The PI3K/Akt and Wnt/β-catenin pathways play an important role in the acquisition of the malignant phenotype in cancer. However, there are few data regarding the role of the interplay between both pathways in colorectal cancer (CRC) progression. The mutational status and the clinicopathological characteristics of PI3K/Akt and Wnt/β-catenin pathways were accessed by bioinformatic analysis whereas that the impact of the interplay between the activity of both pathways to explain tumorigenic potential was performed in vitro using IGF-1 and Wnt3a treatments in CRC cell models. The mutational status of these pathways did not influence the survival of CRC patients, but an association between clinicopathological characteristics in patients with mutations in one, but not in both pathways was observed. A potentiating effect on the activation of both pathways and enhanced cellular migration and proliferation was observed when both pathways were activated simultaneously with IGF-1 and Wnt3a. In addition, these effects were hindered after pretreatment with LY294002, a specific PI3K inhibitor, suggesting some dependence between these two signaling cascades. Our findings show that, regardless of mutational status, there is an interplay between the activity of PI3K/Akt and Wnt/β-catenin pathways that contributes to events related to CRC progression and that the reversal of such events using a PI3K inhibitor highlights the value of targeting these pathways for potential directed therapies in CRC patients. Its high mortality rate is a consequence of various factors including the lack of apparent symptoms in early stages and deficits in cancer prevention strategies, particularly in developing countries.APC, CTNNB1 or AXIN2 reduces the capacity of the destruction complex, composed of APC, AXIN and GSK3β, to commit β-catenin to degradation. As a result, β-catenin accumulates in the nucleus, binds the TCF/LEF transcription factors and induces the expression of target genes that play key roles in tumor progression.3 Activation of this pathway contributes to the onset and progression of more than 90% of CRC cases.4 Studies show that 20% of CRC patients present mutations in genes of the PI3K pathway such as, PIK3RI, PI3KCA and PTEN.6 In particular, PI3KCA plays a role in the activation of PI3K/Akt signaling to promote proliferation, cell growth and survival. Drugs targeting PI3K/Akt activity are currently in clinical trials, however, CRC patients exhibit some resistance to these drugs.7–10 In addition, as inappropriate activation of the Wnt/β-catenin pathway was first linked to CRC, there is an intense interest in developing effective inhibitors.11 Unfortunately, the activation or inhibition of a single signaling pathway is unlikely to result in a substantial improvement in disease progression given the co-activation of numerous oncogenic pathways in this cancer. For instance, it was shown that mutations in genes frequently altered in CRC, including KRAS, TP53, or PIK3CA, do not predict the response to PI3K/Akt pathway inhibitors. Interestingly, multivariate analysis indicated that APC mutations are a risk factor for patients treated with these inhibitors, which suggests that the activation of the Wnt/β-catenin pathway could participate in the mechanism of resistance to PI3K/Akt inhibitors.12The dysregulation of various signaling pathways plays a significant role in the development of CRC. Over activation of the Wnt/β-catenin signaling pathway is one of the most frequent alterations in CRC. Their abnormal activation caused by mutations in 14 However, recent studies have shown that an interaction between these two pathways contributes to the progression of various cancer types, including the ovary and pancreas.16 In CRC patient samples, the presence of nuclear β-catenin and an active PI3K/Akt pathway was associated with an increased risk of metastasis, suggesting a crosstalk between these pathways.17 However, little is known about how these pathways interact to contribute to CRC progression.The individual participation of the PI3K/Akt and Wnt/β-catenin pathways during the CRC progression is well established.This study was designed to identify players that mediate the interaction between PI3K/Akt and Wnt/β-catenin pathways or mutational alterations that might be useful in predicting responses to treatment using modulators of these pathways and to determine their viability as therapeutic targets for CRC treatment.APC, CTNNB1, AXIN1 and AXIN2) and PI3K/Akt pathways  in several open-access CRC clinical cohorts. Of the 705 patients included in the study, 397 (56.3%) had mutations in the APC gene; 11 (1.6%) patients had mutations in AXIN1; 19 (2.7%) patients had mutations in AXIN2 and 22 (3.1%) had mutations in the β-catenin gene. Regarding the PI3K pathway, the PIK3CA gene was mutated in 104 (14.7%) patients and 36 (5.1%) patients also had mutations in PTEN.We analyzed the presence of mutations in the main components of the Wnt/β-catenin  having no mutations in any of the pathway genes; (2) mutated for the PI3K pathway genes and wild-type Wnt pathway genes; (3) having mutations in Wnt pathway genes and wild-type PI3K pathway genes, and (4) having mutations in the genes of both pathways simultaneously. We found that 236 (33.5%) patients had no mutations in any of the pathways. Fifty-six (7.9%) patients had mutations in PI3K pathway genes, and 346 (49.1%) patients had mutations in Wnt pathway genes, while 67 (9.5%) patients had mutations in the genes of both pathways. Then, we verified whether these mutations could influence patient survival by using Kaplan Meier curves . There were no significant differences in overall survival between the subgroups of patients and the mutations analyzed.Next, we analyzed the correlation between the frequency of genetic alterations in these pathways and a subset of clinicopathological parameters. In a comparison of patients without mutations and those with mutations in PI3K alone, PI3K mutations were more frequently associated with early stages (I and II) of CRC progression . Regarding those with alterations in the Wnt pathway alone, we observed that these patients were more likely diagnosed at ages above 50 years ; and in the early stages (I and II) . There was also a positive association with tumor location in the rectum . Furthermore, we detected a higher frequency of patients with mutations in Wnt pathway genes associated with microsatellite stability (MSS) . We did not observe significant clinical differences between patients with mutations in the genes of both pathways and patients with no mutations in these genes . These rp = .05 for PI3K; OR: 0.60; 95%CI: 0.40–0.90; p = .015 for Wnt) were associated with lower odds ratios of mutations in the Wnt or PI3K pathway genes. On the other hand, patients with tumors in the rectum  had greater odds ratios of mutations in Wnt pathway genes. Other patient characteristics were not statistically significant  and this effect persisted for 24–48 h. Since GSK3β is a downstream target of Akt,18 we then monitored p-GSK3β levels and observed a similar increase in phosphorylation from 15 min to 48 h. In HCT-116 cells, we observed a significant increase in Akt phosphorylation from 15 min to 48 h and an increase in GSK3β phosphorylation restricted to the 60 min time point. .To evaluate pathway activity in our study model, we initially verified the activation of the PI3K/Akt and Wnt/β-catenin pathways through treatment with IGF-1 and with conditioned medium of cells that overexpress Wnt3a (L-Wnt3a) or recombinant Wnt3a (Wnt3a-RH). WSince most of CRC cell lines harbor mutations in different genetic components of the Wnt/β-catenin pathway that constitutively activate the pathway, we evaluated whether these cells were responsive to treatment using Wnt3a, a ligand of this signaling pathway. We observed that cells treated with recombinant Wnt3a or with conditioned media rich in Wnt3a increased transcriptional activity of β-catenin in both cell lines . We furtConsidering the small variations observed in the activation status of the PI3K/Akt and Wnt/β-catenin pathways in response to the treatments in HT-29 and HCT-116 cells, we expanded our evaluation to three more CRC cell lines . Treatment with Wnt3a, IGF1, or both combined resulted in a wide range of activation of these pathways , indicating that this cross activation happens in a cell-specific manner. Therefore, we choose HT-29 and HCT-116 cells to continue our analysis.Some studies have shown that when the PI3K/Akt and Wnt/β-catenin pathways are concomitantly activated, synergism in tumor progression is observed. To test this hypothesis in our study models, HT-29 and HCT-116 cells were treated with Wnt3a or IGF1, or simultaneously with both treatments, and the activation of these pathways was analyzed through the phosphorylation of key proteins. Our data show that Wnt3a and IGF1 individually or in combination, all stimulated the phosphorylation of Akt and GSK3β in the cell lines indicating increased activity of these pathways. It is worth mentioning that the activation of the IGFR receptor was noticed only with the IGF1 treatment alone and in the combination with Wnt3a, indicating that Wnt3a acts by increasing the phosphorylation of Akt through a different receptor .Figure The effect of the combination of treatment on the Wnt/β-catenin pathway was then analyzed. As expected, Wnt3a alone was able to activate the pathway in both HCT-116 and HT-29 cells. An increase in the transcriptional activity of β-catenin was also noted after individual treatment with IGF1 when compared to the control. The combined treatment exhibited greater luciferase activity when compared to each individual treatment . AdditioTogether, these results show that IGF1 can activate the PI3K/Akt pathway and, to some extent, the Wnt/β-catenin pathway. Likewise, Wnt3a was able to activate both pathways, indicating a crosstalk between them. Furthermore, the combination of treatment with these ligands potentiated the activation of these pathways.To determine if the different treatments affected the malignant phenotype, we evaluated the effect of the treatments on the proliferation and migration of HT-29 and HCT-116 cells. Individual activation of the pathways using Wnt3a and IGF1 or combined treatment with these agents led to an increase in cell proliferation. . We veriNext, we analyzed cell migration and observed that the combined treatment with both compounds significantly stimulated the migratory potential of both cell lines. In the HT29 cell line the combined treatment also showed a significant increase when compared to the Wnt3a treatment . TogetheOur next step was to assess the impact of PI3K/Akt pathway inhibition, using the specific inhibitor LY294002 (LY), on the effects of the treatments previously described. For this assay, HCT116 cells were chosen because of their high sensitivity to the ligands. We observed that LY prevented the increase in phosphorylation of Akt and GSK3β induced by IGF1 and by the combined treatment using IGF1 and Wnt3a. However, LY did not affect the Akt phosphorylation induced by Wnt3a, indicating that this ligand phosphorylates Akt and GSK3β through a PI3K-independent mechanism . RegardiIn TP53 gene is frequently dysregulated in CRC and is associated with resistance to current therapies leading to poor prognoses.19 Tumors with KRAS and BRAF mutations are also associated with worse prognoses in CRC patients. In addition, a