultrasound exposure: frequency −22 ± 2 kHz, power - 400 W, duration – 60 minutesdetermination of bending stress and flexural modulus [ISO 178: 2010],determination of strength and modulus of tensile elasticity [ISO 527-2: 2012];determination of compressive strength [ISO 604: 2002];determination of impact strength [ISO 179-1: 2010];determination of Brinell hardness [ISO 2039-1: 2001];determination of heat resistance according to Vicat [ISO 306: 2004];change in mass, rate of change in mass and magnitude of thermal effects during the heating of the samples was studied using the method of thermogravimetric analysis with the help of a derivator of the “Paulik – Paulik – Erdei” system of the MOM brand Q-1500D under the experimental conditions: weight - 100 mg, medium – air, heating interval - up to 800 °С, heating rate - 10 °С/min, relative error does not exceed 1%;the study of the surface morphology of the samples was carried out using a Tescan VEGA 3 SBH scanning electron microscope;determination of thermal conductivity and thermal resistance was carried out using the ITP-MG4 “100“instrument [ISO 22007-2: 2015];FT-IR spectroscopy of TEG particles was carried out using the Shimadzu IRTracer-100;X-ray phase analysis was performed using ARL X’TRA X-ray diffractometer;24.determination of the curing kinetics of the epoxy composition was carried out according to the method described inThe research was carried out using the following methods:According to the data of scanning electron microscopy, during thermal expansion graphite transforms into a worm-like structure with an increased interlayer distance and a highly active, branching, uneven surface, Fig. TEG particles have a nano-layered structure, the thickness of the packs of layers is about 100 nm, Fig. −1 and 3400 cm−1, Fig. −1 is due to the presence of sp2-hybridization of C=C in the structure of graphene. The peak at ~2300 cm−1 corresponds to the peak of CO2 molecules absorbed by the TEG. The band between 1106 cm−1 and 1005 cm−1 corresponds to C-O-C (epoxy group). The band at 1384 cm−1 is the deformation vibration of the carboxyl group.A series of signals on the IR-spectra confirm the presence of the form of oxidized graphene. This presence of hydroxyl groups between the graphene layers is the band between 2800 cmThe fractional composition of TEG is characterized by the bimodal distribution of particles and is represented by particles from 1 to 400 μm, with a predominant number of particles with sizes of 15–20 μm and 140–160 μm, Fig. An attempt to use TEG with a particle size of more than 100 µm as a reinforcing filler will lead to the opposite effect – a decrease in the indicators of physico-mechanical properties. Therefore, at the second stage, the method of liquid-phase separation of graphite in the media of the TCPP plasticizer was used to obtain a suspension of graphene particles. Dispersing was carried out using the UZDN-2T ultrasonic disperser with a radiator power of 400 W and a frequency of 22 kHz. Such a combined mechanochemical action led to additional exfoliation and breaking of particles with a large lateral size.The fractional composition of TEG after ultrasonic dispersion is characterized by a monomodal distribution of particles and is represented by particles from 0.2 to 70 μm, with a predominance of particles with sizes of 10–30 μm, Fig. Thus, the analysis of the TEG structure showed that it could be used as a structuring additive for epoxy composites, which should provide an increase in their operational properties.22.As a polymer matrix we used previously developed composition consisting of 100 parts by mass of ED-20 epoxy resin, 40 parts by mass of ORPP and 15 parts by mass of a hardener – PEPA. ORPP performs simultaneously the functions of both a plasticizer and a flame retardant. The bending stress doubles and toughness increases by 2 times, and the flammability index - the oxygen index (OI) - increases from 19 to 28% by volume, which enables the material to become flame-retardantTEG was added to the epoxy composition in the amount of 0.01–1.0 parts by mass.The conducted studies have shown that the most rational content of TEG as a structuring additive, providing maximum values of physical and mechanical properties is 0.05 parts by mass, Figs 25. In this case, a smaller number (compared to the volume of the composition) of crosslinks is formed in the border area of TEG and the epoxy composition, and, therefore, this area will have greater mobility.Fractography of the destruction of epoxy composite samples without TEG, Fig. 24.When assessing the effect of the modifying additive on network polymers, it is necessary to take into account that the curing process takes place in the presence of a developed surface of the solid material (TEG), which can influence the kinetic characteristics of the polymerization reaction during curing, as well as the formation of the phase structure of the material. The role of the adsorption interaction of the components of the oligomeric composition with the solid surface of the TEG is also greatThe study of the curing kinetics of epoxy compositions, Fig. Figure The addition of small amounts of TEG to an epoxy composite