ST6GAL-1 mRNA was assessed in untreated human rectal adenocarcinoma by PCR assays. Samples were further assessed by Western blotting, Caspase-Glo apoptosis assays, and colony formation assays. The presence of functional ST6GAL-1 was assessed via flow cytometry using the Sambucus nigra lectin, which specifically binds cell surface α2,6-linked Sia, and via lectin precipitation. In patient-derived xenograft models of rectal cancer, we found that ST6GAL-1 protein was increased after chemoradiation in a subset of samples. Rectal cancer cell lines demonstrated increased ST6GAL-1 protein and cell surface Sia after chemoradiation. ST6GAL-1 was also increased in rectal cancer organoids after treatment. ST6GAL-1 knockdown in rectal cancer cell lines resulted in increased apoptosis and decreased survival after treatment. We concluded that ST6GAL-1 promotes resistance to chemoradiotherapy by inhibiting apoptosis in rectal cancer cell lines. More research will be needed to further elucidate the importance and mechanism of ST6GAL-1-mediated resistance.Locally advanced rectal cancer is typically treated with chemoradiotherapy followed by surgery. Most patients do not display a complete response to chemoradiotherapy, but resistance mechanisms are poorly understood. ST6GAL-1 is a sialyltransferase that adds the negatively charged sugar, sialic acid (Sia), to cell surface proteins in the Golgi, altering their function. We therefore hypothesized that ST6GAL-1 could mediate resistance to chemoradiation in rectal cancer by inhibiting apoptosis. Patient-derived xenograft and organoid models of rectal cancer and rectal cancer cell lines were assessed for ST6GAL-1 protein with and without chemoradiation treatment.  Rectal cancer and other gastrointestinal cancers, including gastric and esophageal cancers, are treated with chemoradiation including 5-fluorouracil (5-FU), or its oral equivalent capecitabine, and radiation daily for 6 weeks followed by surgery. Rectal cancer accounts for one-third of all colorectal cancer cases with approximately 45,000 Americans diagnosed annually, and this rate is increasing . Around There is a critical need for improved therapies in rectal cancer in order to enhance survival and potentially decrease morbidity by improving the rate of complete response to chemoradiotherapy. In this study, we investigated a novel role for ST6GAL-1 in therapeutic resistance in rectal cancer. We generated and treated three patient-derived xenograft models of rectal cancer with chemoradiation and found that ST6GAL-1 protein was increased in two out of three of the models. We then utilized human rectal cancer samples to assess the prevalence and distribution of ST6GAL-1 in untreated tumors. We also found that ST6GAL-1 was increased after treatment in primary rectal cancer organoids. We subsequently used rectal cancer cell lines to assess functional ST6GAL-1 protein after chemoradiotherapy and found that it was increased. In addition, we transduced rectal cancer cell lines with shRNA against ST6GAL-1 (knockdown [KD]) in order to study the effects of ST6GAL-1 on post-treatment cell survival and apoptosis. We showed that ST6GAL-1 mediates therapeutic resistance in rectal cancer by decreasing apoptosis and TNFR1 as the potential target of this decrease. Taken together, these studies implicate ST6GAL-1 as a potentially important mediator of resistance to chemoradiation in rectal cancer.A). Mice implanted with tumors were treated with capecitabine 100 mg/kg  and radiation or vehicle. Tumors undergoing chemoradiation were significantly smaller in volume compared with vehicle treatment (B). After 2 weeks of treatment and waiting period of 1 week, tumors were harvested and protein was collected. ST6GAL-1 protein was assessed by Western blotting and found to be increased in two of the three models (C). Upper band in Western blot is whole ST6GAL-1, and lower band is cleaved ST6GAL-1; quantification included both bands. In two of the three models, ST6GAL-1 increased significantly after chemoradiation. In NP33, there was an increase from 1.01-fold to 2.40-fold from vehicle to treated ; NP26 showed an increase from 1.07-fold to 2.19-fold from vehicle to treated ; and NP86 showed a nonsignificant decrease from 1.32-fold to 0.88-fold from vehicle to treated .Three patient-derived xenograft (PDX) models were assessed: NP26, NP86, and NP33, which were created from pretreatment biopsy samples from consented rectal cancer patients. Once tumors reached appropriate size in Central Institute for Experimental Animals NOD/Shi-scid IL2gamma(null) (NOG) mice, they were implanted into the flank of nude mice. Histology images show that PDX tumors were similar to their tumor of origin A. Mice ireatment B. After e models C. Upper ualcan.path.uab.edu/index.html) (A). Because these data contained so few normal samples and the comparisons were not matched, we performed quantitative PCR (qPCR) on 12 pretreatment rectal cancer biopsy specimens and their matched normal rectal tissue. We again found heterogeneity between tumors whereby four tumors showed an increase in ST6GAL-1 mRNA compared with matched normal rectum, whereas the other eight samples did not have increased ST6GAL-1 mRNA compared with matched normal samples (B). In order to assess the distribution of ST6GAL-1 protein within individual tumors, we performed immunostaining in untreated human rectal adenocarcinoma samples and normal rectal tissues. We found that ST6GAL-1 protein was only in a subset of cells within the base of the crypts (stem cell compartment) of the normal tissues , but that in the human rectal adenocarcinoma samples, ST6GAL-1 was in some but not in all the adenocarcinoma cells in any given tumor . The number of cells staining positive for ST6GAL-1 between tumors appeared variable but was not quantified.In order to ascertain how commonly ST6GAL-1 mRNA is increased in untreated human rectal samples, the UALCAN database (ex.html)  was usedex.html) A. Becaus samples B. In ordp ≤ 0.0001, N = 3 and SW837: 1.60-fold increase, p = 0.004, N = 3) (A). In order to assess whether the increased ST6GAL-1 was functional, treated and untreated cells were assessed for ST6GAL-1 activity using an FITC-labeled Sambucus nigra (SNA) lectin (SNL), which binds to the Sia on cell surface proteins that have been added by ST6GAL-1 in the Golgi. These cells were assessed via flow cytometry for SNA positivity. The number of cells staining positive for SNA increased in both cell lines after treatment : 1.89–1.97], p < 0.001 and SW837: odds ratio = 2.69 [95% CI: 2.65–2.74], p < 0.001, N = 4 separate experiments per cell line) (B and C). In addition, mean fluorescent intensity also increased in both cell lines after chemoradiation treatment  (D) indicating that the function of ST6GAL-1 also increases after treatment. SW837 and SW620 cell lines were treated with chemoradiation (3 μM of 5-FU and 5 Gy radiation) and then underwent precipitation with SNA-agarose, followed by SNA lectin blotting where equal amounts of total protein were added to the gel (N = 3), in order to identify all sialylated proteins. Sialylation (addition of Sia to cell surface proteins by ST6GAL-1) increased after chemoradiation compared with vehicle (E).SW620 and SW837 rectal cancer cell lines were plated and treated 24 h later with 5-FU and radiation (5 Gy). Protein was collected 5 days after treatment. ST6GAL-1 protein increased after chemoradiation  A. In ordll line) , B and C, N = 4) D indicat vehicle E.Figure p = 0.007; Fig. 4A). We also assessed changes in fluorescent staining of ST6GAL-1 (red) and Sia (green) after chemoradiation  in rectal cancer organoids and found that both were increased in the treated samples (B and C).We also assessed whether ST6GAL-1 increases after treatment in rectal cancer organoids grown from primary rectal cancer tissue by both PCR and immunofluorescent staining. PCR for ST6GAL-1 revealed an increase after treatment. For example, there was a 3.1-fold increase in ST6GAL-1 on day 5 after chemoradiation  compared with a 1.1-fold increase in vehicle . SW620 CV and KD and SW837 CV and KD were stained with FITC-labeled SNA and evaluated for surface Sia by flow cytometry indicative of functional ST6GAL-1. As shown in Figure 5B, the CV cells have more SNA+ cells than the KD cells in each cell line, again indicating successful KD of functional ST6GAL-1. To assess whether the previously seen increase in ST6GAL-1 was causing treatment resistance, SW620 CV and KD cells were treated with chemoradiation to assess for differences in survival via colony formation assay at 5 days after treatment. As shown in C and D, cell survival was decreased in the treated ST6GAL-1 KD cells by approximately 50%, further supporting that ST6GAL-1 is mediating resistance, indicating that resistance is mediated by ST6GAL-1 .SW620 and SW837 cells were transduced with either a control vector (CV) lentivirus  or lentivirus encoding shRNA for ST6GAL-1 (KD). Cells were incubated, colonies were puromycin selected, and Western blots confirmed successful KD of ST6GAL-1 protein in both cell lines A. SW620 via sialylation of TNFR or Fas death receptors. SW620 CV and KD and SW837 CV and KD cells were plated and treated with chemoradiation, and 24 h later, Caspase 3/7 assay was performed to measure apoptosis. Comparing ST6GAL-1 KD cells to CV cells, we identified a 1.58-fold and 1.45-fold increase in apoptosis for SW620 and SW837, respectively  indicating that ST6GAL-1 is protecting cells from apoptosis (A). To boost confirmation for a decrease in apoptosis as the mechanism of resistance, SW620 CV and KD and SW837 CV and KD cells were plated and underwent chemoradiation. Protein was collected after 24 h to assess for cleaved caspase 3. We found an increase in cleaved caspase 3 in the ST6GAL-1 KD cells compared with CV cells consistent with the findings of our caspase assay (N = 3) (B).Apoptosis is the main mechanism of cell death following chemoradiation treatment of adenocarcinomas. We hypothesized that the resistance to chemoradiation we identified in the rectal cancer cell lines was mediated by decreased apoptosis likely poptosis A. To boo (N = 3) B.Figure p = 0.02, N = 3; Fig. 7A). In addition, we collected all proteins 24 h after chemoradiation in SW620 cells, performed Western blotting for TNFR1, and found that there was not a significant difference between treatment groups when we assessed total TNFR1 .In order to further investigate the mechanism of chemoradiation resistance because of ST6Gal-1, we interrogated a known target of sialylation after treatment. SW620 cell lines were treated with chemoradiation (3 μM of 5-FU and 5 Gy radiation) and collected 24 h later. We performed an SNA lectin precipitation to specifically collect sialylated proteins to investigate what proteins are altered by ST6GAL1 after treatment. We performed Western blotting of these sialylated proteins for TNFR1 because prior studies have shown that sialylation of TNFR1 causes the protein to be unable to signal apoptosis , 18. We via immunohistochemistry on our own patient samples. We then used rectal cancer cell lines to further elucidate the role of ST6GAL-1 in resistance to chemoradiation. KD of ST6GAL-1 confirmed its importance in blocking apoptosis and increasing survival after treatment. We also found that sialylated TNFR1 is increased after treatment, which prior studies have shown to decrease its ability to signal apoptosis. Consequently, we have shown that ST6GAL-1 may play a role in resistance to chemoradiotherapy in rectal cancer.Rectal cancer and its treatment result in major morbidity and mortality, but therapeutics have remained stagnant, likely because of poor understanding of the mechanism of resistance. Previous studies have shown that ST6GAL-1 is increased in various types of cancer, but the role of ST6GAL-1 in radiation resistance in rectal cancer has not been elucidated. We found that ST6GAL-1 and Sia on cell surface proteins are increased after chemoradiation in rectal cancer cell lines, rectal cancer–derived organoids, and PDX models using Western blot, flow cytometry, immunostaining, and immunoprecipitation. We also found that ST6GAL-1 appears to be subclonally expressed in individual rectal cancers et al.  and provided by the institutional Tissue