large meta-analysis including over 12,000 patients showed that PIK3CA mutations were not significantly associated with CRC.5 On the other hand, mutations in APC seem to have a central impact on the survival of CRC patients.20 Furthermore, our data showed an association between mutations in the PI3K/Akt or Wnt/β-catenin pathway genes and colorectal cancer age of onset and early stages, suggesting that mutations in the genes of these pathways might be useful as prognostic predictive markers and/or as therapeutic targets to improve outcomes in CRC treatment. Studies in lung cancer indicate that the activation of these pathways may contribute to development of new primaries and chemoresistance in relapsed small-cell lung cancer.22 It is important to acknowledge that these alterations are result of a broad mutational analysis grouping mutations that might have diverging effects in tumoral progression. A larger cohort of patients would enable a more detailed analysis of specific mutations and combinations of such.The prognostic role of genetic alterations of PI3K/Akt or Wnt/β-catenin pathways in order to search for therapeutic candidates that complement activity studies has been few explored in CRC. In the present study, we initially analyzed the mutational status of both pathways and their prognostic implications in a large set of CRC patient data from publicly available databases. Our results showed no differences in overall survival among the groups. However, the accumulation of other mutations, which was not examined in this study, might influence these results. For instance, the PI3KCA continue to be activated in the presence of its ligand. The same effect was observed in other CRC cell lines23 where PTEN plays a dominant role. PTEN reverts PIP3 back to PIP2, shifting PI3KCA activating mutations to an Akt-independent mechanism.24 In addition, cells treated with Wnt3a increased the nuclear β-catenin and its transcriptional activity, indicating that it remains ligand-dependent. Some studies suggest that PI3K/Akt is not involved in the Wnt/β-catenin pathway,26 however our results show otherwise, suggesting crosstalk between these pathways. Corroborating these results, cells overexpressing Wnt have been reported to cause an increase in the phosphorylation of Akt.28 Furthermore, simultaneous treatment with IGF1 and Wnt3a induced a potentiating effect on the activation of these pathways. Indeed, concomitant activation of both pathways potentiates tumor progression in ovarian tumor16 as well as CRC,17 which reinforces our hypothesis that interplay between these pathways contributes to CRC progression. The fact that IGF1 and Wnt3a induced a variable response in other CRC cell lines, as shown here, indicates that mutations in PIK3CA, APC, and CTNNB1, as is the case in HT-29 and HCT-116 cells,30 are necessary for the ligand-mediated potentiating effect to occur.We also analyzed the activity of the PI3K/Akt and Wnt/β-catenin pathways using CRC cell lines, HT-29 and HCT-116, treated with IGF1, Wnt3a, or both combined. We observed that IGF1 increased the phosphorylation of Akt and GSK3β in relation to non-treated cells, suggesting that cells with mutations in 31 The increase in cell proliferation was confirmed by cell cycle analysis, where we observed a major portion of cells in the S and G2 phases after treatment. Also, we showed that when both pathways were simultaneously activated, cells migrated more when compared to the control group. Indeed, PI3K regulates cell migration by direct binding of the protein to its lipid products or indirectly via crosstalk with Rho GTPases.32 On the other hand, the Wnt/β-catenin pathway contributes to cell migration through E-cadherin redistribution, cytoskeleton rearrangement, and an increase in Rho family proteins.33 Additionally, synergism among mutations in the genes of both pathways during the development of intestinal cancers has previously been described promoting tumor aggressiveness in preclinical studies.34We observed an increase in cell proliferation after treatment with these ligands, but this effect was cell-specific, given that Wnt3a was more effective in HCT-116 cells and IGF1 was more effective in HT-29 cells. This differential effect can be explained by the abundance of specific receptors of these ligands.35 and found that it inhibited the transcriptional activity and nuclear localization of β-catenin. The increased transcriptional activity of β-catenin obtained by the association of LY, Wnt3a, and IGF1 might have occurred due to a shift in the activation of IGF1 toward the MAPK pathway, which, in combination with Wnt3a, may induce increased activity. The interaction between Wnt/β-catenin and MAPK pathways has already been described.36 However, further studies are necessary to confirm this hypothesis. In relation to Wnt/β-catenin, it is possible that Wnt3a stimulates the phosphorylation of Akt and GSK3β by an independent mechanism of PI3K. A previous study showed that Wnt3a activates ROCK and directly phosphorylates GSK3β in a dependent form of the non-canonical Wnt pathway.37 Furthermore, our results showed that the PI3K inhibitor prevented the increase in proliferation and migration induced by the individual or combined treatment using Wnt3a and IGF, suggesting that these pathways cooperate during tumor progression. A synergic effect between the Wnt/β-catenin and PI3K/Akt pathways during development and progression has previously been observed in ovarian cancer and CRC.34In order to clarify the crosstalk and potentiating effects observed in this study, we used LY294002, a specific inhibitor of PI3K38 The crosstalk between pathways allows for signaling redirection and ultimately the emergence of resistance. An example of this is the fact that patients with mutations exclusively in one of these pathways are found predominantly in the early stages of tumor progression. Our results reinforce the need for mutational status assessment of WNT and PI3K pathways in clinical biopsies and for those patients with concomitant mutations, the usage of clinical approaches targeting proteins located downstream of PI3K.Several therapies have already been developed that target components of the Wnt/β-catenin or PI3K pathways. Despite showing good results in clinical trials, resistance to these therapies is frequently observed.In conclusion, we have provided evidence for the value of targeting key proteins of the PI3K/Akt and Wnt/β-catenin pathways and their prognostic implications for developing new directed therapies and improve outcomes in CRC treatment. In addition, our findings highlight the existence of inter-connectivity between these two crucial signaling pathways in CRC progression , and thaRabbit anti-pAkt S473, anti-Akt, anti-pGSK3Β S9, anti-GSK, anti-IGF1R and anti-pIGF1R Y1135 monoclonal antibodies were purchased from Cell Signaling . Mouse anti-GAPDH monoclonal antibody was purchased from Ambion . Alexa Fluor® 488 goat anti-mouse IgG was obtained from Molecular Probes . Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG were acquired from Sigma . Mouse anti-β-catenin was purchased from Thermo Fisher . Insulin-like growth factor 1 (IGF-1) was obtained from Cell Signaling and reconstituted in 20 mM citrate, pH 3.0, to obtain a stock concentration of 100 µg/mL. The 2-(4-morpholinyl)-8-phenyl-1(4 H)-benzopyran-4-one LY294002 (PI3K inhibitor) was obtained from Cell Signaling and diluted in dimethyl sulfoxide (DMSO) to obtain a stock concentration of 3 mg/mL. Recombinant Wnt3a was purchased from R&D Systems  and diluted in phosphate-buffered saline (PBS) + 0,1% bovine serum albumin (BSA) to obtain a stock concentration of 200 µg/mL and a work concentration of 50 ng/mL.F12 and LoVo cell line was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium. All mediums were supplemented with 10% fetal bovine serum (FBS), penicillin G (100 mg/L), and streptomycin (60 mg/L) (Invitrogen) and cultivated at 37°C in a humidified atmosphere containing 5% CO2. Cells were passaged weekly by using a solution of 0.05% trypsin/0.02% EDTA in PBS. A table with the most noticeable mutations already described for each cell line is provided as Supplementary Material 5.The human CRC cell lines HT-29 , HCT-116 , Caco-2 , SW480 , LoVo , L-Wnt3a  and the parental line L-cells  were obtained from American Type Culture Collection . The cells were maintained in Dulbecco’s modified Eagle’s medium . SW480 cell line was grown in DMEM/ Ham′s Nutrient Mixture Prior to pharmacological treatments, cells were maintained overnight in DMEM with 1% FBS and then treated with Wnt3a (100 ng/mL), 50% conditioned medium of Wnt3a, or IGF1 (100 ng/mL) for different times. For experiments with the PI3K inhibitor, cells were pre-treated with 15 µg/mL of LY294002 1 h before the treatments described above.39 Briefly, cells at 80% confluence were sub-cultured 1:10 in T75 bottles with 10 mL of DMEM medium and grown for 4 days. The medium was collected, centrifuged for 1000 g for 10 min and filtered (0.22 µm pore). Another 10 ml of medium was added, and the cells were cultured for further 3 days. The medium was collected again, centrifuged for 1000 g for 10 min and filtered (0.22 µm pore). The first and second batch were mixed 1:1 and frozen for posterior use.Conditioned medium (CM) of Wnt3a was produced from L-Wnt3a and the parental L-cells cell lines, both kindly provided from Dr Jose Garcia Abreu from Universidade Federal do Rio de Janeiro. These cells were cultured in DMEM medium, and the CM was collected as previously described.To obtain total protein lysates, the cells were homogenized in RIPA extraction buffer: 1% Triton X-100; 0.5% sodium deoxycholate; 0.2% SDS; 150 mM sodium chloride, pH 7.4; 20 mM sodium fluoride; containing 1 mM sodium orthovanadate; and a cocktail of protease inhibitors (Sigma) for 30 min at 4°C. The extract was centrifuged at 10000 g and 4°C for 10 min. The supernatant was collected and stored at −80°C. The protein extract was dosed using the commercial BCA kit  and the BSA protein (Sigma) was used as standard. Protein lysates were submitted to electrophoresis in a polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE) and transferred to nitrocellulose membranes using the Trans-Blot Semi-Dry Transfer Cell equipment . Subsequently, the membranes were blocked with 5% BSA diluted in 0.1% TBS-Tween for 1 h and incubated overnight with the following primary antibodies: anti-GSK3β (1: 1000), anti-pGSK3β S9 (1:1000), anti-Akt (1:1000), anti-pAkt S473 (1:1000), anti-IGF1R (1:1000) or anti-pIGF1R Y1135 (1:1000). Anti-GAPDH (1:50.000) was incubated for 1 hour. After successive washes, the membranes were incubated for 1 h with corresponding secondary antibodies, anti-mouse or anti-rabbit conjugated to peroxidase, at a dilution of 1:40000 in 0.1% TBS-T, for 60 min. The membranes were washed with 0.1% TBS-T and the signal was detected by chemiluminescence using the ECL kit .To analyze the transcriptional activity of β-catenin, we measured the TCF/LEF transcription-factor reporter activity by using a Dual-Luciferase Reporter Assay System Kit . Two TCF luciferase reporters were used in the assay: an intact wild-type TCF-luciferase reporter construct (Super 8X TOPflash), and a mutated TCF-luciferase reporter construct (Super 8X FOPflash), used as a negative control. The cells were seeded in 24-well plates, grown until subconfluence and then were co-transfected transiently with 2 μg each of Super 8X