provides an increase in heat resistance according to Vicat from 132 to 165–182 °C, Table 16.Thermal conductivity of compounds used in electrical and electronic equipment is an important characteristic. In most cases, epoxy resins have a relatively low thermal conductivity of ~0.1 W/m · K. Consequently, if there is local heating, epoxy materials work as thermal insulation, which requires the use of components with higher heat resistance or the use of special heat sinks to dissipate heat, otherwise it can lead to overheating and thermal decomposition of the compositeThe addition of even small amounts of TEG to the composition of the epoxy composite increases the coefficient of thermal conductivity by 2.6–4.2 times, while thermal resistance decreases, Fig. As a result of the conducted studies, the possibility of directional control of the operational properties of epoxy composites with the introduction of small TEG additives into the composition, ensuring the creation of high performance epoxy composites, has been proved.The rational content of TEG was selected as a structuring additive in the composition of the epoxy composite (0.05 parts by mass), which provides an increase in the studied complex of physical and mechanical properties. The influence of thermally expanded graphite on the process of structure formation of an epoxy composite has been established. The addition of thermally expanded graphite increases thermal, fire and heat resistance as well as the coefficient of heat-conducting epoxy composite. The developed compounds, modified by TEG, do not support combustion in the air and belong to the class of flame-resistant materials.Supplementary Fig. S1 and S2


Human neurons expressing mutations associated with neurodegenerative disease are becoming more widely available. Hence, developing assays capable of accurately detecting changes that occur early in the disease process and identifying therapeutics able to slow these changes should become ever more important. Using automated live-cell imaging, we studied human motor neurons in the process of dying following neurotrophic factor withdrawal. We tracked different neuronal features, including cell body size, neurite length, and number of nodes. In particular, measuring the number of nodes in individual neurons proved to be an accurate predictor of relative health. Importantly, intermediate phenotypes were defined and could be used to distinguish between agents that could fully restore neurons and neurites and those only capable of maintaining neuronal cell bodies. Application of live-cell imaging to disease modeling has the potential to uncover new classes of therapeutic molecules that intervene early in disease progression. This study establishes a new automated live-cell imaging method that was used to analyze human motor neurons in the process of dying after trophic factor withdrawal. Our aim was to provide an alternative to traditional endpoint survival assays by determining morphologic changes that predate death. This was accomplished by tracking large numbers of individual motor neurons over long periods of time. We identified features of motor neurons that distinguish between those neurons that can fully recover from the early stages of degeneration and those that are committed to death. Our research is of clear interest to neuroscientists interested in disease and improved methods for discovering effective therapeutics.Technical advances in the use of human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) technology now allow the production of human cells, especially discrete populations of neurons, for use in disease modeling, regenerative therapy, and drug screening . When coin vitro–derived motor neurons (MNs) from mouse ESCs, hESCs, and patient-derived iPSCs have been employed productively  withdrawal as a well-known stressor to study the early processes that underlie MN death. We also measured to ability of TF addback to rescue neurons at various stages of degeneration. Finally, the effects of kenpaullone, previously identified in a MN survival screen . Vitronectin , laminin , and fibronectin  were used for coating. Neural differentiation was induced with basic fibroblast growth factor (bFGF) and dual SMAD inhibition for 7 days (NIM: DMEM/F12 with N2 (Life Technologies), B27 (Life Technologies), Dorsomorphin /SB431542 /bFGF . Three days after plating, retinoic acid  and brain-derived neurotrophic factor  were added. Smoothened agonist  was added at day 5. For 3D cultures, day 8 NSCs were dissociated with Accutase and cultured at a density of 400,000/ml on low-adherence flasks in suspension medium containing ROCK inhibitor (10 µm) and bFGF (10 ng/ml) to generate EBs. Spherical aggregates of NSCs were maintained up to day 21 with RA (1 µm), SAG 1.3 (1 µm), BDNF (10 ng/ml), glial cell–derived neurotrophic factor , insulin-like growth factor , cAMP , and dATP . AraC  was added from day 17 to 21 to eliminate proliferating cells.All experiments with hESCs were reviewed and approved by Harvard University Embryonic Stem Cell Oversight Committee. The H9-Islet1::GFP cell line  was confirmed to be mycoplasma negative by PCR and ELISA. MNs were