Biorepository as part of our institutional review board–approved protocol. For RNA samples, tissue biopsies were obtained from rectal adenocarcinomas and adjacent normal rectal tissue in consented patients according to our institutional review board protocol prior to undergoing treatment and frozen in optimal cutting temperaure compound. Patient-derived xenograft models were created when adequate tissue was available.3-sized tumor piece was implanted into Central Institute for Experimental Animals NOG mice (Taconic). Once tumors were of appropriate size (10 × 20 mm3), mice were sacrificed, and tumors were removed. Tumor pieces of 2 mm3 were implanted in the flank of Nude mice (Jackson Laboratories). Once tumors reached 150 mm3, mice were split into the following treatment groups: vehicle or capecitabine (100 mg/kg) and 2 Gy (Gy) radiation 5 days per week for 2 weeks. Capecitabine (LC Laboratory) was given via oral gavage. Mice were placed in a custom jig for shielding, and radiation was delivered to tumors via X-RAD 320  daily. Mice were weighed, and tumors were measured with calipers at least twice weekly. Tumor volumes were calculated based on caliper measurements as previously described ; necrotic tissue was removed, and a 2 mmescribed . Tumors g for 5 min at room temperature. Viability and concentration were assessed using a Countess II Cell Counter (Thermo Fisher Scientific). Cells were plated at 50,000 live cells in 15 μl Matrigel Matrix (Thermo Fisher Scientific). Once Matrigel was solidified, cells were cultured with organoid media consisting of 50% advanced Dulbecco's modified Eagle's medium , 50% L-WRN conditioned media  (Sigma), 2 mM GLUTamax, 100 units/ml penicillin and 0.1 mg/ml streptomycin, 10 μM SB 431542, 10 μM Y-27632, and 50 μg/ml gentamicin ). Media were changed every other day.Tumors were excised from mice and dissociated using 2 mg/ml collagenase I (Thermo Fisher Scientific). Resulting dissociated tissue and cells were filtered using a 70 μm nylon cell strainer and centrifuged at 250The SW837 (CCL-235) cell line was obtained from ATCC. The SW620 (CCL-227) cell line was donated by the Leopold laboratory at the University of Michigan. The SW837 cell line was grown in advanced DMEM with 5% FBS with 100 units/ml penicillin and 0.1 mg/ml streptomycin and 0.25 μg/ml fungizone and 2 mM GLUTamax (Thermo Fisher Scientific), and SW620 cells were grown in DMEM with 10% FBS with 100 units/ml penicillin and 0.1 mg/ml streptomycin and 0.25 μg/ml fungizone (Thermo Fisher Scientific). Cell lines were verified by short tandem repeat testing on samples of cultured cells sent to ATCC at regular intervals. Cell were used at low passage numbers only. For chemoradiation treatment of cell lines, cells were treated with 5-FU (Selleckchem) (3 μM) 30 min prior to radiation (5 Gy) , and media were changed the following day.Human rectal adenocarcinoma tissue slides were deparaffinized and rehydrated by washing in xylene and ethanol, and the slides were then placed in boiling citric acid–based antigen unmasking solution (Vector Labs). Slides were washed and then covered in BLOXALL solution (Vector Labs) for 5 min and 0.5% Triton-X in PBS for 25 min. The slides were then blocked with 2.5% horse serum and incubated with primary antibody against ST6GAL-1 1:100 overnight (R&D Systems). ImmPRESS horse antigoat immunoglobulin G (IgG) (Vector Labs) was used as the secondary antibody, with NovaRED peroxidase substrate (Vector Labs) used for the primary stain for 2 min. Slides were counterstained with hematoxylin for 30 s and paraffinized. We assessed immunostaining in four human rectal adenocarcinomas.For immunofluorescence studies, SW620 and SW837 rectal cancer cells and organoids were plated onto coverslips, treated with chemoradiation, washed, and then fixed for 15 min with 4% paraformaldehyde. Slides were washed and permeabilized using PBS with Tween-20 + 0.5% Triton X-100, cells for 10 min, and organoids for 30 min. Slides were blocked with PBS with Tween-20 + 3% bovine serum albumin. Primary ST6GAL-1 (1:150) antibody (R&D Systems) was applied followed by donkey antigoat IgG secondary (1:500) antibody (Invitrogen). SNL conjugated to FITC was used for staining of Sia (Vector Labs) and was added for 1 h, and then 4′,6-diamidino-2-phenylindole nuclear stain (Invitrogen) was placed in blocking buffer, and slides were incubated. Slides were mounted with VectaMount (Vector Labs) and then imaged.RNA was extracted from previously frozen rectal adenocarcinoma and matching normal rectal tissue samples or rectal cancer organoids using the RNeasy kit (Qiagen), and equal amounts of RNA were used to generate complementary DNA with the High-Capacity cDNA Reverse Transcription Kit (Fisher). qPCR was performed using a QuantStudio3 (Thermo Fisher Scientific) instrument. TaqMan gene expression assay primers (Thermo Fisher) and TaqMan Fast Advanced Master Mix (Thermo Fisher) were used to generate reactions.SW620 and SW837 cells were plated with 50,000 cells per well in a 24-well plate and allowed to incubate overnight. Cells were washed with PBS and then lentivirus with shRNA for ST6GAL-1 to cause ST6GAL-1 KD  or CV  was added to each well to obtain a multiplicity of infection of five in serum-free antibiotic-free media. Cells were incubated overnight, media were removed, cells were washed with PBS, and cultured in complete media for 48 h. Cells were cultured in puromycin (1 μg/ml) selection media for 7 days. KD was confirmed by Western blotting for ST6GAL-1 and flow cytometry for Sia using the SNA lectin as described.SW620 and SW837 cells were lysed in radioimmunoprecipitation assay buffer and Halt protease and phosphatase inhibitor cocktail (Fisher Scientific) 1 or 5 days after treatment. PDX model tumors were harvested and dissociated 1 week after completion of treatment. Samples then underwent electrophoresis and protein transfer to polyvinylidene fluoride membranes. Membranes were blocked in 5% nonfat dry milk in 1× Tris-buffered saline and 0.1% Tween-20. Blots were probed with 1:1000 antibodies against ST6GAL-1 (R&D Systems), (1:200) cleaved caspase 3 , and (1:300) TNFR1 . Protein loading was verified using 1:10,000 β-actin (Abcam). Membranes were incubated with horse antigoat IgG secondary antibody for ST6GAL-1 (R&D Systems) and biotinylated rabbit secondary for cleaved caspase 3 (Vector Labs) and rabbit monoclonal antibody for TNFR1 (Santa Cruz) and imaged using enhanced chemiluminescence.Cells were plated at equal densities into a 96-well plate. Cells were treated 24 h after plating with 5-FU (3 μM) and radiation (5 Gy), and 24 h after treatment, Caspase-Glo 3/7 Assay reagent (Promega) was added in equal amounts to the media in each well. Plates were incubated for 1 h at room temperature, and luminescence was quantified with Infinite M200 Pro instrument (Tecan).SW620 cells were plated at 60% confluence and then treated 24 h later with 5-FU (3 μM) and radiation (5 Gy). Post-treatment, cells were trypsinized and then diluted to plate 500 cells per well in 6-well plates. Following plating, cells were grown in appropriate serum until colonies were visible. Cells were washed with PBS and then stained with 0.1% crystal violet for 10 min. Colonies containing over 50 cells were counted.6 cells. Cells were incubated in the dark for 45 min, washed, and resuspended with FACS buffer. Propidium iodide was added (1 μg/200 μl). Flow cytometry was performed at the University of Alabama FACS facility.SW620 and SW837 cells were washed in a fluorescence-activated cell sorting (FACS) buffer (1× PBS + 2% FBS), filtered, and resuspended in FACS buffer. Cells were counted and labeled with FITC-labeled SNA at 20 μg/ml of SNA lectin per 10SW837 cells were plated until 80% confluence and then lysed in radioimmunoprecipitation assay buffer and protease/phosphatase inhibitors 24 h after treatment, and protein was collected. About 50 μl of SNA-agarose beads (Vector Labs) were washed and then incubated overnight with 500 μg of the collected protein. Sample was pelleted, washed, and resuspended in wash buffer. Protein was removed from the beads using glycoprotein eluting buffer (Vector Labs) or heating in 1× sample buffer with beta-mercaptoethanol. Samples then underwent electrophoresis and protein transfer to polyvinylidene fluoride membranes, membranes were blocked in 5% nonfat dry milk in 1× Tris-buffered saline and 0.1% Tween-20. Membranes were probed for SNA (1:1000) with SNA, elderberry lectin, biotinylated (Vector), and incubated with secondary (1:300) with streptavidin peroxidase. Protein loading was verified using 1:10,000 β-tubulin antibody (Abcam).t test for statistical comparison. qPCR was analyzed using the comparative CT method  within each experiment (n = 3 experiments per cell type). Subsequently, the log fold change was fit using a linear mixed model with cell type (KD versus CV) as the fixed effect and a random intercept for experiment to account for correlation among technical replicates. Experiments in cell lines were replicated using different passages at least three separate times. Data cleaning and analysis utilized R software, version 4.0.2 . All mixed models utilized restricted maximum likelihood estimation and the R package lme4 (version 1.1-23). Statistical tests were two-sided with significance at p < 0.05.Western blots were quantified and normalized to control protein using ImageJ  and analyzed using a Student’s T method . For theAll data are contained within the article.10.13039/100000051National Human Genome Research Institute. K. M. H. reports that financial support was provided by the 10.13039/100000002National Institutes of Health. All other authors declare that they have no conflicts of interest with the contents of this article.M. S. reports that financial support was provided by the


Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1β (IL-1β). On the contrary, the expression of IL-1β receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1β production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model. Burkholderia mallei, a gram-negative intracellular bacterium, is the etiological agent of glanders. Equids are natural reservoirs of B. mallei and clinical signs of glanders in equids include chronic nasal discharge, enlargement of the lymph node and the presence of nodules, pustules, or ulcers on the flanks and extremities of infected animals , which closely mimic human infection, is critical. animals . Personn animals . HoweverB. mallei enters the phagosome through phagocytosis. Subsequently, bacteria escape from vacuolar membranes and enter into the host cytosol. Once free, bacteria exploit host factors (HFs) to trigger actin polymerization and induce multinucleated giant cells (MNGCs)  induces the expression of cellular protective reactive nitrogen oxide species (RNOS), multiple cytokines and chemokines  and MCP-1 receptor (CCR2) deficient mice were defective in up-regulating IFN-γ, which may ultimately contribute to their inability to clear B. mallei infection  . Followiemokines . Inducibnfection . Both tyactivity . The rolnfection . In addi. mallei . Importaal times . Taken tB. mallei in multiple animals species including mice (mus musculus), Syrian hamsters (Mesocricetus auratus), and marmoset  showed clinical symptoms that resemble human glanders  assay to generate a comprehensive protein-protein interaction (PPI) map between each of the 49 B. mallei virulence factors (VFs) and either human or mouse proteins  and gain insight toward the underlying molecular network that governs B. mallei pathogenesis. This methodology has identified critical signaling components and molecular pathways that modulate pathogen infections in NHPs  supplemented with 10% (v/v) fetal bovine serum  and 1% penicillin and streptomycin .Ezrin inhibitor (NSC668394) was purchased from EMD Millipore . V-PLEX B. mallei FMH (China 7) was selected for the glanders study. It is a human isolate obtained from a patient with a laboratory-acquired infection. This strain was chosen based on association with human disease, virulence in animal models, availability of genetic analyses, and passage history  at 37°C with shaking at 200 rpm for approximately 18 h. history . B. mall7 aerosolized colony forming units (CFU) of B. mallei FMH23344 (dose