TOPflash β-Catenin Luciferase reporter and pRL-TK (Renilla luciferase) plasmids or Super 8X FOPflash β-Catenin Luciferase reporter and pRL-TK plasmids. Transfections were performed using FuGENE® HD Transfection Reagent . After 24 h of transfection, cells were washed twice with PBS, FBS-depleted for 24 h, and then subjected to treatments. After treatments, extracts were prepared using 200 μL of reporter lysis buffer (Promega). Renilla and firefly luciferase activities were assayed using the Dual-Luciferase Reporter Assay System Kit (Promega), according to manufacturer instructions. The Veritas plate luminometer  was used to perform the reading. The firefly luciferase activity in each well was normalized relative to the Renilla luciferase activity.Cells were cultured on glass coverslips in a 12-well plate, and after treatments, rinsed twice with PBS and fixed with 4% paraformaldehyde in PBS/CM  for 10 min at room temperature. The coverslips were washed in PBS/CM, permeabilized with 0.2% BSA + 0.1% Triton X-100 in PBS-CM for 30 min at room temperature and then blocked with 0.2% BSA in PBS for 60 min. Coverslips were once again washed with PBS and then incubated with primary β-catenin antibody diluted 1:500 in 0.2% BSA in PBS, overnight at 4°C. Subsequently, the cells were washed, and the secondary antibody anti-mouse conjugated to Alexa fluor 488 (1:200) was added for 1 h. The coverslips were rinsed and incubated with DAPI (1:1000) for 1 min, then the slides were mounted in Antifade ProLong Gold reagent (Invitrogen). All images were captured using a confocal laser microscope  and analyzed with the ICY bioimage analysis software. To quantify nuclear β-catenin, we examined merged fluorescence images for β-catenin co-localization with DAPI labeling; at least 100 cells were analyzed per condition, and the nuclear β-catenin amount was quantified using ImageJ software.4/well. The next day, the medium was replaced, and the cells were maintained overnight in DMEM with 1% FBS. Then, cells were treated with Wnt3a or IGF1 in DMEM with 1% FBS for 24 h. After this time, cells were washed with PBS (pH 7.0), fixed with ethanol for 10 min, stained with crystal violet solution  for 10 min, washed twice with water and then, the remaining crystal violet was solubilized with methanol for 5 min. Absorbance was measured at 595 nm with a Spectra Max 190 spectrophotometer .Cell proliferation was measured using the crystal violet method. The cells were seeded in a 96-well plate at a final concentration of 2x104) were cultured in T25 flasks, FBS-depleted overnight and treated with Wnt3a and/or IGF1 for 24 h. Cells were then harvested through trypsinization, washed once with PBS, and then kept in 70% ethanol for 30 min at 4°C. The cells were washed with ice-cold PBS and stained with 75 μM propidium iodide (PI) for 10 min in a buffer containing 3.4 mM Tris–HCl (pH 7.6), 10 mM NaCl, 0.2% Triton X-100, and 3500 U/L RNase. DNA content was examined by collecting 10000 events for cell cycle analyses by using a FACSCalibur flow cytometer  and Mod Fit LT software.For cell cycle analysis, cells  was added for 1 h and then monolayers were scraped by a sterile pipette tip to perform the wound healing assay. For each well, five sites of a unique regular wound were analyzed under an Axio Observer Z1 microscope , selected and marked. Then, the cells were permitted to migrate into the clearing area for 24 h. Cells were photographed immediately after wounding (0 h) and at the end of the experiment (24 h). The lesion area was manually quantified using ImageJ software.40 which is an open-access resource for cancer genomics datasets. The dataset was queried by genes of interest based on the most common mutations in the Wnt pathway  and in the PI3K pathway . The studies DFCI;41 Genentech;42 TCGA Firehose Legacy;43 MSK;44 CaseCCC;45 and CPTAC-2;46 were included with a total of 705 patients in the study. Cases classified as high microsatellite instability (MSI) were removed, as patients with this phenotype have better survival than those with MSS, which could influence the results.47 Metastatic samples were also excluded. Only driver mutations were considered because of their already described biological effects. The study was adjusted by the Cox model, stratified by wild-type or mutated Wnt, wild-type or mutated PI3K and simultaneous Wnt + PI3k pathway mutations. An overview of clinicopathological data might be observed in The CRC data was acquired from the cbioportal.org website,Quantitative data are expressed as means ± SEM from at least three independent experiments. Statistical analyses were performed, and column graphs were created using GraphPad Prism 9.0 software . The normality test (Shapiro–Wilk test and D’agostin-pearson) was used to determine whether sample data had normal distribution using R Project for Statistical Computing software version 3.6.2 (The R Foundation). For parametric analysis, one-way variance analysis (ANOVA) followed by the Bonferroni posttest was used for comparisons between treatments. For non-parametric analysis, the Kruskal–Wallis test followed by Dunn’s test was used for comparisons between groups. Differences were considered significant when *p < .05; **p < .01; and ***p < .001.47 then Kaplan Meier curves were performed for patients whose vital status and survival time data were available (644 patients) and grouped by the mutational status of the Wnt/β-catenin, PI3K/Akt or both pathways. Log-rank test was used to calculated differences among groups.For the CRC data, the statistical tests were performed using R Statistical Software . For descriptive statistics, the absolute number with proportion was used. The chi-square test was applied for comparisons among the groups. The multivariate logistic regression model was done including the following covariates: onset, stage, and location. For the survival analysis, first, known microsatellite instability (MSI) patients were excluded as they show better prognosis compared to patients with microsatellite stable (MSS) tumors,Click here for additional data file.


MRAP2 is a small simple transmembrane protein arranged in a double antiparallel topology on the plasma membrane. It is expressed in the paraventricular nucleus of the hypothalamus, where it interacts with various G protein-coupled receptors, such as the prokineticin receptors, and regulates energy expenditure and appetite. The aim of this work was to analyze the functional role of the specific arginine residue at position 125 of MRAP2, which affects protein conformation, dimer formation, and PKR2 binding. Results obtained with the MRAP2 mutants R125H and R125C, which are found in human patients with extreme obesity, and mouse MRAP2, in which arginine 125 is normally replaced by histidine, were compared with those obtained with human MRAP2. Understanding the mechanism by which MRAP2 regulates G protein-coupled receptors helps in elucidating the metabolic pathways involved in metabolic dysfunction and in developing new drugs as specific targets of the MRAP2-PKR2 complex. It isCentrally, in neurons of the hypothalamic arcuate nucleus, binding of PK2 to PKR1 induces activation of STAT3 and release of melanocyte-stimulating hormone (MSH), leading to anorectic effects and modulation of thermoregulation and energy homeostasis. In contrast, PK2β, an alternative splicing short isoform of PK2 composed of only 47 amino acids, shows limited ability to induce STAT3 phosphorylation and leads to increased food intake ,7,8. In Mutations of arginine 125 in histidine (R125H) or cysteine (R125C) have been detected in the conserved C-terminal region of MRAP2 in humans, where they are correlated with an extreme obesity phenotype ,12,13. Mαi signaling in CHO cell lines expressing PKR2 in the presence of the MRAP2 mutants. The C-terminal soluble domain of the MRAP2 mutant proteins was expressed in Escherichia coli and biochemically characterized. The aim of this work was to analyze the functional role of the specific arginine residue in position 125 of MRAP2 in affecting the protein structure, dimer formation, and conformational changes by comparing the mutants with human and mouse MRAP2.We produced two MRAP2 mutants in which the Arg125 residue was replaced by histidine or cysteine, then analyzed their interactions with PKR2 using yeast as a heterologous expression system. In addition, we examined PK2-stimulated GThe C-terminal MRAP2 domain is involved in PKR2 localization and activation. Recently, we identified a region from asparagine at position 77 to alanine at position 131 involved in specific interaction with the extracellular N-terminal sequence of PKR2 . The argIn CHO-PKR2 cells, we examined the ability of mouse MRAP2, R125H, and R125C mutants to affect the level of glycosylation and signaling of PKR2. The results showed that glycosylation was impaired in the presence of mouse MRAP2 and the R125H and R125C mutants B,C. In Cαi inhibitor pertussis toxin [In CHO cells stably transfected with PKR2, PK2 is able to activate ERK and this activation is abrogated by pretreatment with the Gis toxin . Moreoveis toxin . We therBoth the R125H mutant and mouse MRAP2 further reduced the ERK and STAT3 phosphorylation induced by PK2. In the presence of the MRAP2 mutant R125C, the level of ERK phosphorylation was comparable to that observed in the presence of MRAP2 wt .S. cerevisiae Cy12946. Total membrane proteins were purified by Ni-NTA chromatography and subjected to Western blotting analysis. As shown in E. coli cells expressing GST fusion of the N-terminal region of PKR2 were loaded on glutathione–Sepharose columns and then incubated with mouse MRAP2 and the human MRAP2 mutants R125H and R125C. As shown in We expressed mouse MRAP2 and human R125H and R125C MRAP2 mutants with PKR2 terminal tagged His (PKR2-His) in The C-terminal region of MRAP2 encoded by the fourth exon (MRAP2-CT) retains the ability to dimerize, and the protein migrates as a dimer even under denaturing conditions, as described previously . We perfThese results were confirmed by blue native PAGE. Based on the molecular weights of the bands, we were able to detect the presence of structures that are most likely dimers and oligomers, although the prediction of molecular weights in blue native PAGE is approximate B.To analyze the conformational change induced by the substitution of arginine 125 with histidine, we compared proteolysis analysis of R/H-CT with human MRAP2-CT wt. It was clear that the effects on proteolysis were significantly different C. MRAP2 is a glycosylated protein with a single transmembrane domain with no predicted signal peptide. MRAP2 is specifically expressed in the brain, where it regulates the maturation, transport, and signaling of GPCRs involved in energy balance and food intake. Mrap2-deficient mice develop severe obesity, and many MRAP2 variants have been found in severely obese human patients ,12,14. CIn this paper, we examined the mouse MRAP2 protein, demonstrating that it has the same characteristics highlighted for the R125H mutant.The mouse MRAP2 homolog, which naturally has this substitution, is able to inhibit human PKR2 receptor signaling more effectively than the human isoform . Mouse MThe procedure for obtaining pYESC3-PKR2 has been described previously . For conS. cerevisiae, the p413 mouse-MRAP2 plasmid was constructed. The cDNA of mouse MRAP2 was amplified by PCR using the plasmid OMu00763 as a template, with the oligonucleotides MRAP2 