differentiated from hESCs and grown on Matrigel (Corning) with mTeSR (Stem Cell Technologies), for 21 days. A two-step “hybrid method” was devised in which cells were first maintained on dishes in 2D and later switched to 3D embryoid bodies (EBs). Multipotent neural stem cells (NSCs) were derived from hESCs in 2D. For 2D culture, Accutase-dissociated single hESCs were seeded at a density of 4 × 10In this study, assay development was necessary to determine the optimal cell density to achieve uniform distribution of MNs. We tested the robustness of the experimental system across multiple biological replicates using independent batches of MNs. There is some interexperimental variability, which is a combined consequence of variability in the differentiation efficiency, differential cell survival during dissociation, and deviations in the plating cell densities. Despite this, four  and fiveFigs. 2––) at day 1 (1 day after live imaging initiation). To study the early processes that underlie MN death by TF withdrawal, as well as to distinguish different actions of kenpaullone and TF addback treatment on the MNs deprived of TF at day 1, TFs  were reintroduced to the cultures (defined as “TF addback”) at varying lengths of time  after their withdrawal. For kenpaullone treatment, two different concentrations (2.5 and 5 µm) were supplied to MNs during the entire period in which they were maintained in the absence of TF.Withdrawal of trophic factors  is a well-established method to activate neuronal apoptosis . To initTo prepare MNs for live imaging, day 21 EBs were dissociated with Accutase, triturated until no clumps were visible, and seeded into 96-well µClear black-walled plates  with primary mouse glia as feeder cells, and maintained with BDNF (10 ng/ml), GDNF (10 ng/ml), and CNTF (10 ng/ml). FluoroBrite DMEM (Thermo Fisher Scientific) medium with N2 and B27 was used to minimize background fluorescence.in vitro live-cell imaging. We first evaluated how MNs plated at different initial densities performed in our survival assay. We compared cells plated at 3.5 × 104 (35K) cells and at 6.0 × 104 (60K) cells per well. We tracked an average of 110 individual GFP-positive MNs in the 35K/well plating density and 140 MNs per well in the 60K/well plating density (data not shown). We noted that the number of MNs that were able to be tracked did not increase linearly with the number of cells plated. This reflects an inherent challenge with automated live cell tracking, which is that cells are more difficult to individually distinguish and track as they become more crowded. When plating at a lower cell density onto a glial support layer, we observed that MNs exhibit little clumping, which yields a single neuron distribution for more accurate tracking and measurements. Therefore, a 35K/well plating density was employed for all subsequent experiments.Cell density is a critical variable in neuronal cultures and for Three technical replicate wells per condition per experiment , and Nikon designed specialized viewer software to easily review the analysis results from these data sets. To detect the morphologic properties of the cells in the culture, morphologic filters (or masks) were designed to identify and quantify the different features, including cell body number, neurite length, cell body size, compactness, and intensity, in the raw image. To detect nodes, a combination of the cell body filter and neurite filter was used to detect the overlapping regions between neurite and cell body. This filter combination was then used to identify nodes in the raw images. After the filters for detection of each cell were optimized, the tracking was performed using the filter for cell body. The setting parameters used in CL-Quant for this tracking are as follows: minimum object size: 50, maximum object size: 999999, maximum search range: 200, split threshold: 0.90, merge threshold: 0.70, minimum trajectory length: 5, object split: ignore split, object merge: merge with partition, enable lineage: off, enable robust measurement: off, object-to-object overlap: off, remove short track when merging: off.To track individual cells, a cell ID number was assigned to each identified cell body at the beginning of the imaging period. Comparing the cell body filters between two subsequent time frames for morphology, position, and overlap, scores were given to allow the best assignment of cell IDs (see example images in The tracking data set for the cell body filter was then used as the basis to track the number of nodes over time. A moving average (MA) was employed for smoothing the data set before plotting. The node detection algorithm was further used to define class A MNs (with three or more nodes) and class B MNs (with one or two nodes) and to individually track MNs to determine survival outcomes. The number of MNs tracked for all TF addback and kenpaullone experiments at the 35K seeding density can be found in When it was observed that TF addback rescued some class B MNs to class A MNs, we devised a reverse tracking approach to distinguish rescuable class B MNs from unrescuable class B MNs. Rescued and unrescued class B MNs were identified and individually tracked