range 1.51 × 107 to 3.43 × 107). Briefly, prior to aerosol exposure, NHPs were anesthetized by intramuscular injection with a ketamine/acepromazine mixture (10:1) for African green monkeys or Telazol was used to anesthetize the macaque species. Minute volumes (MV) were estimated using plethysmography. NHPs were exposed to a small-particle aerosol in a well-characterized, dynamic, head-only exposure chamber controlled and monitored by an automated bioaerosol exposure system. Integrated air samples were obtained from each run using all-glass impingers. Individual aerosol exposure doses were calculated as the product of the average aerosol concentration within the chamber and the total volume of air breathed by the NHP  were included in the study. Experimentally naïve NHPs were screened before being placed on protocol  to ensure the general health status of the animals is considered normal and that no underlying infection or abnormal health conditions are present that could adversely affect the study or data interpretation. The NHPs were then randomly assigned and balanced with regards to sex to obtain an approximate equal ratio of males and females in each experimental cohort. Under biosafety level 3 (BSL-3) conditions, the NHPs were exposed to a challenge dose of approximately 2.3 × 10 the NHP .n = 6), African green monkeys (n = 4), and cynomolgus macaques (n = 5) before infection and then post infection either at the time of euthanasia based on clinical score or those that survived till the end of study design (42–45 days post-exposure) (Blood samples were collected from rhesus macaques (xposure) . Bupreno6 PBMCs were collected and lysed in T-per reagent  containing protease inhibitors. After a brief centrifugation step to remove cellular debris, SDS-PAGE sample buffer  containing DTT was added to the cleared lysate. Samples were heated to 95°C for 15 min to inactivate the bacteria. Each PBMCs protein lysate was gel-fractionated by running on a 4–12% Tris-Bis polyacrylamide gel and dividing the gel lane into 10 equal fractions. In-gel trypsin digestion was then performed and the resulting peptides were analyzed by LC-MS/MS on a LTQ Orbitrap Elite mass spectrometer equipped with a Dionex 3000 RSLCnano system (Thermo Fisher Scientific). The system injected 5 μL of each digest onto a pre-column  housed in a 10-port nano switching valve using a flow rate of 10 μl/min. The loading solvent was 0.1% formic acid in HPLC grade water. Peptides were then loaded onto an Easy-Spray analytical column (15 cm × 75 μm) packed with PepMap C18, 3 um particle size, 100A porosity particles . A 2–58% B gradient elution in 60 min was formed using Pump-A (0.1% formic acid) and pump-B (85% acetonitrile in 0.1% formic acid) at a flow rate of 300 nL/min. The column eluent was connected to an Easy-Spray source  with an electrospray ionization voltage of 2.2 kV. An Orbitrap Elite mass spectrometer  with an ion transfer tube temperature of 300°C and an S-lens setting of 55% was used to focus the peptides. A top 10 data dependent MS/MS method was used to select the top 10 most abundant ions in a 400–1,600 amu survey scan  with a full AGC target value of 1e6 ions and a maximum injection time of 200 ms. Higher Energy Collisional Dissociation (HCD) ms/ms spectra were acquired at a resolution of 30,000 (FWHM at m/z 400) with an AGC target value of 5e4 ions and a maximum injection time of 200 ms. MS2 data from all 10 fractions were then merged for the protein database search, performed with MASCOTTM 1.4 , using a mixed taxonomy (NHP and Burkholderia) subset database of the non-redundant protein database from National Center for Biotechnology Information (NCBI) web site1. Search parameters included trypsin enzyme specificity with two missed cleavages, variable modifications included oxidized methionine, precursor mass tolerance of 20 ppm and product ion mass tolerance of 0.5 Da. Only proteins with a minimum of 2 unique peptides were included in the final dataset. Expression levels were based on the Exponentially Modified Protein Abundance Index (emPAI) which is available in the MASCOT software package. The emPAI score offers approximate, label-free, relative quantitation of the proteins in a mixture based on protein coverage by the peptide matches in a database search result. The mass spectrometry proteomics data has been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers listed in Briefly, 1 × 10P-value based on Redundant siRNA Analysis (RSA) was then assigned to each gene and a threshold of 0.01 was applied to identify genes differentially expressed across all donors in the same species | ≥0.673 and paired t-test p ≤ 0.05. A total of 115 unique genes were considered differentially expressed in at least one species and the hits were hierarchically clustered by FC of individual donor.Prioritization process of 3,288 human genes: Genes with neither pre- nor post-infection emPAI for one host were considered absent and not used for the fold change (FC) calculation of the same host; however, they might be retained for other hosts. A noise level of 0.03 was determined by the 5th percentile of all data and any emPAI less than the noise level was set to 0.03 for the following analysis. Fold changes of emPAIs between pre- and post-infection were calculated for remaining genes in 15 donors of three species.  species . We alsop ≤ 0.01 were retained  at a multiplicity of infection (MOI) of 1. After incubation for 2 h at 37°C and 5% CO2, extracellular bacteria were killed by addition of the antibiotic kanamycin (500 μg/ml). After incubation for 16 h at 37°C and 5% CO2, the infected RAW264.7 cells were fixed with 10% formalin for 24 h for subsequent immunofluorescence staining. The mBMDMs at 5 h post infection were processed for RNA isolation and Real-Time PCR studies.RAW264.7 macrophages were seeded at a density of 20,000 cells/well in 96-well imaging plates (Greiner μ clear). mBMDMs were seeded at density of 80,000 cells/well in regular tissue culture treated 96 well plates. Both cell types were then incubated at 37°C and 5% COBMDMs were lysed in TRIzol Reagent and total RNA was isolated according to the manufacturer’s protocol . Total RNA was reverse transcribed with iScript Reverse Transcription Supermix . cDNA samples were amplified using SYBR Green on the QuantStudio 12K Flex Real-Time PCR System . Briefly, the reaction conditions consisted of 2 μl of diluted cDNA with a final concentration of 150 nM primers in a 10 μl of reaction mix. Each cycle consisted of denaturation at 95°C for 15 s, annealing at 58.5°C for 5 s and extension at 72°C for 10 s. The specific primer sequences used for the real-time PCR are listed in Burkolderia mallei polyclonal antibody  at 1:1,000 dilution. After subsequent washing and incubation for 1 h with the anti-goat Dylight 488 secondary antibody (1:500 dilution in blocking buffer), the cells were stained with Hoechst nuclear dye  and CellMask Deep Red dye  for host cell cytoplasmic staining.Formalin fixed plates were washed with Dulbecco’s Phosphate buffered saline (DPBS), the cells were permeabilized for 15 min with DPBS containing 1% Triton X-100, washed and then blocked for 1 h with blocking buffer (Cellomics 1860291). To detect bacteria, infected cells were incubated for 1 h with goat anti-B. mallei, the different NHP species showed different clinical signs. African green monkeys were by far very sensitive to B. mallei. Five of six animals developed lethal disease ; the surviving animal had an abscess and was bacteremic at study endpoint . Cynomolgus and rhesus macaques were resistant to B. mallei and survived the challenge. All animals became febrile after exposure to B. mallei but most of the cynomolgus macaques exhibited the shortest duration of fever. In the African green monkeys and some of the rhesus macaques, body temperature remained elevated above baseline for the duration of the study.The subjects consisted of six rhesus macaque, six African green monkeys and six cynomolgus macaques . FollowiAfrican green monkeys exhibited leukocytosis with neutrophilia and monocytosis beginning 2–3 days post- infection, along with azotemia and elevation of the hepatic enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Cynomolgus macaques had variable elevations of leukocytes during the study. Two of six cynomolgus macaques developed an apparent anemia of inflammatory disease. Leukocyte elevations in rhesus macaques were the most variable during the protocol. Four of six developed an apparent anemia of inflammatory disease.Gross pathology and histopathology findings in the African green monkeys were consistent with reports of acute glanders in humans (data not shown): respiratory infection leading to bacteremia with pustule development in multiple organs and death within 2 weeks. It is important to note that little clinical data on acute human disease exist. Although there was pyogranulomatous or resolving inflammation with fibrosis in the lung and draining lymph nodes in five rhesus macaques, the lesions in other organs were limited, which is not necessarily consistent with acute glanders in humans. Cynomolgus macaques had few lesions in the lung and draining nodes and limited lesions in other systems. These observations are also not consistent with acute glanders in humans.B. mallei infection, it is critical to study the protein expression profile from B. mallei infected primary cells using LC-MS/MS approaches. Profiling the abundance of the protein in primary cells provides a “snapshot” of the complex immune networks that operate throughput the system. The resulting mass spectra were interpreted automatically by querying MASCOT algorithm against NHP database  (p < 0.001) and cytoskeletal protein binding (p < 0.003)  . Further< 0.001) . GO anal< 0.003) . The funB. mallei VFs through a combination of computational and experimental approaches. Using the Y2H assay system, 26 of the B. mallei virulence factors and their interacting HFs were curated. To gain insights toward the relationship between B. mallei VFs versus 115 HFs identified in our LC-MS/MS study, a predictive PPI network based on the binary protein-protein interaction data obtained from the Y2H study was generated , ceruloplasmin (CP) and Reticulon-4 (RTN4). The spectra from LC-MS/MS dataset were also queried against the B. mallei protein database. Twenty-two VFs were found to be present in at least one of the donors in one of the NHPs. Eight of the VFs were able to interact with the major PPI network whereas 14 additional VFs could not be mapped to the PPI network due to the lack of Y2H data. Interestingly, among these 22 VFs, 5 of them were found in all three NHP species , suggesting the importance of these factors in mediating host response as well as bacterial replication during the pathogenesis of B. mallei infection.enerated . The oveB. mallei induced inflammatory responses. Ezrin is a multifunctional protein that connects the actin cytoskeleton to the extracellular matrix through transmembrane proteins  in mediating proteins . Further demonstrated significantly enhanced MNGC formation rate and improved bacterial survival in J774.A1 murine-derived macrophage-like cells. By employing a systems biology approach, we have revealed the topology of a protein interaction network, which reflects the underlying host-pathogen interactions of B. mallei infection in a NHP model.The induction of innate immunity, inflammatory cytokines and chemokines following approach . We idenB. mallei pathogenesis is not well characterized. However, insight could be gained from the infection of B. pseudomallei  can be found in the article/Animal research at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) was conducted under an animal use protocol approved by the USAMRIID Institutional Animal Care and Use Committee (IACUC) in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals .C-YC, RP, and PW conceived and designed the experiments. C-YC, MW, DL, TK, RR-A, BE, ST, TC, DW, LC, and CC performed the experiments. YZn and YZu analyzed the data. C-YC, RM, and RP wrote the manuscript. All authors contributed to the article and approved the submitted version.Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U. S. Army.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.


