The significance of the Wnt/β-catenin signaling cascade in Rotavirus (RV) infection has not been elucidated. In this study, we attempt to elucidate the importance of the Wnt/β-catenin pathway in the RV pathogenesis and investigate a miRNA-mediated approach to regulate the pathway to repress the RV infection in the host. The regulation of the Wnt signaling pathway in terms of β-catenin accumulation and activation was analyzed by Western blotting and Confocal imaging analysis. The expression levels of miR-192 family members and miR-181a were enquired into using qPCR assays, whereas their targets in the Wnt pathway were confirmed using the Luciferase Reporter Assays. Members of the miR-192 family and miR-181a, which target the components of the pathway, were also found to be considerably decreased in expression during RV infection. Ectopic expression of these miRNAs could restrict the RV pathogenesis by targeting the intermediates of the Wnt signaling pathway. The miR-192 family and miR-181a were capable of suppressing the RV infection via targeting of the Wnt/β-catenin pathway. The study not only highlights the role of the Wnt signaling cascade in RV infection but also suggests that miRNAs can synergistically decrease RV replication by a significant amount. Thus, the miR-192 family and miR-181a present themselves as prospective antivirals against RV infection. The global RV detection rate had decreased from 42.5% in 2000 to about 37% by 2013. Nevertheless, India accounted for about 22% of the global death incidences due to RV, while being one of the four countries that account for about half of the total estimated deaths due to RV infection [Before the introduction of the vaccines Rotarix® RV and RotaOn the other hand, since its discovery 38 years ago, the Wnt/β-catenin pathway has been associated with cell survival and proliferation. The Wnt family contains about 19 glycoproteins capable of binding to the members of the Frizzled receptor family of G-protein coupled receptors. This receptor–ligand binding disrupts the destruction complex for β-catenin by restraining the components of the destruction complex at the cell membrane, leading to the accumulation and activation of β-catenin for cell survival. Without the ligand binding interaction, the β-catenin is anchored by the destruction complex comprising of the Axin, APC, and Glycogen synthase kinase 3 beta (GSK3β). The destruction complex component, GSK3β, phosphorylates and destabilizes the β-catenin at Ser33, Ser37, and Thr41, followed by proteasomal degradation of the catenin protein. The accumulated β-catenin localizes into the nucleus, where, along with TCF/LEF, it transactivates the gene expression of Cyclin D, c-Myc, and CD44 to regulate cell survival and proliferation. Therefore, the Wnt/β-catenin pathway is a popular target in anti-cancer therapeutics . The man2, 37 °C humified incubator. The cell-culture-adapted human RV strains Wa, Ku, and Simian RV strain SA11 (H96) were used for this study. These strains were cultivated and propagated in the MA104 cells, extracted, and titrated via the plaque assay to establish the viral titer [Two epithelial cell lines, namely, Caco-2 and MA104, one from the human colorectal adenocarcinoma and another from the African Green Monkey kidney, respectively, and the highly transfectable derivative of the human embryonic kidney cells (293T) were cultured and maintained in 10–20% heat-inactivated fetal bovine serum (FBS), 2 mM sodium pyruvate, 0.1 mM non-essential amino acids (NEAA), and 1% antibiotics supplemented minimum essential medium (MEM) or Eagle’s minimum essential medium (EMEM) in a 5% COn SA11 H9 were use2. A spectrophotometric reading at 490 nm gave a measure of the cell viability. The control/ mock cells were considered to have a 100% viability, and the percentage viability in the transfected cells was calculated in comparison to the mock cells. The cytotoxic effect of the transfected miRNA/siRNA was checked. Cells were transfected with scrambled miRNA or siRNA-Fz9 or mimics of miR-192/215 and/or miR-181a in 96-well plates. As per the manufacturer’s protocol , after the transfection procedure, the reagent solution was added onto the cells in a serum-free medium and maintained at 32 °C, 5% CO−ΔΔCt was used for the determination of the relative miRNA and viral gene expression levels after normalization with their respective endogenous controls. The RNA extraction method using the Trizol reagent  for the isolation of total RNA was carried out, as instructed by the manufacturer. The miR-192/215, miR-181a, and U6- specific primers were used for the cDNA preparations via reverse transcription of the specific miRNAs using the TaqMan miRNA reverse transcription kit . The cDNA for the RV-VP6 and GAPDH mRNAs was prepared via reverse transcription using the SuperScript™ III First-Strand Synthesis System  with random hexamers. For the quantitative real-time PCR for miRNA expression, the TaqMan universal PCR master mix  was used to perform the specific quantification of hsa-miR-192 (Assay ID: 000491); hsa-miR-215 (Assay ID: 000518); hsa-miR-181a (Assay ID: 000480); and the endogenous miRNA control U6 (human origin) (Assay ID: 001973). Specific primers designed to determine viral gene expression of the RV-VP6 gene and the endogenous control GAPDH were used with the SYBR Green Master mix . The formula, 2The targets of miR-192, miR-215, and miR-181a were predicted using miRWalk and TargetScan. The 3′UTR of the Fz9; SMAD4; TCF7; CCND2; and CTNNB1 were PCR-amplified and digested by the MluI and HindIII restriction enzymes. The pMIR-REPORT miRNA expression luciferase reporter plasmid vector  was also digested by the same restriction enzymes so that the PCR-amplified and restriction-digested 3′UTR of the targets were cloned at the MluI/HindIII site of the vector. The 293T cells were transfected/co-transfected with the pMIR-REPORT plasmid vector having the 3′UTR of the predicted targets at the cloning site and/or the mimics of hsa-miR-192, miR-215, miR-181a, siRNA-Fz9, or the scrambled miRNA, or the scrambled siRNA at a final concentration of 10–40 nM with Lipofectamine 2000 , as per the manufacturer’s protocol. The cloned 3′UTR of the miRNA targets contained in the pMIR-REPORT was co-transfected with the mimics of miR-192, miR-215, miR-181a, or scrambled miRNA in order to validate the miRNA targets in terms of measurements of their relative luciferase activities. The relative luciferase activities in the presence or absence of the miRNA were measured by the Dual Luciferase Assay System  post-normalization with the Renilla Luciferase Expression.After the suitable endpoints were achieved in the experiments, the cells that were washed with phosphate-buffered saline (PBS) were then lysed in RIPA buffer. The protein concentrations in the cell lysates were quantified by the PierceTM BCA Protein Assay Kit . A polyacrylamide gel electrophoresis followed by transferring the proteins onto the PVDF membrane marked the first step in the immunoblotting procedure. 5% non-fat containing milk was used to block the untransferred regions on the membranes. The membranes were then probed with antibodies specific to the proteins phospho-β-catenin ser33/37 ; phospho-β-catenin ser552 ; β-catenin ; ICAT (Santa Cruz Biotechnology: 293489); SMAD4 ; TCF1/7 ; CCND2 (Santa Cruz Biotechnology: 56305); Wnt-1 (Santa Cruz Biotechnology: 514531); Wnt-5a (Santa Cruz Biotechnology: 365370); phospho-LRP6 ; LRP6 ; phospho-Axin ; Axin (Santa Cruz Biotechnology: 293190); Naked-1 , RV-NSP1 (a kind gift from Prof. Koki Taniguchi); RV-NSP4 ; and RV- VP6 . These antibodies were either itself tagged with horseradish peroxidase (HRP) or detected via secondary antibodies tagged to HRP, followed by recording of chemiluminescence after the addition of ECL substrate  in the Chemidoc imaging system  or developed onto the BioMax Films . All the PVDF membranes were stripped and reprobed with the internal loading control GAPDH (Santa Cruz Biotechnology: 47724 HRP). The ImageJ software v1.53a  was used to measure the band intensities followed by normalization with GAPDH and represented in terms of relative fold changes. The immunoblots displayed in this manuscript were compliant with the digital image and followed the integrity policies.w/v paraformaldehyde in PBS and processed for the immunofluorescence technique, as mentioned previously [Caco-2 and MA104 cells were cultured on glass coverslips and incubated after infection with RV-SA11 for 6 and 12 h. These cells were then fixed with 4% eviously . Cells wp-value < 0.05, which is considered to be statistically significant, was observed in all the experiments and represented with asterisks. The number of asterisks indicate the significance levels . The significance levels have been analyzed via the Student’s t-test. GraphPad Prism 8.4.3  was used to analyze those figures having more than two experimental groups. The mean of at least two independent experiments plus the standard deviations were calculated and represented as the results of this study in this manuscript. The In order to understand the regulation of cell cycle progression by RV, the effect of RV infection on β-catenin mediated cell cycle control was investigated. The accumulation of β-catenin in the cells was observed in the early hours, i.e., 3 h to 12 h post-RV infection. The β-catenin that phosphorylated at Ser-552 was observed to increase with the progression of infection, whereas phosphorylation at Ser-33/37/Thr-41 decreased as the infection progressed. Decreased phosphorylation at specific