BamHI up and MRAP2 EcoRI dw and cloned into p413 digested with BamHI and EcoRI.For the expression of mouse MRAP2 in For constitutive expression of MYC-DDk-tagged human MRAP2 in mammalian cell lines, RC203668 plasmid was purchased from Origene .The p413 humanMRAP2 plasmid previously described in  was usedE. coli, the DNA fragment coding for the C-terminal domain was amplified by PCR with gene-specific primers T70 BamHI and MRAP2 EcoRI dw, using as templates R125H-pcDNA, R125C-pcDNA, and OMu00763, respectively, and cloned into pBluescript to obtain R125H-CT pBS and R125C-CTpBS and mMRAP2-CTpBS. The resulting plasmids were digested with BamHI and EcoRI, and the obtained fragments were cloned into pET28a and PGEX-2T. All oligonucleotides used for the plasmid constructions are listed in For expression of the carboxy-terminal domain of MRAP2 in 5 per well in six-well plates and grown in Dulbecco’s Modified Eagle Medium/Nutrient mixture F-12 Ham (DMEM/F12) containing 100 U/mL penicillin/streptomycin, 10% fetal bovine serum (FBS), 2 mM L-glutamine, and G418 200 ng/mL  at 37 °C and 5% CO2. When the cells reached 70–80% confluence, transient transfection with plasmid DNA of mouse MRAP2, human MRAP2, and the R125H MRAP2 and R125C MRAP2 mutants was performed using Lipofectamine 2000  according to the manufacturer’s instructions. Briefly, cells were incubated with the lipofectamine–plasmid DNA complexes for 24 h at 37 °C and 5% CO2. Lipofectamine–plasmid DNA complexes were obtained by incubating 2 µg of plasmid DNA of mouse MRAP2, human MRAP2, and R125H the MRAP2 and R125C MRAP2 mutants with Lipofectamine 2000 diluted in Opti-MEM (Invitrogen) for 20 min at room temperature. After transfection, cells were deprived of serum and stimulated with PK2 100 nM for 10 min and 1 h at 37 °C and 5% CO2. At the end of the incubation period, cells were lysed in protein extraction buffer. Proteins were then quantified by the Bradford method and used for Western blot analysis.CHO (Chinese Hamster Ovary) cells, which stably express human PKR2, were plated at a density of 4 × 10Western blot was performed as reported in Fullone et al. 2022 . The folS. cerevisiae strain used for co-expression of PKR2, mouse MRAP2, and the R125H-MRAP2 and R125C-MRAP2 mutants was performed according to the procedure described in Fullone et al. 2022 [Transformation of the Cy12946 Solubilized membrane fractions co-expressing PKR2, mouse MRAP2, and the R125H-MRAP2 and R125C-MRAP2 mutants were co-precipitated by metal chelation chromatography using Ni-NTA His-bind resin after batch purification under native conditions according to the manufacturer’s instructions . The eluates were fractionated by SDS-PAGE on a 12% gel and transferred to a polyvinylidene difluoride membrane. The membrane was coated with a monoclonal anti-His antibody conjugated to horseradish peroxidase (1:5000) and with a polyclonal anti-MRAP2 antibody.E. coli, respectively. Beads were washed as described above and bound proteins were eluted with GSH according to the procedure of GE Healthcare and analyzed by Western blotting with anti-MRAP2 antibodies or anti-HIS antibodies as described in Section 4.3The R2-GST proteins were obtained by fusion of the 69 amino acids of the amino terminus of PKR2 with GST . mMRAP2-Blue native-PAGE of purified MRAP2-CT, R/H-CT, R/C-CT was performed on NativePAGE Novex 3% to 16% Bis-Tris gels  as described in Fullone et 2022  using thv/v). At specific time intervals, the proteolytic reaction was stopped by the addition of SDS and samples were boiled. Samples were then analyzed by SDS/PAGE.Protein samples (5 µg) were incubated with trypsin from bovine pancreas  at 20 °C in 50 mM NaHepes, pH 8.0, containing 300 mM NaCl and glycerol 5% (p < 0.05.Data were analyzed with GraphPad Prism 6 for Windows using One-Way ANOVA followed by Tukey’s post hoc test. Results are expressed as mean ± SEM and differences were considered significant at


This research aims to examine the effect of different fermentation times on the results of scanning electron microscope energy dispersive X-ray (SEM-EDX), chemical, organoleptic, and microbiological quality of banana peel.The design in this study used a completely randomized design (four treatments and four replications). The treatments in this study were different durations of fermentation of banana peels; T0 = no fermentation; T1 = 6 days; T2 = 12 days; and T3 = 18 days. The research parameters were chemical, organoleptic, microbiological quality, composition, and SEM-EDX images. Analysis of chemical, organoleptic, and microbiological quality data was done using analysis of variance, followed by Duncan’s multiple range testing at the 5% significance level, while the elemental composition and SEM-EDX images were analyzed descriptively.p < 0.05) on moisture content, ash, crude fiber, nitrogen-free extract, and all organoleptic quality variables of banana peels. Different fermentation durations had no effect (p > 0.05) on crude protein, ether extract, metabolic energy, total lactic acid bacteria, total bacteria, Gram positive or negative bacteria, and total fungi on banana peels. The analysis showed that the elemental composition of banana peels is C, Na2O, Cl, K2O, MgO, SO3, SiO2, and P2O5.The results of the study showed that the length of fermentation had a significant effect (Eighteen days of fermentation improved the chemical quality, organoleptic, microbiological, elemental composition, and SEM-EDX image of fermented banana peel. Musa paradisiaca L.) is the largest fruit commodity in Indonesia which has increased in production every year. The increase in banana fruit production is in line with the increase in banana peel waste produced because banana peels account for 30%–40% of the total bananas. Unused banana peels can negatively affect the environment and cause disposal problems because banana peels are produced every day at the market or in household activities [Banana  Shirota, pH 7 buffer, aquadest, K2SO4, H2SO4, H3BO3, chloramphenicol antibiotics, de Man Rogosa Sharpe Agar (MRSA), and potato dextrose agar (PDA). The tools used were Camry EB9003 digital scale (accuracy 0.0001), pH meter Eutech™ (accuracy 0.001), aluminum foil, Whatman filter paper number 41,100-ml measuring cup, oven 110°C, furnace 600°C, Merck Millipore Buncher funnel, Kjeldahl tube, Duren desiccator, Pyrex Soxhlet, Iwaki distillate tube, cover paper, Rofa wire, Bunsen burner, Portable Pressure Steam Sterilizer autoclave, Funke Gerber colony counter, mercury thermometer, and 200-ml Erlenmeyer.The research material is banana peel. The ingredients used were banana peel, salt, molasses, YakultResearch design with a completely randomized design consisted of four treatments and four replications. The treatment given is different for the length of fermentation as follows:T0 = no fermented banana peelT1 = banana peel fermented for 6 daysT2 = banana peel fermented for 12 daysT3 = banana peel fermented for 18 days®, 60 gm of salt, 60 ml of molasses, and 100 ml of distilled water were added and stirred until homogeneous. The mixture is then put into a fermenter and fermented according to the treatment, a modification of the research procedure by Utama and Christiyanto [Various types of fresh banana peels  were used, with the criteria that they are clean from dirt and a maximum of 24 h after the contents are taken. Banana peels are cut into lengths of 1–2 cm, then mashed using Cu. The banana peel was weighed as much as 1,000 gm, and then 60 ml of Yakultstiyanto .The research parameters were chemical quality, including moisture, ash, fat, crude protein, crude fiber, nitrogen-free extract (NFE), and metabolic energy (ME); organoleptic quality, which includes odor, texture, color, contamination, temperature, pH, and total acid; and microbiological quality, which includes total lactic acid bacteria (LAB), total bacteria, Gram positive or negative bacteria, and total fungi, as well as elemental composition and SEM-EDX images of banana peels.Data were collected on chemical quality parameters, namely water content using the oven method at a temperature of 110°C, ash content using the furnace ashing method, crude protein using the Kjeldahl method, ether extract using the Soxhlet method, and gravimetric method of boiling acid and alkali treatment to measure the fiber content of the leather fermented banana according to AOAC . The conOrganoleptic testing was carried out using a questionnaire test on 20 semi-trained panelists. Organoleptic testing was carried out by assessing odor, texture, color, and contamination based on the assessment being a modification of the research procedure of Utama and Christiyanto . Odor asTemperature measurements were carried out using a digital mercury thermometer. The potential of the hydrogen (pH) test was carried out using a pH meter measuring instrument; the test followed the procedures of Utama et al. . The totMicrobiological quality parameters consisted of total LAB, total bacteria, total fungi, and Gram positive or negative bacteria. The total plate count (TPC) method with MRSA media was used to figure out how much LAB and how many bacteria are present .Gram positive and negative staining were done by microscopic identification. The identification results are then combined and calculated using the following formula:The identification of Gram positive and negative bacteria obtained was tabulated, and scoring was carried out with the following conditions.Score 1 = there are 0 Gram positive and negative bacteriaScore 2 = there are 4/3/2/1 Gram positive and 2/3 Gram negative bacteriaScore 3 = there are 4/3/2/1 Gram positive and 1 Gram negative bacteriaScore 4 = there is 1 Gram positive and 0 Gram negative bacteriaScore 5 = there are 2 Gram positive and 0 Gram negative bacteriaScore 6 = there are 3 Gram positive and 0 Gram negative bacteriaScore 7 = there are 4 Gram positive and 0 Gram negative bacteriaAnalysis of total fungi colonies was done using the TPC method with PDA media mixed with chloramphenicol.d. Elemental Composition and SEM-EDX Image of Banana PeelThe composition of the elemental content of the banana peel was analyzed using the energy dispersive X-ray method; then, the banana peel image was observed using a microscope with a magnification of 5,000 times using the Rey and Poluakan scanning electron microscope (SEM) method .The research data on chemical quality, organoleptic quality, and microbiological quality obtained were tested using analysis of variance ANOVA; –3. If anp < 0.05). The lowest water content was in the T1 treatment because the fermenting microorganisms still did not work optimally, which made the banana peel texture not yet runny (hard), thereby reducing the water content of the ingredients at T1. Anggraeni and Yuwono [Different fermentation durations affected the moisture content of banana peels (d Yuwono stated thd Yuwono  stated td Yuwono states thp < 0.05). Banana peel ash content, from highest to lowest, was T1 (29.82%), T3 (28.15%), T2 (27.57%), and T0 (24.85%). The study results were higher than those reported by Koni et al. [The ash content of banana peels was also influenced by different fermentation durations . The crude protein of banana peels fermented ranged from 6.43% to 7.42%. The results of the protein content in the study were higher than that reported by Putra et al. [Banana peel protein content was not affected by different fermentation durations . Fat content in the study ranged from 7.39% to 