Epoxy resins are currently used in many areas of construction, such as resistant coatings, anchors, fibre-reinforced polymer (FRP) composites, grouts, etc. This paper deals mainly with epoxy composites that can be applied during the rehabilitation of concrete constructions. The influence of a filler type on epoxy thermoset composites was monitored, whilst three different types of epoxy resin were used in order to achieve a better representation and confirmation of the results. During the testing of fillers, these were mainly secondary raw materials, including pre-treated hazardous waste , representing various shapes and sizes of particle, while their amount in the epoxy matrix was chosen with regard to optimal viscosity and workability. Physical and mechanical parameters, like compressive and flexural strengths, cohesion with the concrete and thermal expansion of the epoxy composites containing various fillers were determined. The microstructure of epoxy composites with a different filler type and chemical resistance against chemical aggressive media were all monitored. The microstructure of epoxy composites was monitored using scanning electron microscopy (SEM) supported by energy-dispersive X-ray spectroscopy (EDX). Computed tomography (CT) was also used for the evaluation of the cohesion of the epoxy composites with concrete and dispersion of the filler in the epoxy matrix. Polymer composites are often used as binding agents of rehabilitation materials of building constructions ,2. EpoxyEpoxy resins are polymeric substances that are mainly formed by cross-linking when an epoxy class enters the reaction .During the curing of epoxide resins, crosslinking between the epoxy molecules and reactive groups on each end of the curing agent occurs .The principle of epoxy resins curing using polyamines is stated in The means of crosslinking fundamentally affects the properties of epoxy thermosets. Generally, epoxy resins have excellent cohesion with a substrate, low shrinkage and good permeability resistance to water, acids, alkalis and other corrosive substances .The monitoring of the compressive strength of polymeric concrete and polymeric mortars based on epoxy resins has already been described in many previous scientific works. It was observed that the compressive strength depends mainly on the resin content . Rebeiz 2O3 and nano-SiC particles) within a range of 5–15 wt.% and monitored thermal properties, also focusing on morphology, and use of mineral fillers in the epoxide matrix. The coefficient of linear thermal expansion is important to analyse the processes of structure formation and the behaviour of epoxy composites, with different amounts and types of fillers, under the influence of a thermal field. [For the selection of suitable fillers for epoxy composites, the maximum possible filler content in the polymer matrix and particle size and shape index are important. . In the l field. .It was found that the addition of waste glass powder at an amount of 7.4 to 35.9% has a positive effect on the pull-off strength of epoxy polymer mortars . An irreHowever, a detailed comparison of the dependence of the influence of the filler component’s particle shape on the behaviour of the polymeric compound in terms of rheologic properties and properties of the resulting product was not summarised in any available literature. Following previous research of epoxy composites containing fine waste from the production of mineral wool board insulation , and theBy using waste products as fillers in epoxy composites, it is possible to save primary resources and achieve the required properties of epoxy composites in a much more environmentally friendly way. The use of pre-treated hazardous waste could, in particular, reduce the volume of hazardous waste in landfills, as tons of unused waste are landfilled worldwide.Proportions between resin (epoxy resin (A), hardener (B)) and fillers are stated in During the experimental verification stage, four types of epoxy resins were used, differing mainly in the type of hardener used—see Based on the differences in the morphology of the grain, the fillers were chosen using the widest spectrum of possible shape properties. Another evaluation criterion for the selection of input raw materials was the maximum possible use of secondary raw materials and waste materials from which a significant reduction of the ecological impacts of industrial production can be obtained.The quartz sand mixtures  were used as the reference filler. They are impurity-free and have the optimal round grain shape mainly used as a filler for polymer concrete (PC), resins, grouts, grouting and backfills. The sand fraction of 0–1.5 mm (ISG A1) was used within the research, which is commonly used for epoxy mortars.2 and have not been polluted by other materials. Currently, it is estimated that the service life of the panels, defined by a 20% reduction in efficiency, will remain at a sufficient quality level for 30–40 years following installation. However, in most cases, the main reason for the disposal of a panel is due to mechanical damage caused during transportation or installation. The biggest problem for lower-quality panels is delamination, whereby the ‘sandwich’ structure of a PV panel comes apart due to the effects of temperature and UV radiation. In order to recycle PV panels, the PV Cycle system was devised. This is a Europe-wide activity engaged in by the manufacturers and importers of photovoltaic (PV) panels based on voluntary responsibility for the product during its whole service life. One of the containers is intended for crystal quartz panels, and the second container is intended for thin-layer panels, for which different recycling is used. Glass accounts for the largest part of the weight of crystal PV panels (60–70%) and the aluminium frame (approximately 20%), while for thin-layer panels the glass and aluminium parts account for at least 95%. During 2010, the highest number of panels were installed in the Czech Republic, namely 160,000 tons of PV panels, for which their service life is estimated to end in 2040 [The solar (photovoltaic—PV) panels, that were used in this work, are manufactured by the QS Solar company (Nantong) in China. These panels are thin-layer modules with a base made of amorphous SiO in 2040 . When usThe waste from the production of insulation boards made of mineral wool and containing a high proportion of recycled glass (>80%) was selected as another perfect filler in the form of a secondary raw material for the rehabilitation masses being developed. This is the dry by-product of the production process that falls off from below a pulper in front of a hardening chamber so it does not contain any organic elements, and it is, therefore, possible to classify this as recycled glass without organics.The filter fly ash from the thermal power plant  from the combustion of hard coal was also selected as a filler. FA was contaminated by the influence of the flue gas denitrification (DeNOx) using selective, Selective Non-Catalytic Reduction (SNCR), when a urea solution (CO(NH₂)₂) is injected into a boiler at high temperature.The neutralisation sludge is created as a by-product during the surface treatment of metal elements . The objective of galvanic plating is to create a metal coating on predominantly metallic base materials. The protective, anticorrosive layer shields the product against the influences of the environment, thus, extending their useful service life. During the galvanic plating process, a number of waste products are generated—sludges and filter cakes from the neutralisation station—that contain dangerous substances. According to the European Waste Catalogue (EWC), the selected hazardous waste (HW) is classified under code 19 02 05—Sludges from physical and chemical treatment containing hazardous substances. These types of sludge are characterised by several dangerous properties such as HP5 (Specific Target Organ Toxicity/Aspiration Toxicity), HP14 (Ecotoxic) and HP15 . This sludge had to be dried and ground by the vibratory disc mill RS 200  down to a suitable granulometry, which would represent finer fillers  and so that it can be successfully uniformly dispersed in the polymer matrix, prior to usage itself.In the Czech Republic, the annual consumption of foundry sands is about 800,000 tons, of which only less than 10% is recycled. Bentonite or cement-bonded sands and water glass mixtures are practically environmentally friendly. Due to their variable nature, natural foundry sands are being increasingly replaced by synthetic sands, into which the specified amount of bonding admixture is added (bentonite on most occasions) . The susThe fillers were characterised by the determination of chemical composition , densityAs can be seen from the size distribution curve , the typTo study the stage of synergic influence of a filler and epoxy resins, fillers were used, based on the different particle shapes  was used for the compressive and flexural strength determination. The specimens were stored in a laboratory environment, during the polymerisation and subsequently until the time of testing. Compressive strength was tested from each formulation on three specimens and flexural strength on fractions of these beams, i.e., six specimens.Compressive and three-point flexural strength was determined pursuant to the EN 12808-3  on speciThe cohesion of epoxy composites was determined according to the EN 1542 standard . The thiDetermination of the viscosity was performed using an MYR VR 3000 V1L rotational viscometer . The temperature of the fresh mixtures was 20 °C at the start of the determination process and a spindle type R6 was used to measure the viscosity. The determination was performed immediately after the mixing of all epoxy composite components. Three repetitions of the dynamic viscosity determination of each epoxy mixture were performed.3 to 50 cm2.The abrasion resistance was determined according to the EN 13892-3 standard [The determination of impact resistance of the epoxy composites was performed according to the EN ISO 6272-1 standard  using faThe surface hardness of epoxy composites was determined according to the standard EN ISO 868 . A D typThe determination of the coefficient of linear thermal expansion (α) was performed using the CLASIC 30/100/15DIL dilatometer . The samples were of the same dimensions as in the determination of flexural strength (20 mm × 20 mm × 100 mm). The measurement was executed within a temperature range of 20–60 °C for approximately 18 h. Three repetitions were performed with each type of epoxy composite.