sites, namely, Ser-33/37/Thr-41, showed that the level of activated β-catenin was maintained at 12 h post-infection and indicated that RV infection was capable of inducing the β-catenin signaling pathway in Caco2 cells A. In addThe accumulation of β-catenin is possible only when it has escaped the destruction complex, which is regulated by Wnt. Therefore, it is acceptable that this accumulation is encouraged by the induction of Wnt signaling. We checked the protein expression levels of Wnt-1 and Wnt-5a, which showed a similar gradual elevation pattern as that of β-catenin. LDL receptor-related protein 6 (LRP6), a co-receptor of Wnt, also exhibited increased phosphorylation at Ser-1490, which was maintained up to 12 h post-RV infection, indicating that the Wnt pathway had been triggered A. On theThe involvement of the Wnt/β-catenin pathway in RV infection involves a number of miRNAs in RV replication within the epithelial cells. In addition, several miRNAs have their targets involved in the Wnt/β-catenin pathway. Thus, the idea of RV infection manipulating the cellular miRNAs to modulate the Wnt/β-catenin pathway was perceived. The Frizzled receptors (Fz9/FZD9) were comprehended to be the target of two-member miRNAs of the miR-192 family, miR-192, and miR-215, clearly depicting around 35% decreased luciferase activity as compared to the control A. SimilaAfter identifying the miRNAs that could interfere with the expressions of their targets in the Wnt pathway, we then looked for changes in the expression levels of these respective miRNAs in RV infection. Real-time PCR results disclosed that the expression levels of miR-192, miR-215, and miR-181a decreased with an increase in RV infection of MA104 (data not shown) and Caco2 cells . Where tConfirmation of the participation of miRNAs in the RV-mediated upregulation of the Wnt/β-catenin pathway led us to investigate its role in assuaging the RV infection. The role of miR-192/215 and miR-181a was confirmed by using mimics of these miRNAs to check the modulations in the Wnt/β-catenin pathway as well as the changes in the virus titer . OverexpTrypanosoma cruzi infection, or viral infections such as HIV [2+/Calmodulin pathway, the epithelial-mesenchymal transition pathway, and suppression of apoptosis [Inhibition of cell cycle arrest is a common mechanism adopted by oncoviruses leading to cancer establishment in the host. Although RV has not been established as an oncovirus, our previous study has reported the involvement of RV in the manipulation of the cancer-associated epithelial-mesenchymal transition (EMT) pathway for efficient viral spread . Apart fh as HIV . As estapoptosis ,9,11. Thpoptosis ,26,27,28In summary, this study highlights the involvement of the cell survival associated pathway Wnt/β-catenin in RV pathogenesis and substantiates the role of prospective miRNA therapeutic agents that target the components of the pathway to extenuate RV pathogenesis and spread . This, a


Rhynchophorus ferrugineus or the red palm weevil (RPW) is a major invasive insect pest that causes severe problems for palm cultivations and, in turn, for the palm industries worldwide. The current practice for controlling RPW in Malaysia mainly involves the use of synthetic pesticides. However, synthetic pesticides should not be encouraged because they are highly toxic and persistent in the environment and as such, can cause deterioration to the ecosystem and human health. Essential oil (EO) derivatives from eugenol and thymol were tested as potential natural compounds for controlling RPW. We evaluated the effect of eight EO derivatives from eugenol and thymol against the fourth instar larvae of RPW. Feeding contact bioassay was conducted for 14 days with different concentrations of these derivatives, in three replications for each treatment. All the derivatives showed feeding deterrent effects against RPW larvae, particularly 4-allyl-2-methoxy-1-(4-trifluoromethyl-benzyloxy)-benzene and 2-isopropyl-4-methyl-2-((4-nitrobenzyl) oxy) benzene, which demonstrated the most effective antifeedant properties in the eugenol and thymol derivative group. Overall, our findings revealed that both eugenol and thymol derivatives showed similar antifeedant activity against RPW larvae. However, the ether derivatives showed a stronger antifeedant effect than that of esters. Both eugenol and thymol are effective against RPW and have the potential to be investigated further as botanical insecticides for the management of RPW in the future.Coconut palms in Malaysia are infested by a destructive invasive pest, RPW since 2007, and the pest’s population is difficult to control. At present, RPW control management mainly relies on the use of monocrotophos, which is administered by the trunk injection method. However, this pesticide can negatively impact human health and the ecosystem. Plant EO that can be used as a bio-pesticide is highly recommended as an alternative to monocrotophos because of its target-specific and eco-friendly properties. The antifeedant activity of eight eugenol and thymol derivatives from clove and thyme EOs were tested against the fourth instar larvae of RPW through oral bioassay for 14 days. Relative growth rate (RGR), relative consumption rate (RCR), the efficiency of conversion of ingested food (ECI), and the feeding deterrent index (FDI) were compared and analyzed. All of the derivatives showed antifeedant activity, particularly the eugenol derivative, 4-allyl-2-methoxy-1-(4-trifluoromethyl-benzyloxy)-benzene (FDI = 54.14%) and the thymol derivative, 2-isopropyl-4-methyl-2-((4-nitrobenzyl) oxy) benzene (FDI = 53.88%). Both of them showed promising results on their ability to be the most effective antifeedant agents in each derivative group. There was no significant difference in the effectiveness of the eugenol-based and thymol-based derivatives, but the ether derivative group (FDI = 45.63%) had a significantly stronger effect than the ester derivative group (FDI = 39.71%). This study revealed that the compound in ether form is more effective than the compound in ester form as an antifeedant agent against RPW larvae, regardless of the plant EO that the compound is derived from. Rhynchophorus ferrugineus Olivier (Coleoptera: Dryophthoridae), is an insect pest that causes damage to a broad spectrum of palm species around the world. This invasive species intruded into Malaysia in 2007 owing to the import of illegal date palm trees from the Middle East [Red palm weevil (RPW), dle East . An intedle East .To date, RPW has been reported in an oil palm smallholding in Tumpat, Kelantan, and there was evidence of the oil palms there being affected . At presCurrently, an integrated pest management (IPM) approach based on biological control rather than insecticides is being strongly promoted globally by policy-makers and international development agencies. In this context, many plant-based natural products are being tested, including EOs. EOs are reputed natural products utilized in many industries such as the food, cosmetics manufacturing, and pharmaceutical industries and in the production of botanical pesticides for use in the IPM approach. EOs perform a variety of functions such as those of a repellent, antifeedant, and attractor and shield from natural predators of herbivores ,14.Investigations on the efficacy of some plant EOs in controlling the RPW have focused mostly on the mortality percentage and antifeedant activity. For example, a study by Shukla et al.  showed tSyzygium aromaticum Linnaeus (Myrtaceae) and thyme, i.e., Thymus vulgaris Linnaeus (Lamiaceae) were tested for their antifeedant activity against RPW larvae.However, to date, the information available about the application of clove and thyme oil against RPW larvae is limited. It is believed that these plants, other than the family Arecaceae, possess certain chemical compounds that are effective in countering RPWs. Thus, this study tested the eugenol and thymol derivatives that were extracted from clove, i.e., Pheromone traps were used to collect RPW adults around coconut plantations in Kuala Nerus, Terengganu (5°22′17.6″ N 103°04′52.5″ E). The method adopted for RPW pheromone trapping was based on the method used by Wahizatul et al.  and YongRearing of RPW adults and larvae was carried out according to the methods described by Zulklifi et al. . The colEight eugenol and thymol derivatives were obtained from Nurul Hazwani, C.A.R., and Asnuzilawati, A., Universiti Malaysia Terengganu ,22. All A series of dilutions for the eight derivatives of eugenol and thymol were prepared using acetone as the solvent to obtain concentrations of 200 ppm, 400 ppm, and 600 ppm. The control was treated with an acetone solvent  only. Each treatment was replicated thrice, and each treatment replication consisted of three larvae. A total of 225 larvae were used in this study.Sago stem was selected as the food substrate as it is locally available and cheap. Sago is also one of the host plants for RPW . Each prRelative growth rate (RGR), relative consumption rate (RCR), efficiency of conversion of ingested food (ECI), and feeding deterrent index (FDI) were calculated according to the following equations :RGR =  therein. All the significance difference levels were determined at α = 0.05. All statistical analyses were conducted using SPSS (version 21.0).p < 0.001). The lowest food consumption amount among all treatments was observed in 4-allyl-2-methoxy-1-(4-trifluoromethyl-benzyloxy)-benzene (WN16) with 400 ppm. This value was significantly different from the other derivatives with