7.90%. This result is lower than that of Putra et al. [Banana peel fat content was not affected by different lengths of fermentation . The values of the highest to lowest crude fiber were T0 (10.01%), T3 (8.75%), T1 (8.18%), and T2 (8.06%). This result is lower than that of Putra et al. [Banana peel fiber content was affected by different lengths of fermentation . The T1 and T2 treatments decreased the NFE levels of banana peels, which could occur because the fermenting microorganisms had utilized the carbohydrates contained in the banana peels, which are the main component contained in NFE. Vitanti et al. [The content of the banana peel NFE was influenced by different fermentation durations . The value of 5.64 can be interpreted as the fermented banana peel having a slight characteristic smell of fermentation. The appearance of a characteristic odor of fermentation and the absence of such a sour or foul odor in the sample indicates that the fermentation process is going well. Marianti et al.  stated tThe best color organoleptic score for fermented banana peel was in the T3 treatment, with a value of 2.76. The value of 2.76 indicates that the fermented banana peel is black. Marianti et al.  foundthap < 0.05) of banana peel. The fermentation temperature at the beginning of the treatment was higher due to the fast fermentation, resulting in a rapid increase in temperature. The temperature of banana peel fermentation is one of the supporting factors for fermentation because it is related to growth, microorganism development, and enzyme production [Different fermentation durations affected the temperature  of banana peel. The lowest total acid was in treatment T0 (0.42%). The longer duration of fermentation will result in the formation of organic acids, and the impact on the total acid will increase. The total acidity of fermented banana peels correlated with the pH value, the treatment having a higher total acidity resulted in a lower pH value. Adriawan et al. [Different fermentation durations affected the total acid .The LAB used as a starter for fermentation is h et al.  stated th et al. stated thTotal bacteria that were not significantly different could occur because the total LAB in the study was also not significantly different. Increasing or decreasing LAB will affect the total bacteria because LAB can suppress the growth of Gram negative bacteria. LAB can damage the outer membrane surface of Gram positive bacteria. Mukodiningsih et al.  feed proEscherichia coli and Salmonella typhi can grow optimally at pH 7–9 conditions.The average score of Gram positive and negative bacteria on fermented banana peels is 5.75–6.75. The low Gram negative bacteria in banana peels indicates the safety of the material as an alternative feed ingredient for poultry. Kukier et al.  stated t5 CFU/gm (5.48 log CFU/gm). The T3 treatment had the fewest fungi, though this was not significantly different from the other treatments, so it was recommended that the T3 treatment (fermentation time of 18 days) be used as a feed ingredient.The length of fermentation did not significantly reduce the total fungi. Uruilal et al. stated thLactobacillus sp., which is a LAB that can inhibit the growth of Gram negative bacteria because it can produce lactic acid.Bacterial culture results from fermented banana peels  showed tBacteria with diplococcus morphology had fewer numbers than solitary yeasts and were only found in Gram positive bacteria. Yusuf et al.  stated tL. casei. This is because the added starter contains a lot of LcS bacteria. Kusumaningsih et al. [L. casei were temperature and pH values, which must be in very acidic conditions, and experience growth inhibition at pH 4.5–5.The type of Gram positive bacteria that dominates the bacteria that grows on fermented banana peels is thought to be h et al.  stated th et al.  stated th et al.  stated tThe presence of LcS in fermented banana peel products can have a positive impact in the form of health benefits for poultry because it can produce and help absorb essential nutrients such as organic acids and vitamins and can suppress pathogenic bacteria in the intestines. Susilaningrum et al.  stated t2O) in the fermented banana peel. The elements of Na2O, from the highest to the lowest, are T0 (6.72%), T1 (4.13%), T2 (2.97%), and T3 (2.56%). The duration of fermentation reduces the Na2O element in the banana peel; this occurs because the element can be decomposed by microorganisms during the fermentation treatment, and the longer the fermentation, the lower the composition of the Na2O element in the material. Widhiyanuriyawan and Hamidi [2O is a component of hydrated aluminosilicate or zeolite compounds derived from alkali metals and alkaline earth, which when heated, releases moisture.Treatments T0, T1, and T2, had elemental carbon (C) compositions in almost the same range because banana peels contain a cellulose C source . Pamungkd Hamidi  stated t2O and COCl2.6H2O [2O) fermented banana peel, sequentially from highest to lowest, is T2 (6.65%), T1 (5.60%), T0 (1.92%), and T3 (0.96%). The lowest K2O element in the T3 treatment can occur because the element has been utilized by microorganisms during the fermentation process as a substrate component. Nasrun et al. [2O levels increase during the fermentation process caused by the cell division of microorganisms.The elemental composition of chloride (Cl) in fermented banana peels, sequentially from highest to lowest, is T2 (7.81%), T0 (6.11%), T1 (5.91%), and T3 (2.58%). The composition of Cl can decrease during fermentation because it has been used by microorganisms as an essential food element for metabolic processes. For microorganisms to grow and develop, they require macro- and micronutrients from nutrients such as CaCl.2HCl2.6H2O . The elen et al.  stated tThe element magnesium oxide (MgO) of banana peels was not affected by different fermentation durations. The elemental composition of fermented banana peel MgO is between 0.12% and 1.18%. This indicates that the fermenting microorganisms cannot digest the MgO element, so the elemental composition is not affected by the duration of fermentation. Silica dioxide (SiO2) in the study is in succession from highest to lowest, namely T0 (2.49%), T3 (0.56%), T1 (0.41%), and T2 (0.02%). SiO2 in banana peels is beneficial as an additive to prevent clumping in the product. Putra et al.  stated t2O5) in banana peels, from highest to lowest, were T1 (1.93%), T2 (0.45%), T0 (0.35%), and T3 (0.03%). The T3 treatment gave the lowest P2O5 results because it increased the activity of microorganisms. The increase in phosphorus-degrading bacteria will reduce the elemental composition of P2O5. Rasmito et al. [2O5. Meanwhile, the element of sulfite (SO3) in banana peels was in the range of 0.26%–2.37%. Based on the parameters of elemental composition, the recommended treatment is T3, that is, banana peel within 18 days of fermentation, because the content of elements that have been decomposed in the fermentation process is more optimal to be safe when consumed by livestock.The elements of phosphorus pentoxide (Po et al.  stated tThe SEM image at T0 still looks like a rough surface; the constituent fractions are still united and agglomerate with one another . The rouThe T1 and T2 treatments had almost the same structure, but both were smoother than the T0 treatments. This can happen because the fermentation that makes the microorganisms can loosen the bonds that make up the banana peel at T1 and T2 . Lin et The T3 treatment had the loosest surface compared to other treatments, as well as fewer components of the material. This can happen because the T3 treatment is the optimal fermentation duration for microorganisms in simplifying the components that make up banana peels. The looser and simpler structure will affect the cells that make up the banana peel. Wei et al.  stated tIn conclusion, 18 days of fermentation improved the chemical quality, organoleptic, microbiological, elemental composition, and SEM-EDX image of fermented banana peel. Fermented banana peel has the potential as an alternative feed and functional feed because it has good nutritional content and health benefits.


Our results showed that EX-4 attenuated neurological deficits, brain edema, infarct volume, BBB disruption, and rtPA-induced HT in ischemic stroke. EX-4 suppressed the production of ROS and the activation of MMP-9 to protect the integrity of the BBB by activating the Wnt/β-catenin signaling pathway. PRI-724, a selective inhibitor of β-catenin, was able to reverse the therapeutic effect of EX-4 in vivo and in vitro. Therefore, our results indicate that the GLP-1R agonist may be a potential therapeutic agent to decrease the risk of rtPA-induced HT after ischemic stroke via the Wnt/β-catenin signaling pathway.Tissue plasminogen activator (tPA) is recommended by the FDA to dissolve intravascular clots after acute ischemic stroke (AIS). However, it may contribute to hemorrhagic transformation (HT). The Wnt/β-catenin signaling pathway plays an important role in regulating the blood–brain barrier (BBB) formation in the central nervous system. We explored whether glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4) reduces the risk of HT after rtPA treatment via the Wnt/β-catenin pathway by using a rat transient middle cerebral artery occlusion (MCAO) model  The recombinant tissue-type plasminogen activator (rtPA) is the only drug approved by the Food and Drug Administration for the treatment of ischemic stroke , 2. NeveExendin-4 (EX-4) is an agonist of the glucagon-like peptide-1 receptor (GLP-1R) with effects on various physiological functions. Namely, EX-4 inhibits glucagon secretion and apoptosis. As a relatively small molecule, EX-4 diffuses across the BBB to directly access the central nervous system . It has The canonical Wnt/β-catenin pathway plays an important role in the BBB formation via β-catenin stabilization , 13. ThiIn this study, the middle cerebral artery occlusion (MCAO) rat model and the oxygen–glucose deprivation plus reoxygenation (OGD/R) model of cerebral microvascular endothelial cells (BMECs) were used to study the role of the Wnt/β-catenin pathway in tPA-induced HT by using EX-4. Meanwhile, the Wnt/β-catenin pathway was inhibited by PRI-724, a selective inhibitor of β-catenin, to further study the mechanism of EX-4. We hypothesized that the administration of EX-4 as a therapeutic drug would reduce the BBB permeability and the risk of HT and improve neurofunctional prognosis through the Wnt/β-catenin signaling pathway.Adult male Sprague–Dawley (SD) rats weighing 250–280 g were included in this study. The rats were raised in the Experimental Animal Center of Tongji Hospital and kept at 22 °C under standard conditions of 50–60% relative humidity and 12:12-h light/dark cycle. Standard food and water were supplied ad libitum. All animal experiments were approved by the Experimental Animal Ethical Committee of Tongji Hospital affiliated with the Huazhong University of Science and Technology.Focal cerebral ischemia was performed by MCAO according to a previously described protocol , 18. In Rats were randomly divided into five groups: sham group rats were treated with the same surgical operation without filament insertion, and they were administered 1 mL 1% DMSO intraperitoneally with 1 mL saline intravenously; vehicle group rats were treated with MCAO and administered 1 mL 1% DMSO intraperitoneally with 1 mL