α) was determined according to the following equation:α is the coefficient of linear thermal expansion in (K−1), L0 is the original length of the specimen in (mm), ΔT is the temperature change in (K), and ΔL is the change in the length of the specimen L in (mm).The coefficient of thermal expansion . The chemically aggressive environment acted on the samples for 28 days at a temperature of 23 ± 2 °C. After this period, the samples were taken out of the aggressive medium, dried and then in accordance with Firstly, the epoxy composites in a fresh state were applied to a thinner layer on an acetone-cleaned and dried laboratory slide. The specimens were left to polymerise for seven days on a clean underlay at a temperature of 20 ± 2 °C, Samples were then immersed into a lockable glass cuvette with a specific aggressive media, which in practice can affect epoxy composites primarily intended for the rehabilitation of concrete structures  due to the use of different filler types. Examination concentrated on looking for possible defects, air pores, microcracks, micro splits and micro blisters, also the means of distribution, the quality of homogenisation and eventual clustering of the filler within the cross-section of the specimen were evaluated. For monitoring the inner structure and microstructure, a TESCAN MIRA3 XMU  Scanning Electron Microscope (SEM), which allows the examination of materials with magnification up to 1,000,000×. Using the SEM, the samples of epoxy composites were examined at a resolution of up to 5000×. The acceleration voltage was 15 and 20 kV. Samples with a thickness of approximately 4 mm were prepared by sputtering by gold in a high vacuum.−1. Within the 4000–2500 cm−1 range, there is valence vibration of hydrogen, and a free binding of O-H absorbs at the highest wavenumbers at approximately 3600 cm−1. To determine the infrared spectrum, the Frontier PerkinElmer IR/NIR type spectroscope with an attenuated total reflection (ATR) diamond crystal as an adaptor was used. A small amount of the sample (10 mg) with a part size <100 µm homogenised with 300–400 mg KBr and a tablet was then extruded, which was then inserted into the FTIR spectrometer to determine its composition. Using FTIR, the spectrum of used treated hazardous waste (NS) and fly ash (FA) was also determined for comparison with individual spectrums.Using FTIR, it is possible to identify especially organic compounds that have a spectrum area within the wavenumbers of 400 to 4000 cmThe computed tomograph (CT) Phoenix v|tome|x m 300  was used particularly to monitor the inner structure of epoxy composites, cohesion of polymer mortar with a concrete underlay, and the distribution of fillers in the epoxy matrix. This is a multi-purpose tomograph used for the analysis and 3D viewing of a wide spectrum of materials that operates with a voltage of 300 kV/500 W.From the obtained results of compressive  and flexThe highest flexural strength was reached in samples using recycled material from the production of glass insulation (RGI). This fact can be explained by the ‘rod-like shaped’ particle shape of RGI fine filler particles. Fly ash worked best with ER3 epoxy binder when almost the same flexural strength was achieved as in the case of a composite containing RGI filler. Neutralisation sludge (NS) showed comparable strengths in all used binders. Waste foundry sand (WFS) reached its highest strength when combined with the ER2 binder.Failure in the sub-concrete layer occurred in all monitored samples, except for those containing NS. The highest cohesion with sub-concrete was reached by the reference filler in combination with the used ER2 binders: fly ash with ER3 and a waste foundry sand with ER3. The comparison of individual resins: WGS filler had the highest cohesion with sub-concrete combined with the ER3 epoxy resin. Recycled material from the production of glass insulation (RGI) reached the highest cohesion with the used ER2 and ER3 binders. Fly ash reached the highest cohesion with the ER3 binder. For samples using the NS filler, the value of cohesion with concrete was the lowest. In terms of the resin used, the highest cohesion with the sub-concrete with the waste foundry sand filler was reached by the ER3 binder. All examined samples met the requirements of the EN 1504-3 standard , which dAs is obvious from the results of the evaluation of dynamic viscosity , its valThe highest abrasion resistance was observed in samples using fly ash (FA) as a filler and the ER1 as a binder, alternatively, the lowest ability to resist the abrasion with the highest volume reduction was observed in composites containing waste foundry sand (WFS) as a filler and in all types of binders. Based on the comparison of the abrasion resistance results  it is poThe highest impact resistance was recorded in the epoxy composites containing fly ash as the filler, whilst the highest values were observed with the samples based on ER1 and ER3 resins—see Shore D hardness was tested on samples aged 28 days and the results are shown in It was verified that the influence of the type and amount of filler can slightly influence the results for material hardness. Furthermore, it was proven that materials containing finer particles as a filler  show higher values of the Shore D hardness than the materials filled by WGS, NS and WFS, in which the particle size ranged up to 1.5 mm. The highest value of Shore D hardness was recorded for samples using the fly ash filler. Moreover, it is possible to see that the type of resin used did not have any influence on the hardness of the composite surface. On the contrary, the lowest values were observed in samples using the reference filler and WGS.−6 K−1 and by the adding of 2.5% (weight) of graphite plates, it reduces to 36–41 × 10−6 K−1, which is approximately 30–40% lower in value [The coefficient of linear thermal expansion (α) is a particularly important parameter of polymers used mainly in engineering applications. A low-value α is often desirable for acquiring dimension stability and this can be achieved by the addition of a solid and fine graphite filler. It was observed that the α value of the hardened epoxy resin (ER) is 60 × 10in value . The maiThe crosslinking point mutually pulls molecular chains under a micro-Brownian motion, thereby preventing the molecular chains from expanding with rising temperature. In the rubbery region 190–250 °C), the coefficient decreases as the crosslinking density of cured resin and on the contrary increases in the glassy region (50–140 °C), when an increase in the crosslinking density occurs  °C, the , that th3COOH and CH3CH2COOH, are quite weak acids and so they are much less dissociated. These acids mainly act as solvents; their effect leads to the creation of surface blisters and the separation of segments of macromolecular chains. It was also proven that an increase in chemical resistance can be achieved by the implementation of fillers that are able to react with a diffusing acid. Inner fillers or pigments , present in the epoxy matrix, increase the diffusion of an aggressive (corrosive) medium since the penetration at the pigment/binder interface occurs along the pigment particles [Organic acids, such as HCOOH, CHarticles .2O2 [3COOH. The higher chemical resistance of the ER3 resin is related to the fact that it contains formaldehyde and phenol in the A component, thanks to which it is possible to rank it among Novolac resins. The added functionality of the phenolic resin increases the ability of the resin to crosslink, creating a stronger polymer network with high resistivities. The high chemical and solvent resistivities and temperature compatibility of epoxy phenolic resins are most useful when used in high-performance applications and in corrosion resistance [Based on the evaluation of the executed accelerated chemical resistance test in 2O2 . The degsistance ,60. SampFrom the optical digital microscope images , it is pThe samples were also microscopically examined  followinFrom the photomicrographs obtained from SEM , it is oFrom −1. In the sample containing NS filler, carbonates stripes (CaCO3) were observed and at the start of the middle area of IR, iron oxides were most likely identified, which were also observed in the chemical analysis of the sludge itself. In the organic component, only compounds typical for epoxides were observed, such as the oxirane ring (C-O-C), C-H bonds and other aromatic compounds, and these findings were observed in all ER used. In all spectrums of the epoxy composites, the hydroxyl (-OH) group with a value of approximately 3400 cm−1 was detected.Due to the incorporation of hazardous waste (NS) into the polymeric matrix , no new In the CT scans , the int3) and epoxy composites (1500 kg/m3), fly ash has a density of approximately 2600 kg/m3, and, therefore, its grains can be clearly seen in the mixture. From the SEM analysis and the CT tomography, it is obvious that finer and coarser particles of fillers are dispersed equally throughout the composite.In It was determined that the type of filler used has a more significant influence on the resulting properties of the mixture than the type of resin itself. Fillers that were used for the experiment were mainly secondary raw materials, which were chosen to have as many different shapes and particle sizes as possible. Regarding the obtained results, it can be stated that coarser grains of a filler with a more equal geometric shape  have a positive influence especially on the compressive strength thanks to less compression of the filler grains in the epoxy composite. Alternatively, epoxy composites containing finer fillers  show higher flexural strength, better abrasion resistance and better impact resistance. Epoxy mortars containing quartz sand as a filler were also tested in order to compare the results of tested materials with the available materials containing only primary row materials. Compared to other commercial epoxy materials, even better mechanical properties have been achieved with epoxy composites containing waste materials as fillers. The highest flexural strength was recorded in samples containing the RGI filler, which was the most heterogeneous in terms of particle shape, and it can be assumed that the ‘rod-shaped’ particles of a filler had a positive influence on the load resistance. Based on the evaluation of the FTIR analysis, no new chemical bonds between the filler and binder particles were observed. The highest strength was recorded in the ER1 and ER3 samples using the waste foundry sand filler. No damage to the polymer structure occurred due to exposure to NaOH, NaCl or distilled water. The best resistance against the chemically aggressive environment was observed in the ER3 Novolac epoxy resin, although composites based on ER1 and ER2 also showed outstanding chemical resistance. The filler type has no particular influence on the chemical resistance. The filler components were only physically bonded to the epoxy matrix. It was also proven that it is possible to incorporate hazardous waste (NS) into the epoxy matrix. Newly developed epoxy materials with a high content of by-products can be used in practice (building applications) due to their high mechanical parameters and chemical resistance, e.g., as polymer mortars, rehabilitation materials, polymer concretes, adhesives and grouts.