the same concentration   .p = 0.169).RGR of the larvae was analyzed after 14 days. Negative RGR values indicated that the weight of the larvae decreased after the treatment. Overall, there was no significant difference between the RGR values of all the treatments and the control or in each treatment with different concentrations  of the larvae during the experimental periods. Most of the larvae that fed on treated food in all the concentrations exhibited lower RCR values than the control within the 14-day duration of the experiment. However, the larvae that fed on 4-allyl-2methoxyphenyl 4-ethylbenzoate (WN14) in concentrations of 400 ppm and 600 ppm had no significant difference compared with the control. ECI was calculated by taking into account the data of RGR and RCR arrived at by the aforementioned methods.p = 0.064). It was found that 4-allyl-2-methoxyphenyl 4-bromobenzoate (WN15) had a significantly higher ECI value in 200 ppm compared with the ECI value of 4-allyl-2-methoxy-1-(4-nitrobenzyloxy)-benzene (WN11) in 400 ppm.The ECI value indicates the rate of the ingested food that gets converted into larval growth rate. A higher ECI value implies that more of the consumed food was ingested and converted into a supplement for growing. There was no significant difference between ECIs of the control and all the treatments with different concentrations  .The effectiveness sequence was arranged by the following steps: a comparison through RGR, RCR, ECI, and FDI; a comparison of FDI among the derivatives (in the same concentration); and a comparison of FDI among concentrations in the same derivative. The results from RGR and ECI were not suitable to compare the effects of these compounds given that the treatments had no significant difference when it came to the control. Further, no significant differences in RCR were detected in the control and WN14 (in 400 ppm and 600 ppm), which indicates that WN14 had the lowest antifeedant activity among the eugenol derivatives. WN16 expressed the highest FDI value (54.14%), and it was the most effective eugenol derivative. The FDI values of WN11 and WN15 were similar, but the FDI value of WN15 in 400 ppm had no significant difference from that of WN14 in 400 ppm. Thus, in terms of effectiveness, these four compounds can be ranked as follows: WN16 > WN11 > WN15 > WN14.p = 0.001), followed by WNT2 in 400 ppm, which was also significantly different from other compounds with the same concentration   oxy) benzene (WNT2) in 200 ppm presented the lowest food consumption rate (0.178 ± 0.003 g/day)  .p = 0.529). The differences in RCR between the control and treated larvae were up to 0.24 g/g day. All the larvae that fed on treated food in all the concentrations showed a lower RCR value than the control. There was no significant difference between the ECI for control and each of the treatment groups with different concentrations .RGR’s range for treatments was between −0.001 g/g day and 0.005 g/g day. There was no significant difference between the RGR values of each treatment with different concentrations and control -1-isopropyl-4-methylbenzene (WNT1) had the highest FDI values, but it showed a significant difference only in the case of 2-isopropyl-5-methylphenyl 4-ethylbenzoate (WNT5).All thymol derivatives were classified into their respective functional groups, which were ether for WNT1 and WNT2 and ester for 2-isopropyl-5-methylphenyl 4-bromobenzoate (WNT4) and WNT5. The mean value of FDI within these groups was calculated and compared . The meaThe antifeedant activities of the four thymol derivatives were evaluated based on the criteria mentioned in All the eight derivatives in each base were grouped and compared with each other as presented in The eight derivatives were arranged based on the effectiveness of their antifeedant activity, and their sequence got listed as WNT2 > WN16 > WN11 > WNT1 > WN15 > WNT4 > WNT5 > WN14. WNT2 was listed as the most effective derivative as its FDI value was the highest in the 200 ppm concentration. WNT2 was followed by WN16, which had the highest FDI value in the 400 ppm concentration, but no significant difference was detected between WN16 and WNT2 in the same concentration.Larval feeding is crucial for its growth and optimal development according to its lifecycle. The quality and quantity of the food administered were important aspects for their development. Therefore, this study focused on regulating or deterring the feeding of the RPW larvae by continually providing food added with the necessary compounds, without any lethal effect (acute toxicity).+ 0.01 g/day (applying WNT2). This proves that eugenol and thymol have deterred the feeding behavior of the RPW larvae.The daily food consumption rate of the control, which was treated with acetone, was compared with the one found by Zulkifli et al. . They haSitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). The repellent action of the tested oils, which was introduced through vapors, affected the targeted pests’ respiratory systems or interacted with their receptors, thereby blocking their sense of smell.According to Price and Berry , eugenolSpodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) and the Mediterranean corn borer, Sesamia nonagriodes (Lefebvre) (Lepidoptera: Noctuidae), through the olfactory system by their antennae or setae. Hence, a tri-fluorinated compound may adversely affect the feeding activity of RPW larvae.The most effective antifeedant agent, WN16 -benzene) may have contributed to the tri-fluorinated structure in this compound . The comCulex pipiens (Linnaeus) (Diptera: Culicidae) [Aedes aegypti (Linnaeus) (Diptera: Culicidae) [Sitophilus granarius (Linnaeus) (Coleoptera) [Sitophilus zeamais [The effect of thymol derivatives has been studied earlier for its insecticidal activity against many types of insects such as licidae) , Aedes alicidae) , and Siteoptera) . Thymol  zeamais . However2) in its molecular structure. The continuous contact of NO2 results in allosteric inhibition of Glutathione S-Transferase (GST) enzyme, which can produce a detoxification function in larval bodies [2. In addition, WN11 also has a composition of NO2 in eugenol derivatives like WNT2, while WN11 was placed second only to WN16 in the effectiveness of the antifeedant activity. This indicates that the NO2 structure may cause a negative impact toward the RPW larvae and in determining the effectiveness ranking of the derivatives. The results implied that eugenol and thymol derivatives were not affecting the growth mechanisms of the RPW larvae, including food digestion and growth hormone inhibition. However, there was a decline in RPW larvae’s food intake, which could possibly be the outcome of these compounds entering them through other sensory systems such as the olfactory and gustatory systems [Carpophilus hemipterus (Linnaeus) (Coleoptera: Nitidulidae), in both adult and larval stages, whereas it also proved that the extract’s antifeedant compound could carry out its function without affecting its digestive system.WNT2 (2-isopropyl-4-methyl-2-((4-nitrobenzyl) oxy) benzene) comprises nitrogen dioxide , and they found that some compounds shared the same antifeedant activity. Eugenol and thymol were also tested on green peach aphid, and both these compounds showed not only behavioral effects but also toxic effects on the insect. From a functional perspective, both eugenol and thymol share the immobilization function of antimicrobials [The antifeedant activity of the eugenol derivatives did not significantly differ from the thymol derivatives. Isman et al.  have repcrobials . Both thcrobials . From thThese derivatives were also classified into a group according to their functional grouping, which involved ether and ester. It was interesting to find that ethers were significantly stronger antifeedant agents than esters. From the effectiveness sequence above, four derivatives  that showed stronger antifeedant activity consisted of ether functional groups, while four weaker derivatives  were esters. The stability of a compound is one of the factors that can alter the effectiveness of its antifeedant activity. All ethers analyzed in this study were found to have more stable characteristics than all the esters analyzed in this study , and theRhynchophorus phoenicis (Fabricius) (Coleoptera: Dryophthoridae) were evaluated, and showed that glutathione peroxides (GPX), glutathione reductase (GR), and GST were found in them [The detoxification action in RPW larvae may also explain why ethers are stronger antifeedant agents than esters. According to Wang et al. , RPW fat in them .Further experiments on these compounds need to be conducted on the other stages of RPW’s life cycle, including the adult and egg stages. It is hoped that the findings of his study will contribute toward exploring the potential of eugenol and thymol as botanical pesticides for developing a sustainable agricultural pest management system for RPW.The findings of this study have revealed the antifeedant activity of eugenol and thymol derivatives against the RPW larvae, with 4-allyl-2-methoxy-1-(4-trifluoromethyl-benzyloxy)-benzene and 2-isopropyl-4-methyl-2-((4-nitrobenzyl) oxy) benzene being indicated as the most effective compounds from the two groups respectively. Furthermore, a similar effect was found in the case of eugenol and thymol derivatives, where the ethers have significantly stronger effects on the RPW larvae than esters. Therefore, both these compounds have the potential to be used as targeted bioinsecticides for the management of RPW.


Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that protects against brain injury. However, little is known about the effect of Ex-4 on kainic acid (KA)-induced seizures and hippocampal cell death. Therefore, this study evaluated the neuroprotective effects of Ex-4 pretreatment in a mouse model of KA-induced seizures. Three days before KA treatment, mice were intraperitoneally injected with Ex-4. We found that Ex-4 pretreatment reversed KA-induced reduction of GLP-1R expression in the hippocampus and attenuated KA-induced seizure score, hippocampal neuronal death, and neuroinflammation. Ex-4 pretreatment also dramatically reduced hippocampal lipocalin-2 protein in KA-treated mice. Furthermore, immunohistochemical studies showed that Ex-4 pretreatment significantly alleviated blood–brain barrier leakage. Finally, Ex-4 pretreatment stimulated hippocampal expression of phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), a known target of GLP-1/GLP-1R signaling. These findings indicate that Ex-4 pretreatment may protect against KA-induced neuronal damage by regulating GLP-1R/CREB-mediated signaling pathways. The kainic acid (KA) mouse model of seizures and epilepsy has been proven to be an important system for understanding seizure pathology and developing therapeutic agents. KA-induced seizures are characterized by neuronal cell death, neuroinflammation, blood–brain barrier (BBB) leakage, and oxidative stress ,2,3. KA Glucagon-like peptide-1 (GLP-1), an incretin hormone, reduces blood glucose levels by stimulating insulin secretion and suppressing glucagon release . In partTherapies that target GLP-1/GLP-1R signaling may have neuroprotective effects in the KA-induced seizure mouse model. Ex-4 is a GLP-1R agonist that shares 53% amino acid homology with GLP-1. Ex-4 mimics the activity of GLP-1R but has a longer half-life due to its resistance to enzyme dipeptidyl-peptidase-4 (DPP-4) degradation . AlreadyIn the current study, we explored the effects of Ex-4 pretreatment on hippocampal neuronal death, neuroinflammation, and BBB breakdown in KA-induced seizure mice. Additionally, we determined whether Ex-4 pretreatment induced GLP-1R/CREB signaling in response to KA treatment in mice. Our findings suggest that activation of GLP-1R by Ex-4 pretreatment protected against hippocampal damage via upregulation of the p-CREB signaling pathway.n = 63) were obtained from KOATECH  and maintained at Gyeongsang National University’s animal facility (GNU). All animal research at GNU was approved by the Institutional Board of Research (GNU-190701-M0033). For laboratory animal care, we followed the National Institutes of Health recommendations. Mice were housed in a 12 h light/dark cycle.Male ICR mice . After washing with PBS, Ex-4-ABD was eluted with elution buffer . The production yield of Ex-4-ABD, as determined by a bicinchoninic acid (BCA) protein assay, was 8.9 mg/L culture, and the purity was above 80% as calculated by densitometry analysis using ImageJ .The pET28a-small ubiquitin-like modifier (SUMO)-Ex-4-ABD vector was constructed by double digestion of the Ex-4-ABD coding gene  with Nde1 and EcoRI followed by ligation into linearized pET28a-SUMO. A colony of pET28a-SUMO-Ex-4-ABD transformed BL21 (DE3) E. coli cells was picked and used to inoculate 50 mL Luria-Bertani (LB) broth containing 80 µg/mL kanamycin. This starter culture was shaken at 250 rpm and incubated at 37 °C overnight. After incubation, the starter culture was added to 1 L fresh LB broth with 80 µg/mL kanamycin and then incubated as described above. When the absorbance at 600 nm (OD600) reached 1.0, isopropyl β-d-1-thiogalactopyranoside (IPTG) was added as the inducer , and the batch culture was incubated for an additional 4 h. After incubation, cells were harvested by centrifugation and then resuspended in 60 mL of 20 mM phosphate-buffered saline (PBS) (pH 7) containing 300 mM NaCl, 1% leupeptin, and 1% soybean protease inhibitor . After sonication and centrifugation of the cell suspension, the supernatant fraction containing soluble SUMO-Ex-4-ABD was placed onto Talonn = 15) were treated with an intraperitoneal injection of KA . Control mice  were intraperitoneally injected with an equivalent volume of 0.9% normal saline. Mice in the experimental group were sacrificed at 2, 6, or 24 h after injection (n = 4 mice per group).Experiment 1: Experimental mice , KA-induced seizure mice , KA and Ex-4-treated mice , and Ex-4-treated (only) mice (n = 10). Seizure activity was evaluated using a 6-point seizure scale during the 2 h observation period after KA administration as previously described [Experiment 2: To assess the neuroprotective effects of Ex-4 pretreatment in mice with KA-induced seizures, Ex-4 (200 nmol/kg) was dissolved in 0.9% normal saline and intraperitoneally injected 72 h before KA injection. We previously determined the neuroprotective concentration of Ex-4 in diabetic mouse models ,23. The escribed . Seizuren = 8 mice per group).To measure Ex-4 in serum and hippocampal tissues, mice were anesthetized with Zoletil  24 h after KA administration. Blood samples were obtained by cardiac puncture of the left ventricle and then centrifuged. Hippocampi were removed from the brain. Ex-4 levels in both serum and hippocampal tissues were quantified using an ELISA kit  according to the manufacturer’s instructions  in 0.1 M PBS. Then, the extracted brains were post-fixed in PFA for 6 h and sequentially immersed in 0.1 M PBS containing 15% sucrose and 0.1 M PBS containing 30% sucrose at 4 °C until submerged. Brains were cut into 40-µm thick coronal sections. The sections were stained with Cresyl violet (Sigma-Aldrich) and imaged with a BX53 light microscope .TUNEL was conducted on frozen tissue sections according to the manufacturer’s protocol using an in situ cell death detection kit . The sections were examined using a BX51 DSU microscope (Olympus), and digital pictures were taken.For total protein isolation, frozen hippocampi were homogenized in tissue protein extraction reagent (T-per) lysis buffer . Homogenized hippocampi were incubated on ice for 20 min and sonicated three times. Samples were centrifuged for 30 min at 12,000 rpm at 4 °C, and supernatants were transferred to clean vials. The cytosolic and mitochondrial fractions were obtained using a mitochondria isolation kit for tissue (Thermo Fisher Scientific). Protein concentrations were determined using a Bio-Rad protein assay , and samples were stored at -80 °C until use. Total proteins (10–20 μg) were separated by SDS-PAGE on 8–12% acrylamide gels and transferred onto membranes. The membranes were probed with primary antibodies . β-actin® in situ PLA  was performed according to the manufacturer’s manuals. Anti-rabbit MINUS (first PLA probe) detected rabbit Ex-4 antibody, whereas anti-mouse PLUS (second PLA probe) detected mouse GLP-1R antibody . After 14 h at 4 °C, slices were washed and treated with a biotinylated secondary antibody (Thermo Fisher Scientific) for 1 h at room temperature. Brain sections were processed with an avidin-biotin complex (ABC) solution  and a 3,3′-Diaminobenzidine (DAB) kit (Sigma-Aldrich), mounted on gelatin-coated slides and coverslipped with Permount (Sigma-Aldrich). The stained sections were visualized with BX53 light microscope (Olympus), and digital images were captured.n = 3–4 mice per group) between Bregma level −1.58 mm to −2.18 mm. For each treatment group, TUNEL-positive cells, Duolink-stained signals, and immunostained positive cells were counted in 8–10 CA3 regions (200 × 200 µm2) using automatic quantitative component image analysis software . For the quantification of albumin in extravascular space, we counted albumin-positive cells, which were detected outside AQP4-positive vessels in the damaged CA3 regions caused by KA using automatic image analysis software (I-solution®).According to mouse atlas , we selected two coronal brain sections per mouse brain . Statistical differences were determined by a one-way ANOVA, followed by Tukey’s test as a post hoc analysis. In addition, two-group differences were determined by Student t-tests. The mean SEM was used to represent all values in graphs. A We first investigated the hippocampal expression of HO-1, an intrinsic factor against oxidative stress in hippocampal neurons, at the same time points after KA injection . HO-1 wap = 0.0007).Given that KA treatment led to decreased GLP-1R expression, we evaluated whether pretreatment with Ex-4, a GLP-1R agonist, would attenuate other effects induced by KA. For 2 h after KA treatment, we evaluated the anticonvulsant effects of Ex-4 by monitoring behavioral seizure activity and percent survival. Ex-4 pretreatment did not reach seizure scores greater than grade 4 in KA + Ex-4-treated mice compared to KA-treated mice A. FurtheNext, we investigated the role of intrinsic apoptotic pathways in hippocampal cell death in KA-induced seizure mice. Intrinsic apoptosis, which is triggered by the release of cytochrome c from the mitochondria into the cytosol, is regulated by a balance of pro-apoptotic factors  and anti-apoptotic factors . Ex-4 pretreatment downregulated the Bax/Bcl-2 ratio was induced by KA D. FinallFirst, we assessed the circulating and hippocampal expression levels of Ex-4 across treatment groups using Western blot and ELISA analyses . In seruKA treatment remarkably increased expression of microglia marker Iba-1 24 h after injection, which was significantly attenuated by Ex-4 A. The IbWe next investigated expression levels of the acute pro-inflammatory cytokine, LCN2. Western blot analyses revealed that KA treatment significantly increased both circulating and hippocampal LCN2 expression levels. Critically, Ex-4 pretreatment significantly reduced these expression levels compared to KA treatment alone A,B. The We verified that KA induces BBB leakage by performing immunohistochemistry analyses of the tight junction protein ZO-1, serum albumin to mark BBB permeability, and the glial water-channel AQP4. KA-treated mice had significantly fewer ZO-1-positive cells. Intriguingly, the number of ZO-1-positive cells in mice treated with both Ex-4 and KA was similar to the numbers observed in control mice A. AdditiThe p-CREB pathway plays a critical role in the process of cellular survival following neuronal injury . WesternIn this study, we investigated the possible neuroprotective effects of Ex-4 in mice with KA-induced seizures. Ex-4 pretreatment led to a decrease in markers of seizure activity, neuronal apoptosis, neuroinflammation, and BBB leakage in KA-induced seizure mice. In addition, increased circulating and hippocampal LCN2 protein levels in KA-treated mice were reversed by Ex-4 pretreatment. Furthermore, our results indicate that Ex-4 treatment activated GLP-1R/CREB signaling, which plays a critical role in protecting against excitotoxicity-induced neuronal damage. Therefore, we propose that Ex-4 pretreatment may exert a neuroprotective effect on seizure-related neurological disorders.GLP-1/GLP-1R signaling regulates the protein expression of Bcl-2 family members . These proteins tightly regulate the intrinsic apoptosis pathway, which is triggered by the mitochondrial release of cytochrome c and ultimately leads to caspase activation ,28. ActiIn models of cerebral ischemia, intranasal administration of Ex-4 reversed the upregulation of caspase-3 in the hippocampus. Silencing expression of GLP-1R using short hairpin RNA reversed the effect of Ex-4 on cell survival . In a diAs shown in Seizures cause hippocampal neuronal cell death and reactive gliosis, which produces pro-inflammatory cytokines ,38. Ex-4Inflammation is a key factor in the progression of BBB leakage, which results in brain injuries such as seizures and ischemic stroke ,41. BBB Ex-4 pretreatment activates the GLP-1R/CREB signaling pathway to exert neuroprotective effects ,47. AccoIn conclusion, our findings show that pretreatment of Ex-4 stimulated GLP-1R/CREB signaling pathway to protect against brain damage including hippocampal cell death, BBB leakage, neuroinflammation, and oxidative stress in mice with KA-induced seizures. Therefore, these data indicate that Ex-4 may have beneficial therapeutic effects on seizure activity and other brain injuries.