saline intravenously; rtPA group rats were treated with MCAO and administered 1 mL rtPA  inserted into the left femoral vein 4 h after MCAO; rtPA + EX-4 group rats were treated with MCAO and administered EX-4  intravenously immediately after rtPA injection; and rtPA + EX-4 + PRI-724 group rats were treated with MCAO and administered EX-4 intravenously and PRI-724 , a selective inhibitor of the β-catenin, intraperitoneally after rtPA injection.An investigator blinded to the experimental groups tested the neurological function in the animals 24 h after MCAO using a modified scoring system as described previously . Each anBrain edema was assessed by measuring brain water content with the wet–dry method as previously reported , 20. TheThe infarct volume was evaluated by the 2,3,5-triphe-nyltetrazolium chloride  staining technique 24 h after MCAO, as previously reported , 22. TheEvans blue  dye was administered through the femoral vein 2 h before cardiac perfusion , 23. AftThe level of neutrophil infiltration in the brain tissue was measured using the measurement of myeloperoxidase (MPO) assay kit  according to the manufacturer’s protocol . The absThe concentration of hemoglobin in the ipsilateral brain tissue was assessed with a QuantiChrom hemoglobin assay kit  as reported previously . The ratThe coronal brain tissues of rats in five groups were stained with hematoxylin–eosin (HE)  as reported previously , 26. BriStreptomycetes working solution was added and incubated for 20 min. Finally, the slices were incubated with DAB solution for 10 min and sealed. Images were acquired using a confocal microscope.Based on our established protocol, the slices were incubated for 30 min at room temperature with 0.25% Triton X-100 in 1% BSA and for another 1 h in 5% BSA. Next, the slices were incubated with primary antibodies  overnight at 4 °C, followed by incubation with biotin-labeled anti-rabbit IgG  for 1 h at room temperature. Then, an HRP-conjugated 2/95% air. One day later, the culture medium was replaced by EC culture medium. After 10–12 days in culture, the cells were collected for further detections.Primary BMECs were isolated from 3–4-week-old rats based on a previous description . Briefly2/95% air atmosphere for 4 h. Then, the medium was replaced by a normal medium containing high-glucose DMEM, 20% FBS, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. BMECs were washed with PBS and incubated with glucose-free DMEM as an OGD medium. Then, the cells were transferred to an anaerobic chamber filled with a gas mixture of 1% O2, 95% N2, and 5% CO2 at 37 °C for 4 h as the OGD treatment (OGD group). After the OGD treatment, the cells were cultured in a normal medium for another 20 h for reoxygenation with or without the corresponding treatment containing 300 µg/ml rtPA (OGD + rtPA group) and/or 50 nM EX-4 (OGD + rtPA + EX-4 group) and/or 4 μM PRI-724 (OGD + rtPA + EX-4 + PRI-724 group) at 37 °C in a humidified atmosphere of 5% CO2/95% air.The sham group was incubated with high-glucose DMEM (HyClone) with 20% FBS at 37 °C in a 5% COThe cells were seeded in 96-well plates . After 20 h of treatments, the cell viability was determined using the CCK-8 assay kit  in line with the manufacturer’s instructions.After the treatment, the sedimented cells were resuspended with PBS and incubated with DCFH-DA  for 30 min at 37 °C to measure intracellular ROS generation. The image was captured by fluorescent microscopy immediately. The mean fluorescence intensity was evaluated by flow cytometry. The results were analyzed by using FlowJo 10 software.Apoptotic BMECs were labeled with terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end labeling (TUNEL) according to the manufacturer’s instructions . The number of positive BMECs was observed by fluorescence microscopy and counted in 8 microscopic fields under 200 × magnification.The brains were cut into 25-μm-thick sections for immunostaining analysis. Based on our established protocol , the sliEight percent to 12% SDS-PAGE gels was electrophoresed with some amounts of protein per lane (20 μg) based on our established protocol , 29. Prop < 0.05.All the data were analyzed by using the GraphPad Prism version 5.04 statistical package and were expressed as mean ± standard deviation (SD). Differences among the groups were determined by one-way analysis of variance (ANOVA). The differences were considered statistically significant at p < 0.01; Fig. p < 0.05). These rats treated with rtPA + EX-4 + PRI-724 performed worse than the MCAO rats treated with rtPA + EX-4 (p < 0.05). Cerebral edema measured by wet-to-dry weight was significantly higher in the vehicle group than in the sham group . The increase in rtPA-induced edema was inhibited by EX-4 administration (p < 0.05), and the MCAO rats treated with rtPA + EX-4 + PRI-724 had more pronounced cerebral edema compared with the rtPA + EX-4 group (p < 0.05) at 24 h after MCAO. The MCAO rats treated with rtPA showed the enlarged infarct compared with the vehicle group . The MCAO rats treated with rtPA + EX-4 + PRI-724 had a larger infarct volume than the rtPA + EX-4 group (p < 0.01) at 24 h after MCAO.The diagram of MCAO anatomy and the schematic diagram of the research design and experimental groups are displayed in Fig. p < 0.05; Fig. p < 0.01; Fig. p < 0.05; Fig. All the coronal sections were observed; we found that the hemorrhage was not significant in the vehicle group and rtPA + EX-4 group. In contrast, there was increased hemorrhage in the rtPA group and rtPA + EX-4 + PRI-724 group 24 h after MCAO Fig. . The hemp < 0.01; Fig. p < 0.01), whereas the rtPA + EX-4 + PRI-724 group reversed this protective effect at 24 h after MCAO (p < 0.01).In this study, HE staining was used to assess the histological changes in the cerebral cortex. The normal brain structure showed integral histological and cellular structure. While the cerebral structure in the vehicle group revealed edema and few inflammatory cells, the rtPA group showed increased edema, inflammatory cells, and hemorrhage Fig. . The trep < 0.01; Fig. Western blotting showed that the band density of ZO-1, Occludin, and Claudin-5 was reduced in the vehicle group at 24 h after MCAO. The treatment with rtPA aggravated the degradation of TJPs compared with the vehicle group (p < 0.05) and GSK-3β (p < 0.01), whereas PRI-724 reversed this effect 24 h after MCAO, suggesting that the activation of the Wnt/β-catenin signaling pathway may reduce rtPA-induced HT 24 h after MCAO in rats.Immunohistochemistry was used to assess the expression level of β-catenin in the brain tissue Fig. . The expTo further analyze the mechanism of rtPA-induced BBB damage after MCAO, we detected the expression levels of MMP-9 by immunofluorescence in the ipsilateral brain tissue Fig. . We founp < 0.01). EX-4 improved the viability of BMECs compared with the rtPA group (p < 0.01) and reduced the rtPA-induced apoptosis of BMECs (p < 0.01). PRI-724 aggravated the BMECs damage and cell apoptosis 24 h after OGD/R.BMECs are important components of the BBB, and they exert a crucial effect on the occurrence of HT. To further analyze the degree of rtPA-induced BMECs damage after OGD/R, primary BMECs were isolated from SD rats and the cell purity was greater than 95%, which detected by immunofluorescence FVIII labeling of endothelial cells Fig. . We alsop < 0.01). EX-4 significantly inhibited the rtPA-induced damage of ZO-1, Occludin, and Claudin-5, whereas PRI-724 reversed this effect at 24 h after OGD/R. These results showed that activating the Wnt/β-catenin signaling pathway may decrease rtPA-induced BMECs damage and tight-junction protein damage.Furthermore, we explored the expression of tight-junction proteins in BMECs 24 h after OGD/R. Western blotting showed that the band density of ZO-1, Occludin, and Claudin-5 was reduced in the vehicle group at 24 h after OGD/R Fig. . rtPA dep < 0.01; Fig. Immunofluorescence was used to detect the expression of β-catenin in BMECs Fig. . The expp < 0.05), which could be reversed by PRI-724 (p < 0.01). These findings suggested that the Wnt/β-catenin signaling pathway may affect rtPA-induced BMECs damage by regulating MMP-9 expression at 24 h after OGD/R.To further analyze the mechanism of rtPA-induced BMECs damage at 24 h after OGD/R, we detected the expression levels of MMP-9 and Claudin-5 by immunofluorescence in BMECs in vitro Fig. . We founp < 0.01; Fig. Since oxidative factors play a crucial role in cerebral ischemia , we furtRecently, many different drugs acting on various mechanisms have been developed to intervene at various steps of ischemia–reperfusion injury or BBB damage, so as to decrease the incidence of HT after AIS. In this study, the risk of HT after rtPA thrombolysis and the effect of EX-4 on a rat MCAO model and on BMECs in vitro were observed. EX-4 played a protective role in reducing neurological deficits, brain edema, infarct volume, neutrophil infiltration, BBB damage, and hemorrhage in the MCAO model treated with rtPA. EX-4 protected the viability of BMECs from rtPA treatment in OGD/R model in vitro. In addition, EX-4 reduced degradation of tight-junction proteins by inhibiting MMP-9 activation and ROS production via activating Wnt/β-catenin signaling pathway. However, PRI-724, a selective inhibitor of β-catenin , was ablGLP-1 is a hormone that promotes insulin release in hyperglycemic conditions . EX-4 mePrevious studies showed that the activation of the Wnt/β-catenin pathway may protect the BBB and reduce the risk of HT in AIS –38. HowePrevious studies have shown possible mechanisms of HT with various treatment targets including MMP inhibition, ROS reduction, BMEC protection, and inflammation regulation –52. The There are some limitations in our article. First, we used corresponding drugs to assess the effects of GLP-1R without knocking out or overexpressing the GLP-1R gene in vivo and in vitro. Second, the component of the BBB is complex. We studied the effects of EX-4 mainly on microvascular endothelial cells and intercellular junction proteins. Whether there are corresponding effects on astrocytes or pericyte deserves further study. Finally, the mechanism of the BBB disruption is very comprehensive in HT; microglial activation and inflammation also require further research.Our study showed the effect of GLP-1R agonists on BBB in HT. Treatment with Ex-4 immediately is effective to decrease the occurrence of HT for patients receiving rtPA thrombolytic therapy after AIS. Clinical trials of rtPA in combination with GLP-1R agonists are needed to reduce the adverse effects of rtPA and protect the prognosis of patients.Our study indicated that the GLP-1 agonist protected the brain against HT and alleviated the rtPA-induced BBB disruption in ischemic stroke through the Wnt/β-catenin signaling pathway. Our results showed that the activation of the Wnt/β-catenin signaling pathway provided a protective effect, which was reflected in reduced ROS levels and leukocyte infiltration, thereby inhibiting the rtPA-induced MMP-9 activity and tight-junction protein degradation in the rat MCAO model and in the BMEC OGD/R model in vitro. These findings have significant clinical implications and could potentially be developed into novel therapies for patients receiving thrombolytic therapy after AIS. Our results suggested that the GLP-1 agonist may be a potential strategy to reduce ischemia–reperfusion injury and rtPA-induced HT via the Wnt/β-catenin signaling pathway.