Bone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs).ADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively.Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA.Salidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results.Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.The online version contains supplementary material available at 10.1186/s13018-021-02598-w. Bone disease causes short-term or long-term physical pain and disability. Osteoblast differentiation is a highly regulated process controlled by a complex signaling pathway , 2. ImbaAt present, drugs for the treatment of osteoporosis can be aimed at promoting osteoblastic bone formation or inhibiting osteoclasts . Among tRhodiola rosea L. have long been used in traditional medicine of China. Salidroside (SR) is a main component of Rhodiola rosea L. and exhibits a variety of pharmacologic properties, including anti-inflammatory, anti-fatigue and anti-oxidant properties, anti-apoptosis, and hypoglycemic effects. Mao et al. [The rhizome and roots of o et al.  found tho et al. . Zhang eo et al.  revealedo et al. . HoweverIn the treatment of many different bone diseases, mesenchymal stem cells were widely used in cell therapy . MesenchIt was also reported that Wnt/β-catenin signaling is anabolic for bone formation . The WntIn this study, we performed RNA sequencing between normal and Salidroside-treated ADSCs. Moreover, we performed a series of experiments to verify that Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.3 in size. Sterile saline (37 °C) was added to the homogenized adipose tissue in a ratio of 3:1 , followed by the addition of stock collagenase solution to a final concentration of 0.5 units/ml. This suspension was placed in a 37 °C shaker at 200 rpm for 60 min. Samples were then passed through a 100-μm filter into a 50-ml conical tube. After washing three times with sterile PBS, cells were then resuspend in high-glucose DMEM containing 20% FBS.Abdominal subcutaneous adipose tissue was obtained from human adipose tissue through simple liposuction. Then, 200–300 mL sterile PBS was added to every 0.5 g adipose tissue to prevent dehydration. Adipose tissues were cut into small pieces about less than 1 mmThe third passage (P3) cells were used for all subsequent experiments. When 80% of the ADSCs were confluent, cells were seeded in 6-well plates and decomposed overnight. Then, ADSCs were changed to induction medium . ADSCs were treated with osteogenic induction medium for 3 weeks and the medium was changed every 3 days.The ALP staining was performed using the ALP staining kit according to the instructions. In brief, ADSCs were fixed by 4% paraformaldehyde and then washed with PBS for three times. Staining was performed using BCIP/NBT chromogenic substrate . An optical microscope  was implemented for the staining result observation and photographing. ALP activity was measured according to the kit instructions. In brief, ADSCs were washed twice with TB buffer and then added 100 μL lysis solution. ADSCs were then separated for 20 min at 12,000 rpm at 4 °C. Then, 45 μL supernatant was added to 100 μL p-nitrophosphatate. After incubating for 30 min, the absorbance at 450 nm was measured with Microplate Reader (BioTek Instruments).The content of calcium was measured by Alizarin Red Staining. In brief, 21 days after induction, ADSCs were fixed by 4% paraformaldehyde for 5 min and then washed by PBS three times. Then, ADSCs were incubated with 0.2% Alizarin Red solution for 30 min. Finally, optical microscope  was implemented for the staining result observation and photographing.ADSCs underwent osteogenic differentiation and then divided into two groups: control and Salidroside-treated groups. ADSCs were treated with Trizol reagent to extract total RNA. Solexa pipeline v1.8  was used to perform image analysis and substrate call. FastQC Software  was used to assess the quality and integrity of the RNA samples. HISAT2 software was used to align reads to the reference genome sequence (hg38). Transcripts from all samples were merged using StringTie to create a new reference file, which was then used to estimate transcript abundance in each sample. Differential expression analysis of the genes was performed using the DESeq package in R software (version 3.5.1). The heatmap of differentially expressed genes was drawn using the Complex Heatmap package in R. Gene ontology of the differentially expressed genes mainly including biological process, cellular component, and molecular function. Gene Ontology (GO) was performed using the clusterProfiler package in R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes was performed using R software (version 3.5.1).For constructing adenoviruses expressing siβ-catenin, siRNAs targeting the coding region of mouse β-catenin were assembled to an adenoviral shuttle vector. The siRNA target sites against human β-catenin-coding region were cloned into the pSES adenoviral shuttle vector to create recombinant adenoviruses. Polybrene (4 μg/mL) was used to increase adenovirus infection efficiency. The effects of the transfection on β-catenin reduction were assayed by Western blot analysis.−△△Ct method. The primers used were as follows: OCN, 5′-TCTTAGAACAAATTCTGCCCTTT-3′ (forward) and 5′-TGCTTTGGTCTTGAAATCACA-3′ (reverse); COL1A, 5′-CCTCCTCAGCTCACCTTCTC-3′ (forward) and 5′-GTTGGGAGCCCAAATAGAAA-3′ (reverse); RUNX2, 5′-AAGGTGTACGGCAAGGCTTC-3′(forward); 5′-CGTCAGAGCGAGTGAACCTC-3′ (reverse); GAPDH, 5′-CAATGTGGCCGAGGACTTTG-3′ (forward) and 5′-CATTCTCCTTAGAGAGAAGTGG − 3′ (reverse).Total RNA was extracted from ADSCs using the Trizol reagent in accordance with the manufacturer’s protocol. The first strand of cDNA was synthesized using the PrimeScript Reverse Transcriptase (RT) (TaKaRa Code: D2680S) and stored at − 20 °C for later use. The 7500 fast RT-PCR (ABI) was used for real-time quantitative PCR (qPCR). The expression of mRNA was calculated normalized to GAPDH with 2After washing human adipose-derived mesenchymal stem cells three times with PBS, total protein was extracted using RIPA protein lysis buffer  with freshly added 1% protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amounts of protein were resolved on a SDS-PAGE gel. After SDS-PAGE, the proteins were transferred to a PVDF membrane by electro-blotting for 1 h at 100 v. Membranes were blocked with 4% (w/v) non-fat milk powder in TBST  at room temperature for 2 h. Then, the membranes were incubated with primary antibodies on a shaker, overnight at 4 °C. After that, the membrane was incubated with HRP-conjugated second antibody at room temperature for 2 h. The determination of the grayscale value was processed using ImageJ software.Statistical analysis was performed using the SPSS 20.0 statistical package . A statistically significant difference between groups was indicated at p < 0.05. Data are expressed as the mean ± standard deviation (SD). Differences were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey’s post hoc test for multiple comparisons. Graphs were made using GraphPad Prism 7 (GraphPad Software).2D structure of Salidroside can be seen in Supplement In order to assess the role of Salidroside in promoting osteogenic differentiation of ADSCs, ALP, and ARS were performed to assess the early and late osteogenic capacity, respectively. As shown in Fig. Western blot analysis was in agreement with the quantitative real-time PCR (qRT-PCR) results, showing that the protein expression of OCN, COL1A1, and RUNX2 was upregulated in the Salidroside group  assay to identify the optimal dose range of Salidroside. Then, we performed ALP and ARS staining assays to identify the osteogenic promotion effects of Salidroside. RNA sequencing was then performed to identify the differentially expressed genes between normal and Salidroside-treated ADSCs. Differentially expressed genes mainly enriched in the Wnt/β-catenin signaling pathway. We finally constructed small interfering RNA of β-catenin to verify the mechanism of Salidroside.Strengths of this study were as follows: (1) we firstly performed RNA sequencing to assess the mechanism of Salidroside in promoting osteogenic differentiation of ADSCs, and (2) results revealed that Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.2O2 [The process of bone formation includes the initial proliferation of osteoblasts and the development and maturation of the extracellular matrix and the final mineralization. The osteogenic differentiation starts from adhesion and proliferation of several cell types, such as mesenchymal stem cells, pre-osteoblasts, and osteoblasts. Firstly, we performed CCK-8 assay to identify the optimal dose of Salidroside. We selected 5 μM Salidroside for further study. Then, ALP and ARS staining were performed to assess the osteogenic capacity of Salidroside. We found that Salidroside significantly increased osteogenic capacity of ADSCs. This study was in accordance with a previous study, which found that Salidroside protect against bone loss induced by H2O2 . Bai et 2O2  revealedOne of the commonly applied methodologies is deep RNA sequencing of proliferating, which indicated changes in the mRNA expression profiles in treatment and control groups. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Most of the differentially expressed genes mainly enriched in Wnt/β-catenin signaling pathway. Thus, we assessed the Wnt/3A and β-catenin expression in control and Salidroside-treated ADSCs. We found that Salidroside significantly increased Wnt/3A and β-catenin expression. Wnt/β-catenin was involved into multiple cells differentiation including neural differentiation , preadipIn order to further identify the Wnt/β-catenin involved into the process of osteogenic differentiation of ADSCs. We constructed siRNA of β-catenin to inhibit the Wnt/β-catenin signaling pathway. We found that silencing β-catenin partially blocked the promotion effects of Salidroside on the osteogenic differentiation of ADSCs. Therefore, we concluded that Salidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.This study has several shortcomings that are worth mentioning. First, the present study did not perform in vivo experiments. Second, receptor of Salidroside did not identify in this study. Third, the optimal dose regimen of Salidroside has not been established.In conclusion, our data indicate that Salidroside promoted osteogenic differentiation of ADSCs via the canonical Wnt/β-catenin signaling pathway. The findings in this study may provide insights into understanding the mechanism of Salidroside for osteogenesis. Salidroside may be a novel therapy for bone-related diseases.Additional file 1: Supplement S1: 2 D structure of Salidroside.