Glutathione peroxidase 4 (GPX4) is a crucial regulator of ferroptosis. We found that intravenous injection of MSC-ex significantly decreased glutathione peroxidase 4 (GPX4) expression in activated HSCs and collagen deposition in experimental mouse fibrotic livers. Mechanistically, MSC-ex derived BECN1 promoted HSCs ferroptosis by suppressing xCT-driven GPX4 expression. In addition, ferritinophagy and necroptosis might also play a role in MSC-ex-promoted LX-2 cell death. Knockdown of BECN1 in MSC diminished proferroptosis and anti-fibrosis effects of MSC-ex in LX-2 and fibrotic livers. MSC-ex may promote xCT/GPX4 mediated HSCs ferroptosis through the delivery of BECN1 and highlights BECN1 as a potential biofactor for alleviating liver fibrosis.Activated hepatic stellate cells (HSCs) are significant in liver fibrosis. Our past investigations have shown that human umbilical cord mesenchymal stem cells (hucMSCs) and their secreted exosomes (MSC-ex) could alleviate liver fibrosis via restraining HSCs activation. However, the mechanisms underlying the efficacy were not clear. Ferroptosis is a regulatory cell death caused by excessive lipid peroxidation, and it plays a vital role in the occurrence and development of liver fibrosis. In the present study, we aimed to study the proferroptosis effect and mechanism of MSC-ex in HSCs. MSC-ex were collected and purified from human umbilical cord MSCs. Proferroptosis effect of MSC-ex was examined in HSCs line LX-2 and CCl4 induced liver fibrosis in mice. Gene knockdown or overexpression approaches were used to investigate the biofactors in MSC-ex-mediated ferroptosis regulation. Results: MSC-ex could trigger HSCs ferroptosis by promoting ferroptosis-like cell death, ROS formation, mitochondrial dysfunction, Fe Liver fibrosis is a pathophysiological process caused by chronic viral hepatitis and metabolic disorders. It is characterized by the deposition of many collagen fibers and the proliferation of hepatic stellate cells (HSCs) . Current4-induced liver fibrosis [Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewal and multidirectional differentiation capabilities. MSCs have been increasingly applied to clinical treatment, including acute and chronic liver injury , 5. Manyfibrosis , 17. TheFerroptosis is a new type of programmed cell death that is iron-dependent and different from apoptosis, cell necrosis, and autophagy . FerroptIn the present study, we aimed to investigate the function of MSC-ex in HSCs ferroptosis and the mechanism of action. We observed that MSCs-ex promoted ferroptosis of HSCs. In addition, MSC-ex-derived BECN1 decreased xCT/GPX4 expression and inhibited HSCs activation, providing a novel insight into the proferroptosis effect of MSC-ex in liver fibrosis.The human umbilical cord was gained from informed, healthy parturients, and MSCs were isolated as previously reported . IsolateAfter reaching 80–90% confluence, the media of MSCs was changed to an FBS-free medium (Bioind) for 48 h and centrifuged at 10,000 × g for 30 min to remove cells and cell debris. MSC-ex was isolated and purified as reported previously . The supProtein was extracted from MSC-ex, LX-2, or tissues utilizing RIPA . The concentrations of protein samples were determined by BCA assay . Proteins were separated in 10% SDS-PAGE denaturing gel and transferred to the nitrocellulose membrane. Next, the membrane was blocked with 5% milk at room temperature. The membrane with blotted protein was incubated with primary antibodies against β-actin , TSG101 , Calnexin , CD9 , CD63 , Phospho-MLKL , LC3B , GPX4 , α-SMA , xCT , BECN1 . The membrane was further probed with horseradish peroxidase-conjugated secondary antibodies (Invitrogen). Finally, the protein bands were visualized utilizing enhanced chemiluminescence reagents (Millipore).−ΔΔCt method. All primers were synthesized by Invitrogen and listed in Table RNA from LX-2 or liver tissues was collected utilizing Trizol (GIBCO) following the manufacturer’s instructions. cDNA was synthesized from the RNA using SuperScript Reverse Transcriptase Kit . Then qRT-PCR assays were performed using SYBR green (CWBIO). Relative gene expression normalized to β-actin was calculated using the 2LX-2 was planted on coverslips in a 24-well plate and treated with PBS or MSC-ex (400 or 800 μg/ml). After MSC-ex treatment, LX-2 was fixed in 4% (w/v) paraformaldehyde (Sigma-Aldrich) for 15 min. LX-2 was then permeabilized with 0.1% Triton X-100 for 10 min. 5% BSA was utilized to block the cells at room temperature for 1 h. After that, LX-2 was hybridized with primary antibodies against α-SMA, GPX4, and xCT (Abcam) at 4 °C overnight and visualized with fluorescence-labeled secondary antibodies  for 45 min at 37 °C. Hoechst 3342 was used to stain nuclei at room temperature. Images were captured using a confocal microscope .All experiments involving animals were conducted according to the ethical policies and procedures approved by the ethics committee of the Jiangsu University ethics committee . The use of human umbilical cord tissues was approved by the Jiangsu University ethics committee (2012258).n = 6) or PBS (n = 6). Liver samples were collected for further analysis at two weeks post-MSC-ex injection.Mice  were from Cavens animal laboratory Co., Ltd from Changzhou, China. Following the protocols approved by the Jiangsu University ethics committee (UJS-IACUC-AP-2020033127), a mouse model of liver fibrosis was established randomly with an intraperitoneal injection of 10% CCl4  for eight weeks, twice a week. To analyze the effect of MSC-ex on GPX4, BECN1, and xCT expression and liver fibrosis, mice were injected with MSC-ex twice a week at a dose of 20 mg/kg body weight . To analyze hepatic collagen distribution, fibrotic septa randomly selected from the right and left liver lobes of 6 individual mice/groups were assessed. Images were acquired using a digital slide scanner . Collagen extent was expressed as a percentage of stained area in each liver section.The liver sections were deparaffinized and rehydrated for immunohistochemical staining for BECN1 and GPX4; the endogenous peroxidase was inactivated by 3% H2O2 for 30 min. Next, the liver slides were immersed into preheated antigen retrieval solution  for 30 min. After blocking with 5% BSA, the liver slides were probed with primary antibody against BECN1 (Proteintech) and GPX4 (Abcam) overnight at 4 °C and incubated with biotin-conjugated anti-rabbit IgG and streptavidin-biotin. Finally, liver tissue sections were visualized with DAB Horseradish Peroxidase Color Development Kit  and counterstained with hematoxylin. Images were acquired using a pathological section scanner .MSC-ex, MSC-ex/NEC-1 or NEC-1s , MSC-ex/NAC  or Erastin (MedChemExpress) treated LX-2 or L-02 were cultured in medium supplemented with 10 µM FDA at 37 °C for 20 min. Finally, the cell viability of MSC-ex treated LX-2, or L-02, was detected by Cytation 5 Imaging Reader . There were three replicates per group.MSC-ex, MSC-ex/NEC-1 or NEC-1s, MSC-ex/NAC  or Erastin (MedChemExpress) treated LX-2 or L-02 were cultured in medium supplemented with 10 mM fluorescent probe DCFH-DA  for 20 min at 37 °C. After fixing with 4% paraformaldehyde, LX-2 or L-02 were counterstained with Hoechst 3342. Images were captured using a confocal microscope . Mean fluorescent intensity was calculated using Image-Pro Plus 6.0 software. There were three replicates per group.JC-1 staining was performed to analyze the mitochondrial membrane potential in LX-2 or L-02 . JC-1 probe was diluted in fresh medium at 1:1000. Cells were incubated with a diluted JC-1 probe in the dark at 37 °C for 20 min. After fixing with 4% paraformaldehyde, LX-2 or L-02 were counterstained with Hoechst 3342. Images were captured using a confocal microscope . Mean fluorescent intensity was calculated using Image-Pro Plus 6.0 software. There were three replicates per group.BECN1 cDNA treated LX-2 or L-02 were lysed utilizing RIPA , and iron concentrations were determined using an iron assay kit following the manufacturer’s instructions (Abcam). There were three replicates per group.MSC-ex (400 or 800 μg/ml) or Levels of GSH and MDA in MSC-ex (400 or 800 μg/ml) treated LX-2 or L-02 were detected with GSH assay kit  and MDA assay kit  following the manufacturer’s instructions, respectively. There were three replicates per group.PBS or MSC-ex (800 μg/ml) treated LX-2 was plated in 60 mm culture dishes. For intracellular mitochondrial ultrastructure observation, LX-2 was collected and fixed with 2.5% glutaraldehyde in 3 mM CaCl2 and 0.1 M cacodylate. After cutting and staining with uranyl acetate and lead citrate, images of ultrathin sections were acquired with a transmission electron microscope .BECN1 cDNA (NM_003766) was constructed into pBECN1 expression vectors (pCDH-CMV-pBECN1-EF1a-GFP-T2A-Puro). Small interfering RNA (siRNA) targeting BECN1 and control scrambled siRNA were designed . LX-2 was seeded to 80% confluence in 6-well plates in triplicate. Lipofectamine 2000 (Life Technologies) reagent was used to transfigure BECN1 expression vector or synthetic small interfering RNA (siRNA) oligos into LX-2. Cell protein or RNA samples were harvested at 48 h after transfection and processed for western blot or Quantitative RT-PCR analysis.Full-length human shBECN1) or lenti-control shRNA transfected MSCs (Exsh-ctr) were collected and purified. To investigate the regulation of Exsh-ctr and ExshBECN1 on GPX4, mice were injected with PBS (n = 6), Exsh-ctr (n = 6), and ExshBECN1 (n = 6) at a dose of 20 mg/kg body weight twice a week. At two weeks post ExshBECN1 injection, liver samples were collected for further analysis.Recombinant lentiviruses were produced by transient transfection in HEK293T cells. BECN1 shRNA sequences were synthesized and cloned into