backward to the time point of addback . Then, morphologic parameters such as node number, cell body size, compactness, and velocity at days 6, 7, and 8 were tallied and averaged.t test. Statistical details  are provided in t test. Normal (Gaussian) distribution was determined by column test. A p value of <0.05 (*), <0.01 (**), and <0.001(***) was used to denote statistical significance. Superscript letters listed with figure legends and p-values correspond to the statistical tests shown in Statistical analysis was performed using GraphPad Prism 5. All data , in which GFP expression is regulated by Islet1, an MN-specific transcription factor . PrelimiVideos 1–+), the number of MNs was relatively unchanged over time (–), neurite length per cell, number of nodes per cell, and cell body size per cell did not increase, and the number of MNs gradually decreased . Subsequent Bonferroni posttests indicated that all the descriptors have significant differences between TF+ and TF– conditions at the endpoint (b).Neurons have a complex morphology, with many neurites (axons and dendrites) extending from the cell body A. We usever time B, while ver time C, numberver time D, and cever time E continuecreased B–E. Two- time a, a. Subseqendpoint B–E. Diffendpoint Fb.+ and TF– conditions . The greatest absolute difference was observed when measuring neurite length per cell; however, the node number measurement proved to be a more reproducible value between biological replicates and therefore had the lowest p-value. For this reason, we concluded that quantifying the number of nodes is a more sensitive indicator of the presence of TF support than measuring MN survival itself.We measured the fold change in the different morphometric parameters between days 1 and 12. Our results demonstrated that number of nodes per cell exhibited significant differential responses between TFd; n = 5, p < 0.05). At each time after TF withdrawal up to 8 days, TF addback increased neurite length per cell  and number of nodes per cell , although the extent of recovery decreased with longer periods of TF withdrawal. Importantly, changes in these morphologic properties of MNs were again much more sensitive measures of cellular responses to the presence of TFs than was the number of cell bodies.We sought to define critical points in the neuronal degeneration and regeneration processes using population analysis of all cultured cells. We tracked and averaged the number of MNs, as well as their morphometric features, over time following TF withdrawal. We specifically aimed to determine for how long after TF withdrawal were MNs capable of being rescued by restoring TFs to the medium (TF addback), another paradigm that has been used for many years . MNs undf; n = 5, p < 0.05), and, to some degree, neurite length and the number of nodes, although this was not statistically significant . m kenpaullone, and TF withdrawal.We also determined the effects of kenpaullone, an agent previously identified in an MN survival screen , using M+ , number of nodes (magenta), and cell body size (green) of MN over time. Because of the increasing complexity of the neuritic network, the number of nodes metric is better than neurite length for tracking at the single-cell level. Images were captured every 6 hours for 14 days. 10.1523/ENEURO.0001-18.2018.video.2Video 3.A)(. TF was withdrawn at day 1 (1 day after live imaging initiation), and TF was added at day 6 C)( rescues both neurite length and number of nodes. Kenpaullone C)( is less protective compared to TF addback B), number of nodes (magenta), and cell body size (green) of MN in four experimental conditions: TF 10.1523/ENEURO.0001-18.2018.video.3n = 0, 1, 2, 3, >4; + . At the end of the analysis (day 12), the number of MNs with 4 or more nodes was significantly higher in TF+ compared to TF– .Thus, we divided MNs into five categories according to their number of nodes . TF addback at days 7 and 8 appeared to increased class A hours somewhat as well, but those effects were not statistically significant. Next, we used the same class A hour measurement to evaluate kenpaullone’s effects on MNs, using the same experimental conditions as in j; n = 5).We next collected data on single MNs classified according to their number of nodes: class A , class B (one or two nodes), and class C (no nodes). We wanted to determine if neurons with more nodes were more likely to survive TF withdrawal than neurons with fewer nodes. We started by individually tracking the MNs with the most nodes (class A). We defined a metric, “class A hours,” as the length of time in which MNs remained as class A B. After k; n = 5, TF addback at day 6: p < 0.001, TF addback at day 7: p < 0.05, TF addback at day 8: p < 0.05). Similarly, tracking class B MNs indicated that more class B MNs were rescued to class A MNs (class B to A) after TF addback at day 6, 7, and 8 compared to the TF withdrawal condition, with the largest rescue occurring at day 6 . We also tested the effects of kenpaullone under the same conditions as in m; n = 5, p < 0.01), but in contrast to TF addback, kenpaullone did not cause class B MNs to develop into class A MNs . Meanwhile adding kenpaullone