Adenomyosis is defined as the presence of ectopic nests of endometrial glands and stroma within the myometrium. Adenomyosis is a common cause of dysmenorrhea, menorrhagia, and chronic pelvic pain but is often underdiagnosed. Despite its prevalence and severity of symptoms, its pathogenesis and etiology are poorly understood. Our previous study showed that aberrant activation of β-catenin results in adenomyosis through epithelial–mesenchymal transition. Using transcriptomic and ChIP-seq analysis, we identified activation of TGF-β signaling in the uteri of mutant mice that expressed dominant stabilized β-catenin in the uterus. There was a strong positive correlation between β-catenin and TGF-β2 proteins in women with adenomyosis. Furthermore, treatment with pirfenidone, a TGF-β inhibitor, increased E-cadherin expression and reduced cell invasiveness in Ishikawa cells with nuclear β-catenin. Our results suggest that β-catenin activates TGF-β-induced epithelial–mesenchymal transition in adenomyosis. This finding describes the molecular pathogenesis of adenomyosis and the use of TGF-β as a potential therapeutic target for adenomyosis. Tgf-β2 gene is directly regulated by β-catenin in the uterus. TGF-β2 levels were elevated in human adenomyosis lesions, suggesting the protein could be a therapeutic target.A regulatory link between two proteins involved in the progression of a debilitating uterine condition highlights a potential therapeutic target. Adenomyosis involves the invasion of cells from the inner lining of the uterus (the endometrium) into the uterine muscle wall (the myometrium), resulting in heavy, prolonged periods and chronic pain. The aberrent activation of a protein called β-catenin triggers adenomyosis, but the precise mechanisms are unclear. A team led by Jung-Ho Shin at the Korea University Medical Center, Seoul, South Korea, and Jae-Wook Jeong, Michigan State University, Grand Rapids, USA, used sequencing techniques in mice and human tissue samples to identify the pathways governed by β-catenin in adenomyosis. They found that the  Adenomyosis is diagnosed in 10 to 66% of women at the time of hysterectomy3. It is systematically associated with menorrhagia, dysmenorrhea, chronic pelvic pain and dyspareunia5, and can interfere with embryo implantation and cause subfertility7. Currently, adenomyosis is more frequently diagnosed in infertile patients by transvaginal ultrasonography and magnetic resonance imaging8. However, early diagnosis of adenomyosis is difficult because of the absence of pathogenomic symptoms and biomarkers. Therefore, most women are not diagnosed until later stages of the disease, and severely symptomatic women who do not respond to pharmacological therapy require invasive surgical intervention (hysterectomy). It has been suggested that adenomyosis is an ovarian steroid hormone-dependent disorder resulting from high estrogen levels unopposed by progesterone, similar to endometriosis, endometrial hyperplasia, and endometrial cancer10. However, the precise etiology and pathophysiology of adenomyosis is still unknown.Adenomyosis is a common benign heterogeneous gynecological disorder defined by the presence of endometrial glandular and stromal tissue found in the myometrium11. Indeed, there have been several studies in mice using hormonal treatment that have shown an increased incidence of adenomyosis14. Nevertheless, the molecular mechanism for the development and progression of adenomyosis is still unclear. Mice with uterine conditional activation of β-catenin (Pgrcre/+Ctnnβf(ex3)/+) develop adenomyosis15. Pgrcre/+Ctnnb1f(Ex3)/+ mouse has stabilization of β-catenin in Pgr-expressing cells and is a useful mouse model to determine the effect of constitutive activation of β-catenin in the mouse uterus16. Our previous data showed that Pgrcre/+Ctnnb1f(ex3)/+ mice exhibited infertility, hormone insensitivity, and endometrial glandular hyperplasia16. Although the PgrCre mouse shows Cre recombinase activity in the pituitary, ovary, and mammary glands, Pgrcre/+Ctnnb1f(ex3)/+ mice revealed normal ovarian functions and did not develop breast cancer. Therefore, it is a useful model system to investigate the genetic and molecular events involved in the transition from normal uterine structure to adenomyosis.Studies using animal models in many different species, including mice, rats, rabbits, dogs, cats, and nonhuman primates, have provided insight into this disease17. The Wnt/β-catenin pathway is important in tissue differentiation during embryonic development and tissue homeostasis and function in adults. In the absence of Wnt signaling, β-catenin is degraded by the β-catenin destruction complex, including adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK-3β)21. Mutations and excessive activation of β-catenin are associated with many cancers, including hepatocellular carcinoma, colorectal carcinoma, lung cancer, malignant breast tumors, and ovarian and endometrial cancer23, as well as epithelial–mesenchymal transition (EMT)25.β-catenin has a dual function, regulating both the coordination of cell–cell adhesion and gene transcription, depending on its localization in the cell. At the plasma membrane, β-catenin is a component of the E-cadherin-catenin unit, where it maintains cell differentiation and normal tissue architecture. β-catenin nuclear translocation depends on Wnt signaling, and nuclear β-catenin can act as a transcriptional activator to regulate the transcription of target genes responsible for cell proliferation and differentiation25. EMT is important for several developmental processes, including embryogenesis, organ fibrosis, and wound healing27. EMT is also critical in the initiation of metastasis for cancer progression28, as well as in uterine diseases such as adenomyosis, endometriosis, and endometrial cancer30. Serum estrogen levels are negatively correlated with E-cadherin expression levels in the epithelial components of the eutopic endometrium and adenomyotic lesions compared to controls30. EMT is initiated by a number of transcription factors, including Snail, Slug, Twist, ZEB1, and SIP1, via the repression of E-cadherin expression31. Furthermore, EMT is induced or regulated by growth and differentiation factors such as TGF-β32. Crosstalk between TGF-β/Smad signaling and Wnt/β-catenin signaling is important in developmental and pathological events34. TGF-β signaling influences the Wnt pathway by regulating the activity of β-catenin through the interaction of activated Smad complexes with APC/GSK-3β, β-catenin, and TCF/LEF135. In the context of EMT, Smad2 and Smad4 form a complex with LEF1 and suppress the expression of E-cadherin by binding to the E-cadherin promoter. These complexes induce the expression of the mesenchymal markers vimentin and fibronectin36.EMT is a biological process by which epithelial cells lose cell polarity and cell adhesion and gain migratory and invasive properties to become mesenchymal cellsTgf-β2 is directly regulated by activated β-catenin in the murine uterus. The levels of TGF-β2 were higher in endometrial epithelial cells of human adenomyosis lesions and the corresponding eutopic endometrium than in control women. Furthermore, we found a positive correlation between β-catenin and TGF-β2 proteins in the endometrium from women with and without adenomyosis. These results suggest that TGF-β2 plays a critical role in adenomyosis development as a direct target of β-catenin.In this study, we performed transcriptomic and ChIP-seq analyses under in vivo conditions of aberrant β-catenin activation to identify genome-wide β-catenin targets in the mouse uterus. We found that Pgrcre/+ and Ctnnb1f(ex3)/+) and mutant (Pgrcre/+Ctnnb1f(ex3)/+) mice37. Uterine tissues were immediately frozen at the time of dissection and stored at −80 °C for RNA extraction and ChIP analysis or fixed with 4% (v/v) paraformaldehyde for histology and immunostaining analysis.Mice were maintained in the designated animal care facility according to Michigan State University’s institutional guidelines. All animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University. Uterine tissues were collected from control (38. Eutopic endometrium (4 proliferative and 4 secretory phases) and adenomyosis lesion (15 proliferative and 15 secretory phases) samples were from women with adenomyosis, and control endometrium (n = 8 proliferative and n = 13 secretory) samples were from women without adenomyosis. Samples used for immunostaining were fixed in 10% buffered formalin prior to embedding in paraffin wax.Human adenomyosis samples were obtained from Korea University Guro Hospital of Biobank with the approval guidelines instituted by the Institutional Review Boards of Korea University. Written informed consent was obtained from all participants. Adenomyosis tissue samples with their corresponding eutopic endometrium were collected from surgical hysterectomy specimens. Controls were regularly cycling premenopausal women undergoing an endometrial hysterectomy for benign conditions with no history or evidence of adenomyosis, who were documented not to be pregnant and who had not been on hormonal therapies for at least 3 months before tissue sampling. Histologic dating of endometrial samples was performed based on the criteria of Noyes39. Microarray data are deposited in the Gene Expression Omnibus (GEO) database, with accession number GSE88911. Differentially expressed genes were classified according to canonical pathway analysis by Ingenuity System Software .Total RNA was extracted using the RNeasy Total RNA Isolation Kit . RNA was pooled from the uteri of 3 mice per genotype at 1 month of age. All RNA samples were analyzed with a Bioanalyzer 2100  before microarray hybridization. Microarray data analysis was performed as previously described40) peak-finding algorithm was used to normalize ChIP against the input control. Genes associated with intervals were assessed using three increasingly less stringent requirements; if it was within 10, 25, or 50 kb upstream or downstream of a gene, it was counted. Sequence conservation to identify phastCons scores, analysis of enriched motifs, and cis-regulatory element annotation system (CEAS) were performed using the Cistrome Analysis Pipeline software (http://cistrome.org/ap/root) under default settings41. Ingenuity Systems Pathway Analysis software (http://www.ingenuity.com/products/ipa) was used for gene functional annotations.