pPLK/GFP-Puro lentiviral plasmid to generate pPLK/GFP-Puro-BECN1 shRNA/control shRNA. Sequences of BECN1 shRNA are designed using the following: GTGGACACGAGTTT CAAGA. Then HEK293T cells were co-transfected with three lentiviral plasmids of psPAX2 (packaging vector), pMD2G (envelope vector), and pPLK/GFP-Puro-BECN1 shRNA (transfer vector). GFP expression of HEK293T cells was evaluated, and supernatants were collected at 72 h post-co-transfection. MSCs were transfected with lenti-BECN1-shRNA/lenti-control-shRNA lentivirus. BECN1 expression in MSCs was examined with qRT-PCR and western blot. Exosomes of lenti-BECN1 shRNA transfected MSCs (ExANOVA) followed with Dunnett was used for studies involving more than two groups. A two-sided P < 0.05 was considered statistically significant.Data are presented as means ± SD (standard deviation). The student’s t-test used GraphPad Prism version 5.0 software to analyze statistical significance between two groups. One-way analysis of variance , glutathione (GSH), and mitochondria morphology were determined to observe the level of cell ferroptosis. As expected, MSC-ex induced ferroptosis was revealed by increased intracellular ROS production  ferroptosis were also investigated with FDA assay and ROS assay. The FDA and ROS assay results showed that L-02 cell death and ROS production were decreased by MSC-ex Fig. . MSC-ex xCT and SLC7A11 are components of the xc system amino acid antiporter, which import cysteine and export glutamate into cells . BECN1 i4 group , lipid peroxidation, membrane damage, and cell death . To furtsh-ctr) or BECN1 knockdown (ExshBECN1) in the regulation of GPX4 and a-SMA expression, ROS production, and hepatic collagen deposition in vitro and in vivo. Compared with Exsh-ctr, the level of BECN1 was decreased in ExshBECN1  was a critical event of necroptosis . MSC-ex This study found MSC-ex-derived BECN1 induced HSCs ferroptosis and ameliorated liver fibrosis. In vitro, MSC-ex increased BECN1 expression in LX-2 through exosomal BECN1 transfer and subsequently inhibited the expression of xCT and GPX4 in HSCs. Then the word of α-SMA in LX-2 was decreased following ferroptosis. Our results showed that overexpression of BECN1 in LX-2 enhanced ferroptosis, whereas knockdown of BECN1 in LX-2 abolished ferroptosis, further suggesting that BECN1 was crucial in HSCs ferroptosis induction. In vivo, the ability of MSC-ex to induce ferroptosis and ameliorate liver fibrosis was weakened following BECN1 knockdown. Briefly, MSC-ex could enhance ferroptosis in HSCs and subsequently reduce CCl4-induced liver injury through exosomes/ BECN1/xCT/GPX4 pathway.Liver fibrosis is a progressive disease. MSC-ex exerted therapeutic effects in mouse liver fibrosis. However, the mechanism of MSC-ex reducing liver fibrosis remains obscure. It has been confirmed that ferroptosis is essential for defending against various liver diseases, such as liver fibrosis , hepatocExosomes contain complex RNAs and proteins and have aroused widespread interest in the regeneration and repair of various tissues , 8, 37. We further explored the underlying mechanisms by which MSC-ex derived BECN1 can induce ferroptosis and present hepatoprotective effects. BECN1, xCT, and GPX4 inhibition are all reported to trigger ferroptosis . BECN1 cFinally, NAC has significant protective activity against liver fibrosis through attenuation of oxidative stress. Our previous study also showed that MSC-ex could act as an antioxidant to reduce oxidative stress and rescue liver failure in CCl4 induced liver injury mouse model , 17. Our4-induced liver fibrosis, and HSCs ferroptosis may be a new therapeutic approach for liver fibrosis treatment in the future.In summary, we demonstrated that MSC-ex targeted hepatic stellate cells activation in vitro and in vivo. This may be mediated by the delivery of BECN1 to induce ferroptosis via the downregulation xCT/GPX4 pathway Fig. . These fSupplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5Supplemental Figure 6Supplemental Fig. 7.Supplemental Fig. 8.Supplemental Fig. 9.aj-checklistSupplemental figure legends


However, how ER stress interacts with this pathway in TNBC has not yet been understood. Here, the tunicamycin and LiCl have been applied to MDA-MB-231. The related proteins' expression was measured by western blotting. Moreover, acridine orange/ethidium bromide (AO/EB) staining was applied to test the apoptosis degree of the cells, and cell viability was tested by MTT experiment. Then, we found the ER stress and apoptosis degree of MDA-MB-231 were induced after treatment with tunicamycin. Besides, tunicamycin dose dependently inhibited both Wnt/β-catenin pathway and cells viability. Licl, an activator of Wnt/β-catenin signaling pathway, could significantly inhibit cell apoptosis. In conclusion, our study found that the activation of ER stress could promote the MDA-MB-231 apoptosis by repressing Wnt/β-catenin pathway, which provides some promising prospects and basic mechanism to the further research.Triple negative breast cancer (TNBC) has significantly threatened human health. Many aspects of TNBC are closely related to Wnt/ In a pathway , 6, whicfor TNBC . The redof cells , 9.Endoplasmic reticulum (ER) stress has a certain toxic effect on cells, and it can be activated by a variety of stimuli, such as large amount of unfolded protein accumulation in cells, disorder of redox response, and calcium regulation disorder , 11. Excβ-catenin pathway for TNBC. Furthermore, ER stress, as a regulator in the initiation process, is involved in cancer cell apoptosis through multiple pathways. Nevertheless, whether ER stress can reverse the apoptosis of TNBC through the inhibition of this pathway remains unknown. So, we intended to reveal the association between the injury of MDA-MB-231 induced by tunicamycin and Wnt/β-catenin pathway.The studies mentioned above explain the significance of Wnt/2 and at 37°C.The breast cancer cells MDA-MB-231 were acquired from the Chinese Academy of sciences , and Dulbecco's Modified Eagle Medium (DMEM) obtained from the Healthcare Life Sciences (HLS) were used to culture MDA-MB-231 cells ; DMEM was included 10% fetal bovine serum (FBS) which acquired from the Biological Products Technology of Tianjin Haoyang. The culture condition of these cells was in the 5% COμL MTT was applied, respectively, to the well 24 hours after treatment. After four hours of incubation, the medium in each well was removed and purple precipitation was dissolved with 180 μl DMSO. Finally, the spectrophotometer was used to detect the optical density at a 450 nm wavelength.The cells were transferred onto 96-well plates, and there are 5000 cells in each well. 20 β-catenin pathway-associated proteins β-catenin  and phosphorylated β-catenin , key molecules of ER stress GRP78  and CHOP , and regulatory gene c-Myc  and GAPDH . After TBST washing, the primary antibody which combines with the protein functioned as a platform to receive the combination of the secondary antibody. Lastly, an enhanced chemiluminogenic enhancer reagent was bought from Beijing Kangwei Century Biotech (cat. no. CW0049M). An autoradiography system bought from Bio-Rad Laboratories  was used to detect the blot. Every result was obtained by repeating the experiment three times and measured by Image Lab .RIPA lysate is added to the collected cells and is thoroughly mixed to obtain the proteins. The proteins of each group were electrophoresed on 10% gel in equal quantities. Then, the PVDF membrane was used to receive all the proteins and the PVDF membrane was sealed by 5% skim milk. The primary antibodies were applied subsequently onto the PVDF membrane to detect the expression of apoptosis-associated proteins Bcl-2  and Bax , Wnt/μl of TBST containing AO/EB at 1 : 1 ratio was added to each well. After 5 minutes, the medium of AO/EB and TBST was discarded and the plates were washed using TBST three times. Finally, a fluorescence microscope was used to detect the fluorescence intensity.In 24-well plates, cells with treatment were cultured in 24-well plates for 24 hours. After the medium was removed from each well, 500 P < 0.05 represents statistical significance.GraphPad Prism 6.0 was used to analyse all results. One-way analysis of variance (ANOVA) was selected to analyse the differences among each group, experiments were repeated three times; μmol/L  related to the formation of breast tumor [β-catenin signaling pathway, c-Myc affects the tumor cell cycle by inhibiting growth suppression gene transcription and was significantly increased in Tun + Li group compared with Tun treatment (β-catenin and ER stress-related protein expression [In order to identify whether tunicamycin-induced ER stress can block the Wnt/ Figures . Indeed,st tumor , 21. Howreatment , which cpression . Collectβ-catenin pathway and endoplasmic reticulum stress in TNBC. Thus, further research should be conducted on the project of in vivo experiment and the verification of mechanism in different cell lines. Collectively, this study inspected the specific mechanism that the β-catenin signaling pathway affected the apoptosis process which was introduced by ER stress in MDA-MB-231 cells, which proved a potential treatment target and reduction of side effects.Although the study lacks a contrasting experiment between different cell lines of breast cancer and animal experiment, it firstly and sufficiently verified the relationship between the Wnt/β-catenin signaling pathway, which could be reversed by LiCl (This study investigated whether activation of ER stress by tunicamycin could induce apoptosis of TNBC in MDA-MB-231 cells. The processes were initiated through repressing the Wnt/ by LiCl . In conc