at day 6 did not improve survival (data not shown). We next sought to examine the morphometric changes of individual MNs over time after TF withdrawal using computational single-cell tracking. We could not track class C MNs, as they quickly died and disappeared from view. Thus, we tracked the individual fate transitions within and between class A A and cla°; n = 5, p < 0.001) and a larger cell body  compared to class B MNs that did not recover. Thus, MNs with more nodes and larger cell bodies are the subset of MNs that are most likely to recover after a death stimulus, whereas class C MNs cannot be rescued with any of the treatments or compounds we used.Results from the previous section established that many class A MNs could retain class A features after both TF addback and kenpaullone treatment, but only TF could permit class B MNs to mature into class A MNs. However, not all class B MNs were rescued to class A MNs, even after TF addback. Live-cell imaging of individual cells may be much more informative than population-based methods of studying cell behavior . This isin vitro–derived cultures, because the terminally differentiated cells of interest arise and mature at different times. We used the morphometric analysis assay to detect distinct modes of action underlying MN rescue seen with TF addback versus kenpaullone treatment. While both treatments were efficacious, only TF addback restored class B MNs to class A. In contrast, kenpaullone primarily acted to improve survival of the class A MN population. The robust, beneficial effect of TF addback is consistent with reports that TFs can salvage unhealthy neurons in animal models of ALS and have a neuroprotective effect on axotomized MNs (The software we employed is highly customizable, so any morphologic attribute can be selected to categorize and track cell outcomes. Importantly, these time-dependent measurements do not necessarily need to be predefined. Because each cell is individually tracked, its “lifetime” can be computationally annotated by specific morphologic features. This ability is especially relevant to ized MNs .Changes that occur early in neurodegenerative processes might provide key, clinically relevant insights into how to identify cells that possess the capacity for full functional restoration. In this study, we showed that MNs exposed to stress-inducing conditions rapidly exhibited changes in their neurite length and number of nodes. Changes in these parameters appeared to occur early in the death process and constituted sensitive indices of neuronal health. Neurite retraction and reduced soma size are known to accompany neurodegeneration, even in patients . We therOur live-cell analysis platform possesses flexibility and analytical power, making it broadly applicable to a wealth of cell biology inquiries. Live-cell analysis of individual cells is also strongly complemented by the current growing trend in single-cell RNA sequencing . These t


IGF-I was shown to increase biglycan expression (p ≤ 0.01), whereas biglycan-deficiency attenuated significantly both basal and IGF-I induced cell proliferation of MG63 cells . These effects were executed through the IGF-IR receptor whose activation was strongly attenuated (p ≤ 0.01) in biglycan-deficient MG63 cells. Biglycan, previously shown to regulate Wnt/β-catenin pathway, was demonstrated to induce a significant increase in β-catenin protein expression evident at cytoplasmic (p ≤ 0.01), membrane (p ≤ 0.01), and nucleus fractions in MG63 cells (p ≤ 0.05). As demonstrated by immunofluorescence, increase in β-catenin expression is attributed to co-localization of biglycan with the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) resulting in attenuated β-catenin degradation. Furthermore, applying anti-β-catenin and anti-pIGF-IR antibodies to MG-63 cells demonstrated a cytoplasmic and to the membrane interaction between these molecules that increased upon exogenous biglycan treatment. In parallel, the downregulation of biglycan significantly inhibited both basal and IGF-I-dependent ERK1/2 activation, (p ≤ 0.001). In summary, we report a novel mechanism where biglycan through a LRP6/β-catenin/IGF-IR signaling axis enhances osteosarcoma cell growth.Biglycan, a small leucine rich proteoglycan (SLRP), is an important participant in bone homeostasis and development as well as in bone pathology. In the present study biglycan was identified as a positive regulator of MG63 osteosarcoma cell growth ( The small leucine-rich proteoglycans (SLRPs) are a family of proteoglycans (PGs) characterized by a relatively small protein core 36–42 kDa), containing a number of leucine-rich repeats and able to undergo post-translational modifications, notably substitution with various types of glycosaminoglycan side chains , 2. The  kDa, conBiglycan is a canonical class I SLRP member containing two chondroitin or dermatan sulfate side chains which are covalently bound to attachment sites at the N-terminal . RecentlRecombinant human biglycan (2667-CM) and IGF-I  were obtained from R&D Diagnostics. Selective inhibitor of ERK1/2  and allosteric inhibitor of IGF-IR  were used in this study. Primary antibodies from Santa