Upper gastrointestinal (UGI) symptoms are common in East Africa but there is limited diagnostic endoscopy availability. Surgical camps are a recognised method of providing intensive service provision and training. We describe a novel application of the camp model for diagnostic UGI endoscopy in eastern Uganda.H.pylori testing.A 7-day camp took place in an existing endoscopy department of Mbale Regional Referral Hospital. Patients with symptoms warranting investigation were invited for free diagnostic UGI endoscopy, biopsy and H.Pylori. A resident surgeon was trained and performed 55 supervised unassisted procedures.148 patients underwent endoscopy. 47 were deemed to have significant pathology, 7 with malignancy. 61% had Our pilot has illustrated that camps are a safe and efficient way of providing intense endoscopy service and training in an established department. Camps can be utilised for scaling up much needed endoscopy services and training in low- and middle-income countries. Upper gastrointestinal (UGI) symptoms warranting endoscopic evaluation are common in UgandaSurgical camps are a recognised method of providing intense surgical service and training. Camps typically involve volunteer clinicians and support staff performing a large number of free procedures in a short time period on a local patient population, normally at a location with limited existing surgical provision. Surgical service delivery in this manner has previously been found to be safe and effective in Ugandahttps://pont-mbale.org.uk). Services have run uninterrupted for 9 years, but with a low referral frequency - performing approximately 8 procedures per month. A camp providing free diagnostic UGI endoscopy was designed to boost capacity, increase local service awareness and train an additional endoscopist. We describe this camp - the first of its kind in the literature.Mbale Regional Referral Hospital (MRRH) is a government institution in eastern Uganda. In 2009, a diagnostic UGI endoscopy service was established through partnership between the hospital and Wales based organisation PONT The resident surgeon, clinical officer and three additional nurses were all new to the department - a full induction and orientation was provided prior to the camp start date for all staff who were new to the department.The resident surgeon underwent training in diagnostic UGI endoscopy during the camp. The trainee was enrolled in a residency programme as a member of the College of Surgeons of East, Central and Southern Africa . The resident was provided with appropraite learning materials several weeks in advance of the camp start dateHelicobacter pylori (H.pylori) rapidtest with gastric antral biopsies. Where necessary tissue biopsies were taken for histological analysis and patients invited back to a follow up appointment to receive results.Two donated Olympus endoscopy stack systems were used interchangeably. Patients were formally consented and had their findings explained in their first language where possible and were provided with health advice and/or prescription as per national management guidelines. All patients underwent Data was collected prospectively using handwritten admission forms and procedure reports, then entered into an electronic MS-EXCEL database. Variables recorded included, but were not limited to; age, gender, indication, findings and H.pylori status.Prior to the camp a quality assessment of the endoscopy department was conducted according to the pilot JAG Global Rating Scale. A resident surgeon was trained in UGI endoscopy during the camp, utilising JAG international Direct Observation of Procedural Skills forms as a framework for training and feedbackOne-hundrend and fourty eight (148) patients underwent diagnostic UGI endoscopy . Mean age was 50 years.In Fourty-seven (47) patients were classed as having a significant diagnosis  - 7 of whom had a histologically proven malignancy or dysplasia  and were offered onward referral to specialist institutions in Kampala. Two cases of histologically proven Barrett's metaplasia of the oesophagus were identified.H.pylori were positive. For the remaining 8 patients, either testing was deemed unsafe, or the antrum could not be accessed.61% (86/140) of those tested for Morbidity and Mortality: There were no immediate complications of endoscopy. Two patients reattended the unit the day after their endoscopy; one suffered pleuritic chest pain which settled spontaneously and one complaining of worsening dysphagia (which was present pre-procedure) from an oesophageal malignancy who had been referred on to the Uganda Cancer Institute at the time of diagnosis. Four patients died within 30-days post-procedure, all of whom were found to have oesophageal or gastric malignancy. While none were able to afford formal diagnostic staging, their deaths were attributed to their underlying illness by clinicians in all cases.Cost: The camp cost was 6,746,100 Ugandan Shillings ($1839) all of which was provided by international donations to the PONT charity. This donation covered the cost of all endoscopy procedures and histopathology processing for the duration of the camp. The only out-of-pocket expenses for the patient were for personal transport and any prescribed treatment.Training: A local surgeon was trained over 50 hours of the camp  through short hands-on-competence building sessions. Basic UGI endoscopy competences were attained and the surgeon performed 55 successful procedures unassisted. The number of unassisted procedures successfully performed was found to increase each day of training with 5/14 (26%) on the first day and 7/8 (88%) on the final half day. An independent assessment of competence by clinicians at a separate established Ugandan endoscopy department has been arranged prior to the trainee starting independent practice.The surgical camp model was safely and successfully replicated for diagnostic UGI endoscopy. Mobilisation proved effective with direct referral from a clinician or community health worker as the most successful methods.The one-off method of training we describe here has both advantages and disadvantages. The resident surgeon did not formally achieve competence in diagnostic UGI endoscopy over the course of the camp, partly as they were unable to attain the evidence-based threshold of 200 procedures, as described by JAG and other international training programmesOur patient cohort represents a small geographical and chronological snapshot with vulnerability to selection bias. Patients were not reimbursed for transport costs and thus were perhaps less likely to be co-morbid and or to have come from remote surrounding districts. Attendance of younger, better-connected patients is likely given the simple, short method of camp advertising.H.pyloriprevalence was found to be similar to previous African estimatesWith these limitations in mind, we can draw tentative conclusions on the burden of UGI pathology in eastern Uganda. There is a high incidence of squamous cell carcinoma of the oesophagus, in line with previous studiesIntense endoscopy training programmes have previously been found to be successfulScale up of diagnostic UGI endoscopy services in Uganda is urgently required in order to formally diagnose a range of important conditions that are currently treated empirically including peptic ulceration and oesophageal malignancy. We have described the first successful diagnostic UGI endoscopy camp of its kind.While we recognise that camps are not a replacement of established healthcare services, we do feel that camps can be a safe and effective method of providing intensive service provision and training. Further camps of this kind should be considered to help scale up endoscopy services in low- and middle-income countries like Uganda.


Barnesiella and favored Alloprevotella. Body weight, fattening, food intake and feeding behavior as well as plasma lipid and uric acid were unaffected by glyphosate-based herbicides. Taken together, our results show possible disturbances of the cecal microbiota associated with plasma oxidative stress and accumulation of glyphosate in metabolic tissues in response to dietary glyphosate-based herbicides exposure in broiler hens. Luckily, glyphosate-based herbicides at this concentration does not hamper growth and most of the effects on the phenotypes are reversible.Glyphosate-based herbicides (GBHs) are massively used in agriculture. However, few studies have investigated the effects of glyphosate-based herbicides on avian species although they are largely exposed via their food. Here, we investigated the potential reversibility of the effects of chronic dietary exposure to glyphosate-based herbicides in broiler hens. For 42 days, we exposed 32-week-old hens to glyphosate-based herbicides via their food  corresponding to half glyphosate’s no-observed-adverse-effect-level in birds. We compared their performance to that of 75 control animals (CT). Both groups  were then fed for 28 additional days without glyphosate-based herbicides exposure . Glyphosate-based herbicides temporarily increased the plasma glyphosate and AMPA (aminomethylphosphonic acid) concentrations. Glyphosate and aminomethylphosphonic acid mostly accumulated in the liver and to a lesser extent in the leg muscle and abdominal adipose tissue. Glyphosate-based herbicides also temporarily increased the gizzard weight and plasma oxidative stress monitored by TBARS (thiobarbituric acid reactive substances). Glyphosate-based herbicides temporarily decreased the cecal concentrations of propionate, isobutyrate and propionate but acetate and valerate were durably reduced. The cecal microbiome was also durably affected since glyphosate-based herbicides inhibited  It is a broad-spectrum herbicide with a generalized effect on all types of crops. Since it is a non-selective product, its commercialization is often coupled with that of genetically modified crops designed to resist the action of the herbicide, enabling farmers to spread increasingly large amounts on their fields without destroying their own crops , while weductase . Gly’s heductase , act as eductase , and indeductase . The useeductase .Beside their controversial hypothetic effects on human health, GBHs are suspected to have ecotoxicological effects and to be part of the numerous factors leading to the current biodiversity crash. The fate of bird populations is of particular concern. European wild birds are going through a massive decline  and pestThus, the aim of our study was to investigate the effects of a GBH-enriched diet on adult hens’ metabolism and on different biomolecular stress markers, using a dose equivalent to 47 mg Gly/kg body weight/day, half EFSA’s NOAEL, for 6 weeks. We also assessed the reversibility of the potential effects detected, by following the animals for 4 weeks after withdrawal of GBH from their diet. Meanwhile, Gly and AMPA were assayed in plasma and metabolic tissues . Detoxication processes were assayed by measuring mRNA expression of the enzymes cytochrome P450 (CYP) and GST (glutathione S-transferase) in animals’ liver, which is the main tissue of biotransformation. We also studied the impact of this diet on gut microbiome composition and diversity. To our knowledge, it is the first study evaluating the potential reversibility of changes in metabolism-related parameters after chronic dietary exposure to a GBH in broiler hens.All experimental procedures were performed in accordance with the French National Guidelines for the care and use of animals for research purposes .2. The design of the experiment is summarized in All animals  were obtained at 1 day of age from a local hatchery  and reared at “Pôle Expérimental Avicole de Tours”  according to traditional breeding conditions. In our experiment, all 150 hens (32 weeks old) were used. They were divided into groups of five birds in 30 pens, each pen with an area of 3 mHens (32 weeks old) received a restricted laying diet according to Hendrix Genetics recommendation. The composition of the diet is shown in Feeding behavior was quantified by counting pecks at the diet over the daily diet distribution. This count was carried out in three hens per pen during the 8 secondes on D31, D32, D35, D36 (during GBH exposure) and D49, D50, D51 and D52 (after exposure).g for 10 min at 4°C) and stored at −20°C before use for Gly and AMPA assays . Tissue samples were obtained at different points in the experiment [day 42 of the protocol during GBH exposure (n = 9 GBH and n = 9 CT) and day 70 after GBH exposure (n = 12 Ex-GBH and n = 12 Ex-CT)] by dissection after slaughtering.Blood samples from 10 hens were collected from the occipital sinus into heparin tubes on different days during the experiment . Blood samples were centrifuged , in collaboration with Dr S El Balkhi  as previously described .Plasma concentrations of triglycerides, uric acid, phospholipids and cholesterol were determined by enzymatic assay using specific kits from Biolabo SAS : triglycerides (reference: LP80519), uric acid , phospholipids  and cholesterol . The measurements were performed according to the manufacturer’s protocol. For