Clostridiodes difficile colitis during autologous stem cell transplantation (https://www-ncbi-nlm-nih-gov.proxy.libraries.uc.edu/pubmed/29594489). To biological validate these findings, we sought to evaluate the development of chemotherapy-associated Clostridiodes difficile infections by assessing the effect of C.difficile toxin B (TcdB) and of using melphalan in beta-catenin protein expression in Caco2 cells. METHODS/STUDY POPULATION: To determine the effect of melphalan and/or C.difficile toxin B on expression of Beta-catenin from human gut epithelial cells:Adenocarcinoma cells (Caco-2) cells were seeded and allowed to grow into monolayersMonolayers were treated with PBS, TcdB, melphalan and/or TcdB + melphalan for 24 hours and then washed with PBSImmunofluorescence was measured on the monolayers to visualize three markers -DAPI-Nuclear Stain (blue),Actin-ccytoskeletal stain (red), B-Catenin (green)Analysis of images with ImageJ (NIH). Statistical analysis of the effect of TcdB and/or melphalan on β-catenin protein levels was determined by One-way ANOVACells stained with a primary anti-β catenin antibody and an Alexa-488 secondary antibody were evaluated by flow cytometry to quantify the effect of melphalan and/or C. difficile toxin B on Caco2 cells. RESULTS/ANTICIPATED RESULTS: Immunofluorescent intensity was higher in the control (PSS exposed) cells when compared to melphalan, TcdB and mephalan+TcdB exposed cells  DISCUSSION/SIGNIFICANCE OF IMPACT: A significant difference was seen in β catenin expression in Caco-2 monolayers exposed to TcdB and/or melphalan. These data support the a role of β-catenin in the pathophysiology of CDI during chemotherapy and support GWAS findings reporting a difference in CDI susceptibility based on β-catenin genotype.OBJECTIVES/GOALS: We previously reported that genetic polymorphisms in the beta-catenin gene (CTNNB) are associated with the development of


In addition, larger semicircles were registered in Nyquist plots. These results suggest a strong inhibition effect of the electron transfer reaction by LNPs layer, especially in the case of KLNPs. The modified electrodes, properly assembled with concanavalin A (ConA) and glucose oxidase (GOx), were successively tested as biosensing platforms for glucose, showing a sensitivity of (4.53 ± 0.467) and (13.74 ± 1.84) μA mM−1 cm2 for Au/SAMCys/OLNPs/ConA/GOx and Au/KLNPs/ConA/GOx biosensors, respectively. Finally, different layers of the KNLPs/ConA/GOx-modified Au electrode were tested, and the three-layered Au(KNLPs/ConA/GOx)3 showed the best analytical performance.Novel nanoarchitectures based on lignin nanoparticles (LNPs) were designed and realized for electrochemical eco-friendly biosensing development. Two types of lignin nanoparticles were utilized for the modification of a gold bare electrode, namely organosolv (OLNPs) and kraft lignin (KLNPs) nanoparticles, synthetized from a sulfur-free and a sulfur lignin, respectively. The electrochemical behavior of LNP-modified electrodes was studied using two electrochemical techniques, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Compared to the gold bare electrode, an evident decrease in the faradaic current and increase of the ΔE In the last decade, significant effort has been devoted to the design and development of biosensors with improved characteristics in terms of selectivity, sensitivity, simplicity and response time. One of the most critical issues in biosensing design is the achievement of high sensitivity and selectivity together with an easy-to use format. To this aim, the choice of a proper biosensing interface is fundamental.Several nanomaterials have been successfully used as electrochemical platforms for the immobilization of bioreceptor molecules with the desired orientation, allowing the retention of their biological activity. In addition to the immobilization step, the nanoplatforms perform as an analyte recognition site and a signal transduction and amplification probe for the enhancement of biosensor performances ,3,4,5,6.A variety of nanostructured materials have been utilized in the construction of biosensors, such as carbon-based materials like carbon nanotubes ,13,14,15In the last decade, lignin nanoparticles (LNPs) ,30,31,32There are two types of lignins based on different extraction processes: sulfur lignin, produced by chemical pulping processes, including kraft lignin (KL) and ligno-sulfonates (LSs), and sulfur-free lignin, including alkaline lignin and organosolv lignin (OL), produced by bioethanol production processes . OL and In this work, we describe and compare the use of organosolv lignin nanoparticles (OLNPs) and kraft lignin nanoparticles (KLNPs) as novel nanoarchitectures for the modification of a bare gold electrode for electrochemical biosensor development. In particular, we discuss the preparation and the electrochemical characterization of LNPs-modified platforms and, as a proof-of-concept, their application in the development of a glucose biosensor. The electrode was assembled by the layer-by-layer (LbL) procedure, and the electrochemical and biocatalytic electrode performance was evaluated depending on the number of deposited layers, as well as on the role played by Con A in the orientation of the GOx immobilization. To the best of our knowledge, no reports are present in literature on electrode platforms based on LNPs-assembled multilayer films composed by LNPs/ConA/GOx for glucose detection.Aspergillus niger  and Con A from jack beans  type VI were purchased from Sigma-Aldrich, Buchs, Switzerland, and stored at −18 °C.Glucose oxidase from 2HPO4), sodium dibasic phosphate (NaH2PO4), potassium chloride (KCl), potassium ferricyanide (III) (K3[Fe(CN)6]), potassium ferrocyanide (II) (K4[Fe(CN)6], exaammineruthenium(III) chloride ([Ru(NH3)6]Cl3), cysteamine (Cys), (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, commercial grade (EDC) and N-hydroxysuccinimide, 98% (NHS), purchased from Sigma-Aldrich . The complex [Os2(4-aminomethylpyridine)Cl]PF6 (OsCmplx) used as an electrochemical mediator was synthesized according to a previously published protocol 3+ is known to be insensitive to surface modification within monolayer thickness as it is able to exchange electrons by tunneling Cl3  was followed in the mediated oxidation of the FADHH3)6]Cl3 , curve bThe kinetic and analytical parameters of the two proposed platforms are summarized in The sensitivity resulted to be three times higher with KNLPs than OLNPs. This result, apparently in contrast with data previously reported, can be explained by taking into account the larger electrode coating of the KNLPs layer, which allows an immobilization of a much larger amount of enzyme. Moreover, the presence of Con A favors a correct orientation of the GOx enzyme during the immobilization process , enhanciMapp value with KNLPs, suggesting a larger enzymatic affinity for glucose when KNLPs are used.The apparent Michaelis–Menten constants, obtained by fitting the calibration curves, show a reduction of the KThe best electrode platform was Au/KLNPs/Con A/GOx, and therefore this platform was used for further experiments.Different layers of the KNLPs/CoA/GOx were assembled by repeating the steps described for the preparation of the Au-modified electrode . The pro3 biosensor showed an enhanced sensitivity of 15.85 µA/mM cm2, a linear range between 0.1 and 2.5 mM and a lower LOD of 0.02 mM, compared to the one-layered same platform. In addition, the increase in the current value clearly demonstrates the important role played by Con A, which allows a well-oriented immobilization of GOx and by LNPs due to their boosting effect in the activity of the overall electrocatalytic system, as previously hypothesized in our previous work [Comparing the voltammograms of the three-layers system, a two-times increase in the current signal was observed between the first and the third layer. Thus, the assembling of the three layers exhibited a remarkable improvement on the catalytic properties in the recognition of glucose, confirming that the LbL assembly process is able to enhance the performances of the biosensors in terms of sensitivity, reproducibility and stability ,63. In pous work .In this work, a study on the use of organosolv and kraft lignin nanoparticles for the design and development of novel nanoassembled electrochemical platforms is presented. The data reported that the properties of the nanoparticles are governed by the type of lignin used. A blocking behavior was shown by both LNP-gold-modified electrodes because of the insulating nature of the LNPs. This effect is more pronounced with KLNPs compared to OLNPs, most likely because of the formation of a thicker KNLPs layer, which allows a better coating of the electrode surface. In addition, OLNPs were anchored to the Au electrode surface through a SAM molecule, which increases the distance between the LNPs and the electrode surface, with a consequent lower loading of the nonconductive LNPs and enhancement of the electron-tunneling effect through the layer.The Au/KLNPs/Con A/GOx platform showed better electrocatalytic results compared to Au/SAMCys/OLNPs/Con A/GOx due to the efficient coating of KNLPs, which allows an immobilization of a larger amount of enzyme. Moreover, the presence of the lectin Con A favored the correct orientation of the GOx enzyme during the immobilization process, contributing to the enhancement of the electrocatalytic performance of the system. A self-assembly-driven modification strategy consisting of immobilizing three layers of KNLPs/CoA/GOx allowed the construction of a nanostructured film with the maximum catalytic current. This result confirms that LbL technique is a powerful tool to create functional nanocoatings with enhanced properties. These findings hold great application prospects for future applications of lignin nanoparticles in eco-friendly biosensor developments.