Cruz Biotechnology used, anti-biglycan , anti-β catenin , anti-IGF-IR total , anti-pERK1/2  and anti-fibrillarin . In addition, anti-actin  human osteosarcoma cell line of moderately differentiated fibroblastoid-type cells and high metastatic capacity was utilized. Cells were grown in DMEM  supplemented with 10% fetal bovine serum , glutamine , gentamycin  and penicillin/streptomycin . Before addition of treatments (biglycan 10 μg/ml or IGF-I 10 ng/ml), cells were cultured in serum free medium for 24 h at 37°C and 5%CO2. Inhibitors, when used (ERK inhibitor or IGF-IR inhibitor), were added 1 h before growth factor treatment.In this study, MG63 2 in 0% FBS. The cells were then lysed and their number was calculated using the CyQUANT fluorometric assay  according to the manufacturer's instructions. Fluorescence was measured in a Fluorometer (Biotek) using the proposed excitation (485 nm) and emission filters (528 nm). A separate standard curve was used to convert fluorescence units to cell numbers. All experiments were performed in triplicate.Growing cells from non-confluent cultures were harvested and seeded in black 96-well plates  at a density of 3,500 cells per well in 200 μl of DMEM (10% FBS). The cell density number was chosen from optimization experiments (data not shown). The cells were allowed to rest overnight. If necessary, transfection with siRNAs was performed in a serum-free medium without antibiotics for 6 h. This was then replaced with fresh medium (0% FBS) with antibiotics. Treatments were added for the next 48 h at 37°C and 5% COFor transfection experiments, the cells were plated in serum and antibiotic free medium in either 96 well plates , or 24-well plates  or T25 flasks (1:8 dilution of a 90% confluent T75 flask). Short interfering RNA specific for biglycan (siBGN)  or β-caTotal ribonucleic acid isolation was performed using TRIzol , according to the manufacturer's instructions. One microgram of total RNA was added for cDNA synthesis using the TAKARA (RR037A) RT cDNA synthesis kit. For semi-quantification of the genes of interest, real-time reactions were performed in an Mx300P cycler using the Universal qPCR kit  in a total volume of 20 μL. PCR conditions for amplification and primers used are listed in Table Total protein secreted into the serum-free culture medium was concentrated using Amicon Ultra 15 mL  centrifugal concentrator tubes. The initial volume of 3 mL serum-free medium collected from culture, to isolate secreted proteins, was concentrated to final volume of 500 μL whereas, harvested cells were lysed with RIPA solution . Equal amounts of protein, either cell extracts or secreted, were subjected to SDS-PAGE using 8% polyacrylamide gels under reducing conditions. Separated protein bands were transferred to nitrocellulose membranes in 10 mM (pH 11), containing 10% methanol. Membranes were blocked overnight at 4°C with PBS containing 0.1% Tween-20 (PBS-Tween) and 5% (w/v) low-fat milk powder. The membranes were incubated for 1 h at room temperature (RT) with primary antibody in PBS containing 0.1% Tween-20 (PBS-Tween) and 1% (w/v) low-fat milk powder. The immune complexes were detected after incubation with the appropriate peroxidase-conjugated secondary antibody diluted  in PBS-Tween, 2% low-fat milk, using the LumiSensor Chemiluminescent HRP substrate kit , according to the manufacturer's instructions. Protein expression of Actin was used to correct for the amount of each sample analyzed.2, 0.5% NP-40 with protease and phosphatase inhibitors). Tubes were incubated on a rotating platform at 4°C for 10 min and centrifuged strictly at 1,000 rpm for 5 min at 4°C. The supernatants (cytoplasmic protein fractions) were kept at −80°C. The pellets were resuspended in 100 μL of RIPA solution , vortexed and incubated for 60 min on a rotator at 4°C. Samples were centrifuged to pellet insoluble fraction at 13,000 rpm for 30 min at 4°C and after the supernatants (nucleus protein fractions) were kept at −80°C as well as the pellets (membrane fractions).Treated cells in T25 flasks were detached using trypsin-EDTA , deactivated with PBS and centrifuged at 1,100 rpm for 5 min. Supernatants were discarded and the pellets resuspended in 250 μL of ice cold PBS supplemented with protease and phosphatase inhibitors. After centrifugation at 1,100 rpm for 5 min at 4°C the pellets were resuspended in 200 μL of 5x CPV NP-40 lysis buffer  or Alexa Fluor 488 . TO-PRO-3 iodode  diluted 1:1,000 in de-ionized H2O was applied for 10 min to stain nuclei. The coverslips were then placed onto slides using glycerol as a mountant and visualized using confocal microscopy.MG63 cells were seeded on round coverslips placed in 24-well plates, at a concentration of 50,000 cells/well and incubated in complete medium for 24 h. After a 24-h serum starvation, treatments were added and the cells incubated for 48 h at 37°C and 5% COt-test, or ANOVA analysis of variance with Tukey's post-test, using GraphPad Prism (version 4.0) software.The statistical significance was evaluated by student's p ≤ 0.001; Figure We have previously shown that biglycan is a potent modulator of osteosarcoma cells' migration . In thisp ≤ 0.01), was demonstrated  upregulated, as shown by real-time PCR analysis  for 48 h and performing western blot analysis to supernatant and cell extract, a statistically significant increase of secreted biglycan  was abolished in biglycan-deficient cells  in biglycan-deficient MG63 cells  in β-catenin protein expression after the addition of biglycan ; to the nucleus  as well as to the membrane  was demonstrated in biglycan treated cells as compared to control. Therefore, biglycan regulates the protein expression and localization of β-catenin in MG63 osteosarcoma cells.