β-catenin and input ChIP were performed by Active Motif, Inc.  on control and mutant mouse uteri at 1 month of age. ChIP and input DNA were amplified using the Illumina ChIP-Seq DNA Sample Prep Kit . DNA libraries were sequenced by Illumina’s Hi-Seq Sequencing Service. The sequences were aligned to the mouse genome . Alignments were extended in silico (using Active Motif software) at their 3′ ends to a length of 150–250 bp and assigned to 32-nucleotide bins along the genome. Peak locations were determined by applying a threshold of 18  and storing the resulting intervals in Browser Extensible Data (BED) files . These files were analyzed using Genpathway proprietary software that provides comprehensive information on genomic annotation, peak metrics, and sample comparisons for all peaks . The model-based analysis of the ChIP-Seq . Eluted DNA was amplified with specific primers using SYBR Green Supermix . The β-catenin-binding site (CBS) for the ChIP assay was determined by ChIP-seq, and primers for quantitative reverse transcription PCR (RT-qPCR) were designed to query the β-catenin intervals on Tgf-β2. One negative control (NC) site on the Tgf-β2 gene was designed in an area where no enrichment of β-catenin was observed in mutant mouse uteri. The PCR primers were as follows: CBS  and negative control . The resulting signals were normalized to input activity.ChIP assays were performed in the uteri of control and mutant mice at 4 weeks of age as previously describedRpl7 RNA. The sequences of the primers used for mouse Tgf-β2 were 5′-TAAAATCGACATGCCGTCCC-3′ and 5′-GAGACATCAAAGCGGACGAT-3′ and for mouse Rpl7 were 5′-TCAATGGAGTAAGCCCAAAG-3′ and 5′-CAAGAGACCGAGCAATCAAG-3′.Complementary DNA was produced from 1 μg of total RNA using random hexamers and MMLV Reverse Transcriptase . Real-time PCR was performed using an RT-PCR SYBR Green detection system  according to the manufacturer’s instructions . mRNA quantities were normalized against the housekeeping gene 42. Uterine sections from paraffin-embedded tissues were preincubated with 10% normal serum in phosphate-buffered saline (PBS) and incubated with anti-TGF-β2 , anti-E-cadherin , anti-F4/80 , anti-CD68 , anti-CD4 , and anti-vimentin  antibodies in 10% normal serum in PBS. On the following day, sections were washed in PBS and incubated with a secondary antibody  for 1 h at room temperature. Immunoreactivity was detected using the Vectastain Elite DAB Kit (Vector Laboratories). Images were captured with a confocal microscope .Immunohistochemistry and immunofluorescence analyses were performed as previously described2. Transient transfection of the exon 3-deleted β-catenin vector21 into Ishikawa cells was performed using Lipofectamine 2000 .Cells from the Ishikawa uterine endometrial epithelial cell line  were cultured in DMEM/F12 medium  supplemented with 10% fetal bovine serum  and 1% penicillin streptomycin . Cells were cultured in monolayers at 37 °C in 5% CO43. Samples, which were lysed in buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, 0.125% Nonidet P-40 (v/v), and protease inhibitors, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes . Membranes were probed with anti-β-catenin , anti-TGF-β2 , anti-E-cadherin , anti-vimentin , and anti-actin  antibodies. Immunoreactivity was visualized by autoradiography.Western blot analyses were performed as described previously5 per 200 μl serum-free culture medium into the insert chamber of a BioCoat . The cells were incubated at 37 °C in 5% CO2 for 48 h, and noninvading cells were removed with a cotton swab. Invading cells on the lower surface of the membrane were fixed with 100% methanol and stained with 1% crystal violet . Stained cells were observed using fluorescence microscopy  using software from NIS Elements, Inc. .For the transwell invasion assay, post-transfected cells (24 h) were trypsinized and seeded at a density of 2.5 × 10t-test for data with only two groups or analysis of variance (ANOVA) for data containing more than two groups, followed by Tukey’s post hoc test for multiple comparisons using Instat . p < 0.05 was considered statistically significant.Statistical analyses were performed using Student’s 15. An integrated bioinformatics approach combining chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analysis was applied to identify direct target genes of β-catenin in the uterus of control (Pgrcre/+ and Ctnnb1f(ex3)/+) and mutant (Pgrcre/+Ctnnb1f(ex3)/+) mice at 4 weeks of age. To determine the β-catenin cistrome in the uterus, ChIP-seq results were used to create a genome-wide profile of in vivo β-catenin-binding sites in control and mutant mice. More than 20 million tags of each sample were mapped to unique locations in the mouse genome. A model-based analysis peak-finding algorithm was used to normalize immunoprecipitated chromatin against input with a value of p = 1 × 10−7 . This high-confidence cutoff produced 2092 intervals in the control uterine samples and 3524 intervals in the mutant uterine samples. A total of 606 β-catenin-bound intervals were in common between the control and mutant uteri. This approach identified 2918 binding target sites unique to the mutant uterus that are potential β-catenin target genes in the mutant mice (Supplementary Table Our previous study showed that adenomyosis developed in mice with uterine-specific β-catenin activationp < 0.05) as differentially expressed in the uteri of mice with uterine-specific β-catenin activation compared to controls. Of the 2079 genes, 1103 were increased, and 976 genes were decreased . It has been reported that TGF-β1 levels are elevated in uterine washings from patients with adenomyosis44, as well as in a mouse model of adenomyosis45. However, Tgf-β1 expression was not altered in our transcriptomic analysis, and Tgf-β3 was significantly decreased in the mutant mice compared to the controls  and vimentin  in the uteri of control and mutant mice at 4 weeks of age. The levels of TGF-β2 were higher in endometrial epithelial cells of mutant mice compared to the control, while E-cadherin levels were decreased in endometrial epithelial cells of mutant mice Fig. . The exp15. To determine whether TGF-β2 is dysregulated during adenomyosis development, we examined the level of TGF-β2 in the uteri of control and mutant mice at 2, 4, and 6 months of age using immunohistochemistry analysis. The levels of TGF-β2 were higher in endometrial epithelial cells of mutant mice at 2, 4, and 6 months of age compared to controls . After transfection of exon 3-deleted β-catenin, Ishikawa cells were treated with pirfenidone, and the levels of E-cadherin and invasiveness of Ishikawa cells were examined. The levels of E-cadherin in Ishikawa cells transfected with activated β-catenin were decreased at 48 h. However, pirfenidone treatment abrogated this effect Fig. . Further49, as well its pathological role in adenomyosis development15. The integrated uterine transcriptome and ChIP-seq analyses presented here define pathways governed by β-catenin during adenomyosis development. The present study found many candidate genes and pathways that are likely positively regulated by β-catenin in the development of adenomyosis in the mutant uterus. From the pathway analysis using Ingenuity Systems Software, we found altered pathways, including Wnt/β-catenin signaling, TGF-β signaling, embryonic stem cell pluripotency, basal cell carcinoma signaling, ErbB signaling, and ERK/MAPK signaling. Future mechanistic studies will be needed to understand their biological roles in adenomyosis development.This study represents the first comprehensive analysis of the β-catenin cistrome in the uterus, which is important due to β-catenin’s critical role in uterine development and functionThe molecular mechanism for the development and progression of adenomyosis is still unclear, and an appropriate genetically modified mouse model has not yet been developed. Mice with uterine conditional activation of β-catenin were used to explore how the dysregulation of β-catenin signaling can lead to the development of adenomyosis. The major advantage of using such models is spontaneous adenomyosis development and the presence of an intact immune system, which is lost in xenotransplant models. Our animal model constitutes a novel model system that investigates the genetic and molecular events involved in the transition from normal uterine structure to adenomyosis.As with any mouse model for human disease, the data should be properly interpreted within the boundaries of its context. As such, questions remain as to whether women with activated TGF-β signaling in their eutopic endometrium are more prone to developing adenomyosis. Transgenic models such as the one presented in this study provide evidence that if there is an inherent defect in the eutopic endometrium, this induces the development of adenomyosis. Although there is no perfect model for human adenomyosis aside from the human disease condition, studies such as these provide proof-of-principle evidence to explore and consider potential targets of therapy for adenomyosis.50. TGF-β superfamily members are key regulators of female reproduction, including ovulation, uterine decidualization, and embryo development53. TGF-β signaling has been shown to play an important role in EMT54, and it influences Wnt/β-catenin signaling through the interaction of activated Smad complexes with APC/GSK-3β, β-catenin, and TCF/LEF135. Liu et al.55 reported the increased expression of TGF-β1 in adenomyotic lesions. However, our results revealed that TGF-β1 is not a target of β-catenin. Interestingly, we found a strong positive correlation between β-catenin and TGF-β2 in the endometrial epithelial cells of women with and without adenomyosis. The levels of TGF-β2 were remarkably strong in endometrial epithelial cells of mutant mice compared to controls. Our immunofluorescence results confirmed that vimentin-positive epithelial cells express TGF-β2 proteins in the mutant mice, which suggests that aberrant activation of β-catenin and TGF-β2 plays an important role in the pathogenesis of adenomyosis.In this study, we demonstrated that TGF-β2 overexpression plays an important role in the pathogenesis of adenomyosis with aberrant activation of β-catenin. TGF-β superfamily signaling plays a pleiotropic role in fundamental cellular and developmental processes56. TGF-β signaling is widely known to be associated with multiple signaling pathways, such as cell homeostasis, immunomodulatory functions and EMT formation. TGF-β cooperates with Wnt/β-catenin to forward complete EMT and regulate the mesenchymal phenotype of invasive/metastatic tumor cells57. TGF-β can upregulate canonical Wnt signaling58. TGF-β1 induced β-catenin nuclear translocation in primary porcine valve interstitial cells through TGF-β receptor I kinase59. However, this is the first report that β-catenin induces EMT process through TGF-β2 in adenomyosis development.The regulation of EMT is mostly initiated by several signaling pathways, including TGF-β, Wnt/β-catenin, and growth factors60. This cancer cell line is modestly responsive to estrogen but has lost its inhibitory response to progesterone despite the expression of receptors for both hormones. Another limitation is the lack of β-catenin-mediated TGF-β2 secretion in our study.Furthermore, Ishikawa cells with nuclear β-catenin expression induced the expression of TGF-β2 and vimentin but decreased the expression of E-cadherin. Overexpression of TGF-β2 in human adenomyosis and the ability of a specific TGF-β inhibitor to decrease cell invasion indicate that the TGF-β pathway is critical for the establishment of adenomyosis. Treatment of Ishikawa cells with the TGF-β inhibitor pirfenidone decreased the EMT process and preferentially inhibited cell invasion, demonstrating the major role TGF-β plays in adenomyosis. One limitation of our study is that Ishikawa human endometrial adenocarcinoma cell line was used to study benign disease. However, it is one of the few endometrial cell lines that expresses functional ERα and PR62 and is approved by the Federal Drug Administration (FDA) for the treatment of idiopathic pulmonary fibrosis63. Our results show that pirfenidone treatment suppresses EMT and cell invasiveness in Ishikawa cells with β-catenin activation. Notably, pirfenidone also inhibits TGF-β1 as well as TNFα and IL-1β64. Therefore, the effect of pirfenidone was confirmed by using two other TGF-β inhibitors, GW788388 and R268712. Although these TGF-β inhibitors used in the present work are not specific for TGF-β2, our results suggest that TGF-β2 is a potential therapeutic target for adenomyosis-related β-catenin activation and EMT. Therefore, our study improved the understanding of the molecular mechanisms involved in the early pathogenesis of adenomyosis and will help develop strategic therapies for nonsurgical treatment.Pirfenidone (5-methyl-1-2-[1H]-pyridone) is a small synthetic molecule that inhibits TGF-β productionTgf-β2 as a direct target gene of β-catenin in the uterus using transcriptomic and ChIP-seq analyses. The levels of TGF-β2 are higher in endometrial epithelial cells of β-catenin-stabilized mice compared to control mice, as well as in epithelial cells of human eutopic endometrium and adenomyosis lesions compared to women without adenomyosis. We demonstrated a strong positive correlation between β-catenin and TGF-β2 protein levels in women with adenomyosis. β-catenin activation leads to EMT in endometrial epithelial cells through TGF-β2, and TGF-β inhibition suppresses EMT-related β-catenin activation effects. These results provide significant insights into our understanding of the pathophysiological function of β-catenin in adenomyosis development and suggest the therapeutic potential of a TGF-β inhibitor for adenomyosis.In summary, we identified Supplementary Information File