all these assays, the inter- and intra-assay coefficient variations were <15%.Chickens were individually weighed on days 0, 21, 42 and 70. Body weight was recorded and, based on the differences, the body weight gain per day was calculated [/number of days)] for each period D0–D21, D21–D42 and D42–D70 . The weig for 10 min at 4°C, then transferred to 1.5 ml microcentrifuge tubes and stored at −80°C. The TBARS values of the EDTA-treated plasma were measured using the modified method of Lipid peroxidation, as determined through measuring the amount of MDA  that reacts with 2-thiobarbituric acid, was used to estimate oxidative stress . Blood s2PO4, 0.5% Triton) and an Ultra-Turrax instrument for grinding, according to the manufacturer’s recommendations . Lysates were centrifuged for 20 min at 16,000 × g and 4°C, and the supernatants collected. Lysate protein concentrations were then measured using the bicinchoninic acid (BCA) protein assay . The ATP assay was performed using a Promega CellTiter® Luminescent Cell Viability Assay. Briefly, the assay buffer and the substrate were equilibrated to room temperature, then the buffer was transferred to the substrate and gently mixed with it to obtain a homogeneous solution. After a 30 min equilibration of the cell plate to room temperature, protein lysates (100 µL) were put into a 96-well plate and CellTiter-Glo reagent (100 µL) was added to each well. The plate was orbitally mixed for 2 min and incubated at room temperature for 10 min. The ATP concentration was then measured using a luminometer. Luminescence at the integration time 1,000 (ms) was read using an Ascent Luminoskan Luminometer . Lysates’ ATP concentration was normalized with the previously determined total protein concentration.Liver total proteins from 9 CT, 9 GBH, 12 Ex-CT and Ex-12 GBH hens were extracted using lysis buffer (1 M Tris (pH 7.4), 0.15 M NaCl, 1.3 mM EDTA, 1 mM EGTA, 43–23 mM VO, 0.1 M NaF, 1% NHGAPDH , ACTB (actin B) and EEF1α ). For each target gene, expression was calculated according to primer efficiency (E) and quantification cycle (Cq), where expression = E − Cq. Then, relative expression of the target gene to the three reference genes was analyzed.Total RNA from 9 CT, 9 GBH, 12 Ex-CT and 12 Ex-GBH hens was extracted from hens’ liver, gizzard, proventriculus, cecum and spleen using TRIzol RNA Isolation Reagents and an Ultra-Turrax instrument for grinding, according to the manufacturer’s recommendations . The purity and concentrations of the obtained RNA were checked via their A260/A280 ratios using a Nanodrop machine. cDNA was obtained by reverse transcription of 2 µg of the total RNA in 20 µL of a mix containing each deoxyribonucleotide triphosphate , 2 M RT Buffer, 15 μg/μL oligodT, 0.125 U of ribonuclease inhibitor and 0.05 U of Moloney murine leukemia virus reverse transcriptase (MMLV); the mixture was kept for 1 h at 37°C. Quantitative PCR was performed using a mix of 3 µL of cDNA and 8 µL of SYBR Green Supermix 1X Reagent  with 250 nM of specific primers  given in 1H NMR spectra for the metabolic fingerprinting of plasma samples were obtained at 300 K on a Bruker Avance III HD 600 MHz NMR spectrometer  operating at 600.13 MHz for 1H resonance frequency using an inverse detection 5 mm 1H-13C-15N-31P cryoprobe attached to a cryoplatform (the pre-amplifier cooling unit). Plasma samples were prepared, and the analyses performed as previously described  flow rate was 10 ml/min. The oven temperature was initially set at 100°C for 10 min, then increased from 100 to 180°C at a rate of 20 C/min and held for 2 min. The detector temperature was 240°C. Samples were analyzed in duplicate. The peaks obtained were integrated using OpenLAB Chemstation software .Approximately 35 mg of cecal content was weighed, snap-frozen and stored at −80°C until analysis. The samples were extracted with water and proteins precipitated with phosphotungstic acid. 2-Ethylbutyrate was added to supernatants at a ratio of 1: 4 as an internal standard. The SCFA content was determined from a 0.3 µL volume of supernatant by gas chromatography  equipped with a split-splitless injector, a flame-ionization detector and a fused silica capillary column . The carrier gas , GBH (n = 9), Ex-GBH (n = 10), Ex-CT (n = 5)] using a G’NOME DNA isolation kit  . The V3–® software (version 6) was used for all analyses . All data are reported as means ± standard error of mean (SEM). Outliers were identified by the Rout method and removed. We performed t-tests to compare means at D42 and D70, or two-way ANOVA followed by Tukey’s HSD tests. Regarding gut microbiome analysis, all statistical analyses were performed using R software . R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.). The microbiota composition analysis was performed using the mia package . For the variability within bacterial communities , Chao, Shannon and Faith indices were computed. The effect of exposure to GBH on $\alpha$ diversity was investigated using Kruskal–Wallis tests followed by Dunn’s post-hoc tests and was considered significant if p < 0.05. For the differences between bacterial communities ($\beta$ diversity), the Bray–Curtis matrix and Unifrac distance were computed and then visualized with non-metric multidimensional scaling (NMDS) plots.GraphPad Prismp-values were adjusted with the Benjamini–Hochberg correction. A difference is significant if the adjusted p-value is below 0.05 and if the size effect is above 1 for ALDEx2 or if the logFC (log fold change) is above 2 for DESeq2. Finally, sparse partial least-squares discriminant analysis (sPLS-DA) was computed on the centered log ratio (CLR)-transformed data using the mixOmics package . Then, OTU counts were agglomerated at the genus level and relative abundances were computed at each taxonomic level. As recommended by  package . The choAll analyses were first performed considering all four groups  separately. Because no significant difference was detected between CT and Ex-CT groups, they were combined and renamed CT + Ex-CT for the microbiome analysis.p < 0.001).One parameter of the hens’ feeding behavior was estimated by counting the number of times hens pecked at the diet during and after GBH exposure. As shown in p < 0.05). Furthermore, after exposure (D70), spleens and kidneys were heavier and lighter (p < 0.05), respectively, in Ex-GBH hens compared to Ex-CT hens.Weight gain determined at D0–D21, D21–D42 and D42–D70 is presented in Plasma Gly and AMPA concentrations were assayed during GBH exposure and normal diet. The results are presented in p < 0.05) higher in GBH hens than in CT hens. No significant difference was observed between Ex-GBH and Ex-CT hens after GBH exposure at D70.Plasma oxidative stress was measured using the TBARS index, which quantifies the MDA concentration in plasma. The results are presented in Plasma triglyceride, cholesterol, phospholipid and uric acid concentrations were assayed at D0, D21 and D44 . No signIgA) was significantly (p < 0.05) enhanced by GBH treatment after exposure in Ex-GBH hens as compared to Ex-CT hens whereas no significant effect was detected during exposure (CT vs. GBH hens). We next determined the expression levels of genes involved in the inflammatory and immune responses. No significant effect was detected for chemerin, CMKLR1, IFNα, IFNβ, IL-1β, IL-6 and IL-8 (iNOS expression was significantly (p < 0.05) higher in the Ex-GBH group than in the Ex-CT group after exposure and that of HSP70 was significantly (p < 0.05) higher in the Ex-CT group than in the Ex-GBH group after exposure. No significant effect on any of these genes was detected during exposure (CT vs. GBH hens). No significant effect on the mRNA expression of some digestive genes (PGA5 and PGC) was detected in the proventriculus (The mRNA expression of liver biotransformation enzymes was measured by RT-qPCR. The results are presented in and IL-8 . mRNA extriculus . The TNFp < 0.05), while plasma glycine and leucine concentrations were significantly reduced in GBH as compared to CT animals (p < 0.05).Metabolites whose concentrations in hens’ plasma were significantly affected by GBH exposure are mentioned in p < 0.05) reduced during exposure in GBH as compared to control animals (CT). All of them were restored in Ex-GBH chickens’ cecum after GBH exposure, as compared to their respective controls (Ex-CT), except for acetate and valerate, which were still significantly (p < 0.05) reduced.Cecal contents were subjected to gas chromatography to measure the cecal SCFA concentration. SCFAs in hens’ cecum were assayed and the results are shown in Synergistes (family Synergistaceae) were favored by GBH at the final time point only. The other groups that showed a delayed impact were: Akkermansia (family Akkermansiaceae), DTU089 (family Clostridiaceae), Paraprevotella (family Paraprevotella), S50 (family Rikenellaceae) and Treponema (family Treponemataceae). Perhaps unsurprisingly, nine taxa were immediately impacted by GBH: the amounts of Muribaculaceae family, Alloprevotella (family Prevotellaceae), Porphyromonadaceae and Candidatus Vestibaculum were significantly higher after exposure in GBH and Ex-GBH groups than in the CT + Ex-CT group. The abundance of Barnesiella was significantly decreased whereas that of Colidextribacter was significantly higher in the GBH group than in the CT + Ex-CT group. The abundance of Coprobacter was different in all three groups: it was significantly lower in the GBH group than in the CT + Ex-CT group and was significantly higher in Ex-GBH than in the other groups. DTU089 abundance was significantly lower in the Ex-GBH group than in the CT + Ex-CT group. GCA-900066575 abundance was significantly higher in the Ex-GBH group than in GBH hens. Ruminococcus abundance was significantly higher in Ex-GBH and CT + Ex-GBH groups than in the GBH group. Abundance of the S50 wastewater-sludge group was significantly higher in Ex-GBH than in the GBH group. Synergistes abundance was significantly higher in Ex-GBH than in the CT + Ex-GBH group. Clostridia vadin BB60 abundance was significantly higher in the CT + Ex-CT group than in the GBH group. sPLS-DA analysis was performed to explain group variations. Two components have been identified. Most of the differential taxa identified by sPLS-DA analysis was already identified by the previous analysis. The first component (X-variate 1) explains 16% of the variations and allows to separate GBH and CT + Ex-CT hens from Ex-GBH hens. Second component (X-variate 2) explains 13% of the variations and allows to separate GBH hens from CT + Ex-CT hens. For each component, most contributing taxa were identified based on their abundance. The first component has 5 most contributing taxa. Its most contributing taxon is Treponema, more abundant in Ex-GBH group, followed by Prevotellaceae, more abundant in GBH group, Paraprevotella, a non-identified F082 family bacterium, more abundant in Ex-GBH group and Butyricicoccus which is more abundant in CT + Ex-CT group. The second component has 5 most contributing taxa. Its most contributing taxon is Muribaculaceae, more abundant in GBH group, followed by Bacteroidales, more abundant in GBH group too, itself followed by Flavobacteriales, more abundant in CT + Ex-CT group and Candidatus Vestibculum and Synergistes, more abundant in GBH and CT + Ex-CT group.GBH durably changed the composition of the microbiota, which was not completely resilient . SurprisOur results show for the first time possible disturbances of the cecal microbiota associated with plasma oxidative stress and the accumulation of Gly in metabolic tissues in response to chronic dietary GBH exposure in breeder broiler hens . FurtherIndeed, when we monitored hens’ body weight and fat thickness during and after chronic dietary GBH exposure, we did not observe any differences in either of these two parameters between the hens fed with GBH and those fed without GBH. These results are in accordance with those obtained after a 13-weeks study performed on rats . Moreovein ovo injections of Gly and GBH trigger modulations in the mRNA expression of cytochromes and other biotransformation-related genes in chick embryos  and D70 (after exposure), where they were accompanied by an increase in oxidative stress only at D42. Oxidative stress induction has widely been demonstrated in mammals exposed to Gly and AMPA . This ef embryos . Yet, in embryos , which c embryos . However embryos . Studies embryos  which arAkkermansia muciniphila  is part of the gut bacteria able to produce SCFAs from dietary carbohydrates  producer in humans  . Ruminochydrates  that we hydrates  where Run humans . Our resn humans . A 2017 n humans . HoweverHere, we show that half the current NOAEL of dietary Gly does not induce a direct impact on hens’ metabolism , though it reversibly triggers oxidative stress in blood plasma and impacts several gut microbes. Investigating the link between GBH and fertility is a direction that future works should probably take, in order to constitute a robust database allowing characterization of the actual impact that the molecule and its formulations have on animal and human health.