β-catenin is known to regulate gene expression following Wnt pathway activation which affects cell proliferation –32. The p ≤ 0.001) and mRNA levels  for 48 h and mRNA and protein expression of β-catenin were analyzed using Real Time PCR and Western blot analysis, respectively. This strategy resulted in an efficient downregulation of β-catenin at both protein (p ≤ 0.01) in β-catenin deficient cells was shown , cells transfected with siβ-catenin and cells transfected with scrambled siRNAs (siScr). Interestingly, a strong decrease in the phosphorylation levels of IGF-IR  downregulated in biglycan-deficient cells, suggesting that biglycan affects both canonical and non-canonical signaling in osteosarcoma cells.Wnt-mediated transcription through β-catenin—T-cell factor (TCF)/Lymphoid enhancer-binding factor (Lef) transcription factors, is characterized as the canonical wnt/β-catenin signaling pathway. To assess the modulation of this pathway by biglycan we examined the expression of a β-catenin downstream target molecule cyclin D1 , 35. As p ≤ 0.01 and p ≤ 0.001, respectively; Figure p ≤ 0.001) inhibited in biglycan-deficient (siBGN) cells. These results suggest that ERK1/2 is a downstream mediator of IGF-IR/biglycan signaling dependent cell growth.IGF-I/IGF-IR are known to activate several downstream signaling pathways that regulate cell functions including that of the MAP kinases , 37. ThuBiglycan, an important component of bone ECM, has been correlated to the emergence of mesenchymal tumors , 21, 38.We examined the possibility of a cross-talk between IGF-IR and Wnt/β-catenin pathways in the biglycan effect on osteosarcoma growth. Interestingly, in this study β-catenin deficient MG63 osteosarcoma cells were shown to have abrogated both basal and IGF-I-dependent IGF-IR activation, and strongly attenuated growth. Likewise, we demonstrate for the first time that treatment of MG63 osteosarcoma cells with recombinant biglycan increases β-catenin expression and mediates its cellular deposition and enhancingβ-catenins' cell membrane and cytoplasmic localizations. Previously, it has been shown in mouse calvarial osteoblasts model, that biglycan interacts with both the canonical Wnt ligand Wnt3a and the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). Furthermore, this co-localization enhanced downstream Wnt-induced β-catenin/T cell-specific factor-mediated transcriptional activity . In ordeActivation of Wnt signaling was previously shown to enhance the survival of osteoblastic cells, as demonstrated by the reduction in osteoblast and osteocyte apoptosis in mice not expressing the Wnt inhibitor sFRP1 (soluble frizzled-related protein 1) . MoreoveOur present data demonstrate that biglycan, by binding to the LRP6 receptor, inactivates inactivates the formation of the β-catenin destruction complex enhancing its cytoplasmic, membrane as well as nuclear deposition. Increased β-catenin pool interacts with IGF-IR, strongly enhancing pIGF-IR expression at different subcellular compartments. Furthermore, ERK1/2 was found to be a downstream mediator of biglycan/IGF-IR/β-catenin signaling axis facilitating MG63 cell growth. However, we do not exclude the possibility that ERK1/2 may be a downstream mediator of other membrane receptors including Wnt receptors  or that in vitro/in vivo models as well as interpolation with data obtained from patient biopsies could provide more refined data to the mechanism presented here.Our study presents some limitations. First, we utilized a single cell line (MG63), and second, we have not identified the components of the IGF-IR/β-catenin co-localization complex, an issue currently under investigation. Future studies in different In summary we report a novel mechanism where biglycan, an ECM proteoglycan, through a LRP6/β-catenin/IGF-IR axis enhances osteosarcoma cell growth JA, AB, DN, DP, AT, and GT conceived and designed the experiments and wrote the paper. JA, AB, DN, DP, AP, and AT performed the experiments and analyzed the data. All authors approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries . Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At Xcr, the size of cluster aggregates and regular distributions reach a local maximum. Before a similar model was developed for cholesterol/DMPC mixtures (Sugar and Chong (2012) J. Am. Chem. Soc. 134, 1164–1171) and here the similarities and differences are discussed between these two models.When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl- Bacillus brevis. The native gramicidin, designated as gramicidin D, has 15 alternating D- and L-amino acids with three different components: 80% gramicidin A, 5% gramicidin B, and 15% gramicidin C , bu, bu23], Second, our model assumption came from the results of molecular dynamics simulations for cholesterol/DMPC  and gramAreg values are in the range of 0.98–1.0  mixtures with increasing gramicidin ratio. It is assumed that instead of condensation of DMPE to gramicidin, the aggregation of gramicidin takes place in the bilayer [The results obtained from this study can be extended to other phospholipid membrane systems. However, it should be pointed out that condensation of phospholipid to gramicidin depends on the type of the polar headgroup of the lipid. For example, there was no decrease in transition enthalpy detected in gramicidin/1,2-dimiristoyl- bilayer . This ki bilayer .The mole fraction dependent behavior revealed in this study reflects how lipids and peptides are laterally organized in gramicidin/DMPC liposomal membranes. Since lateral organization is key to understanding membrane structure, this study paves the way to deepen our knowledge of the structure-activity relationship between gramicidins and phospholipids in model membranes. The results obtained from this study may also have significant functional implications. It has been proposed that, in addition to increased membrane permeability, the main cause of gramicidin’s killing of Gram positive bacteria is the induction of hydroxyl radicals through Fenton reaction and the subsequent depletion of nicotinamide adenine dinucleotide (NADH) from the tricarboxylic acid cycle . Since t−1cm−1 [−3 mbar) for ~12 h using a Labcono freeze-dry system . The dried DMPC/gramicidins were hydrated using a pre-warmed buffer , followed by vortexing for 2–3 min at 40 °C to make multilamellar vesicles (MLVs) and annealing through three heating (40 °C for 0.5 h)/cooling (4 °C for 0.5 h) cycles. Unilamellar vesicles (LUVs) were made from MLVs by passing the vesicles 10 times through two stacked 400-nm Nucleopore polycarbonate membranes under nitrogen gas pressure at 40 °C using an extruder . The accuracy of the determination of gramicidin content in DMPC is estimated to be ~0.001–0.002 mole fraction, and the procedures to achieve such a high mole fraction accuracy have been described previously [Gramicidin A  and gramicidin D  were dissolved in either 2,2,2-trifluoroethanol (TFE) or methanol to create separate stock solutions. The concentrations of the gramicidin A and D stock solutions were determined by measuring absorbance at 280 nm in methanol on a Beckman DU-640 spectrometer  using the extinction coefficient 21,577 (our determination) and 20,700 M−1cm−1 , respect−1cm−1 . Gramicieviously . The parM) data are presented using phasor plots, which are a convenient and model independent way to describe complex fluorescence decays [M sinφ) against G (=M cosφ) [Lifetimes of intrinsic gramicidin fluorescence in gramicidins/DMPC mixtures were determined on an ISS K-2 multifrequency phase-modulation fluorimeter using a 285-nm LED as the light source . The emission was observed through a Hoya U360 filter. Phase and modulation values were determined relative to an ethanol solution of 2,5-diphenyloxazole . For a given liposome sample, the phase and modulation values were measured as a function of modulation frequency three times. For each measurement at a given modulation frequency, hundreds of data acquisition iterations were employed until the maximum estimated standard error reached 0.2%. Typically, 15 modulation frequencies in the range 1–200 MHz were used. The phase (φ) and modulation  ,54. Eachex = (I435 − I500)/(I435 + I500)) [435 and I500 are the fluorescence intensities at 435 nm and 500 nm, respectively. The sample was excited at 340 nm.Laurdan is a polarity sensitive fluorescent probe with its chromophore located near the diester phospholipid polar headgroup region. For fluorescence labeling, 1.1 µL of 1.055 mM Laurdan in dimethyl sulfoxide (DMSO) was added into 3 mL of liposome sample containing 450 nmol of lipid (probe-to-DMPC ratio ~1/400) at 37 °C and the samples were incubated at 37 °C for one hour prior to fluorescence measurements. The amount of DMSO added has previously been shown not to perturb membrane structure to any significant extent [+ I500))  was thenAreg values are in the range of 0.98–1.0 (Areg values are in the range of 0.65–0.90 [The novelty of the current study is to reveal that the mole fraction of gramicidin relative to the matrix lipids is of fundamental importance for the behaviors of gramicidin/PC mixtures because membrane lateral structure can be altered significantly in a biphasic manner in the vicinity of a critical gramicidin mole fraction. In the long run, the finding of this kind may deepen our understanding of the structure-activity relationship between membrane lipids and membrane proteins/peptides. Specifically, in this study, we present a statistical mechanical model, which describes how gramicidin molecules are distributed in the plane of phospholipid, DMPC, membranes. This model is supported by our fluorescence phasor data and in good agreement with the data from NMR, flash photolysis and molecular dynamics simulations by others ,14,36. A0.98–1.0 , while i.65–0.90 ,19. Othe