In this article, a new manner of cogging a forging (type: shaft), consisting in the application of a two-stage process composed of preliminary shaping in convex anvils, and also principal forging in flat or shaped anvils, is presented. A new manner of forging brought about the formation of favorable conditions for achieving the maximum values of the effective strain in the central part of a forging, accompanied by a simultaneous absence of tensile stresses, which was exerting a favorable influence upon reforging the axial zone of an ingot. What was determined, was the effective geometric shapes of convex anvils; the efficiency of different technological parameters in the case of the intensity of reforging the axial zone of an ingot was analyzed as well. The investigations were complemented by means of predicting the formation of ductile fractures in the course of forging with the application of three different ductile fracture criteria. The comparison of theoretical and experimental outcomes of investigations indicates a good level of being commensurate. The objective of the new forging technologies is to achieve forgings which can boast a very high quality, and that means products having the required shape and dimensional tolerance, and also to achieve such properties and structures of a material which are the most favorable in the aspect of the material in the question’s intended application and functional properties of a product ,2. StreaThe issue of selecting the best possible technological process of forging austenitic steels for the purpose of achieving forgings, which can boast high mechanical properties and which are free of internal defects, constitutes an extremely significant problem ,6. From The issue of the deformability of austenitic stainless steels is a very significant problem in the processes of thermal-mechanical shaping of cogged forgings, and the process of the forging those steels may be, to a significant degree, rendered more difficult by a low level of plastic properties and rapid strengthening of the forgings in question. Nevertheless, in the case of a certain combination of strain, strain rate, and temperature, a good level of deformability is possible to be achieved in the case of the investigated austenitic steel. That fact is assigned to the phenomena of dynamic recrystallization (DRX) and dynamic recovery (DRV), which change the formed microstructure, together with the applied combination of thermal-mechanical parameters. The forming microstructure is closely connected with mechanical properties of the following types: strength, ductility, and fracture toughness ,11.Determining the boundary deformability of a material in the forging processes is a significant issue from the point of view of designing the best possible technologies. One of the directions of investigations into that problem are attempts to determine the properties of the austenitic steel subjected to deformation, the best possible shapes of anvils, and also the parameters of the cogging process, which do not bring about the loss of cohesion by a material and the formation of ductile fractures. The outcome of the conducted investigations demonstrates a significant influence exerted by state of stress, strain rate, temperature, the parameters of microstructures, and also the conditions of friction on the contact surface between a material and a tool, upon the boundary value of the effective strain, after exceeding which material cracking is observed ,13. As tMetal flow, the continuity of internal construction, and the structure and properties of a forged material are determined by the properties of an initial billet, and also, within the scope of forging technologies, are dependent upon the shape and the dimensions of forging tools, and also upon applied technological parameters ,17. AchiIn order to maintain, throughout an entire volume of a forging, a comparatively small gradient of mechanical properties, in the case of simultaneously maintaining high values of them, the forging process ought to be conducted in a manner permitting the axial zone of an ingot to reach the required forging ratio ,21. The The objective of this paper was to analyze a new manner of cogging a forging (type: shaft) made of the X5CrNi18-10 austenitic stainless steel, consisting in the application of a two-stage process: a preliminary shaping of an ingot in convex anvils, and also principal forging conducted in flat or shaped anvils. The outcomes of the investigations into the distribution of the effective strain, effective stress, mean stresses, temperature, stress triaxiality, and also predicting crack formation in the course of plastic shaping in three original special convex anvils, and also in flat and V-shaped anvils, are presented.iF represents surface tractions, iu is the velocity field, S is the surface, V is the volume of a sample, K is a penalty constant (K = 106).In this study, the thermal-mechanical finite element model is used. The solution is based upon a thermo-mechanical approach, combined with solving the Fourier equation for non-stationary heat flows. According to the variational principle, the basic equation for the finite-element formulation is expressed as ,23:(1)δ∏c denotes specific heat, k denotes thermal conductivity, T is the temperature, ρ denotes density, α represents the fraction of mechanical energy converted into heat (assumed to be 0.9).The analysis of heat-transfer can be obtained by solving the energy balance equation, expressed by :(2)k∇2T+nq is the heat flux normal to the boundary surface. The temperature distribution of the forgings can be obtained readily by solving the energy balance equation, Equation (4). The energy equation, Equation (2), can be rewritten using the weighted residual method as:τ is the local flow stress in shear, m is the friction factor, su is the velocity vector of the workpiece relative to the anvil, ou is a very small positive number (10−5) compared to |su|. The frictional boundary condition is given by the vector form :(5)f=−mτδT. The finite element method is then adapted to solve Equations (1) and (4) which define the thermo-mechanical state of a body in the thermodynamic process.Equation (4) as well as the boundary conditions for any arbitrary variation of temperature, f on the interface of the billet and the anvils can be expressed approximately as an arctangent function of its relative sliding velocity su. The contact pressure can be calculated to an approximate value using the correlation of the surface gap and the penalty constant . Computation is carried out until the complete bonding of the two surfaces is achieved. In the processes of forging ingots, most lubrication is not used. In order to maintain similar conditions in the performed compression tests to the conditions prevailing in the experiment, the investigations were performed without lubrication. According to the experimental setup, in numerical simulation, the shear friction factor between the workpiece and anvils equal to 0.7 was chosen in order to reproduce the friction conditions between anvils and the workpiece. In forging experiments, the friction factor was determined from additional ring compression tests. The rigid-viscoplastic thermal-mechanical finite-element method was used in the simulation of metal flow and heat transfer during the cogging process. A three-dimensional model simulation was performed with Deform 3D software . Workpiece was modeled as a free plastic object and it was meshed with about 100,000 tetrahedral elements. In the cogging process of modeling, the contact conditions are constantly updated, reflecting the movement of the anvils and the deformation of the material, which allows to simulate the slip between the anvils and the material of the processing billet. The contact between the anvils and the workpiece is modeled by the shear friction model (Equation (5)). This states that the friction is a function of the shear yield stress of the deforming body. The friction stress φ30 mm × 30 mm were prepared from the as-cast billet. The investigations encompassed the compression of cylindrical specimens on a Japanese cam plastometer . The specimen was subjected to deformation at different temperatures, ranging from 1123 K up to 1473 K, for the following scope of true strains: 0.105–0.693 and for the following strain rates: 0.10–3.0 s−1. All the specimens were heated up to the temperature of 1473 K with a heating rate of 30 K/s, homogenized at that temperature for 160 s, cooled at 10 K/s to different temperatures, held for 30 s and deformed at constant strain rates. All the specimens were subjected to deformation until reaching a true strain of 0.693. Thermocouples were welded on the surface of a compression sample to measure temperature. In −1, as well as for 1273 K and 0.1 s−1, it is observed that, for true strains exceeding 0.2, the flow curves are nearly stable. The flow curves exhibit a broad stress peak. For a larger value of strain rates, continuous flow softening is observed after reaching a peak stress, which may as well be attributed to the dynamic recrystallization (DRX) [The chemical composition of the X5CrNi18-10 stainless steel was as follows (wt. %): 0.07 C, 0.80 Si, 1.55 Mn, 18.05 Cr, 10.15 Ni, 0.025 P, 0.020 S, and Fe in balance. The cylindrical specimens of φ800 mm and the length of 2000 mm; the ingot was made of the X5CrNi18-10 stainless steel. As an initial temperature of an initial billet, the following values were assumed: 1423 K, anvils 523 K, and that means the temperature of heating them in industrial conditions. In order to achieve a good reforging of the axial zone of an ingot, a new effective approach, consisting in the application of convex anvils for the purpose of preliminary forging, was applied. Principal forging was conducted in flat anvils in eight consecutive passes with 90° rotating of the billet, or in V-shaped anvils in four or six passes, determined by the applied reduction ratio , respectively). In the cogging process, the pressing speed of anvil was 20 mm/s. The collation of the applied anvils is presented in In numerical calculations, the following ingot dimensions (after eliminating convergences) were assumed: the diameter of 2K) and the emissivity 0.7 N/(s mm K4) were assumed in order to reproduce the thermal gradient due to the deformation material cooling time [φ80 × 100 mm), six holes were made (diameter: φ1.2 mm), at a homogeneous distance from a frontal plane of a specimen (every 15 mm). In these holes, the sheathed thermoelements (K-type), the diameter of which is 1.0 mm, were made and the lateral surface of cylindrical specimens was insulated. The distribution of temperature in the heated specimen was calculated with the application of the finite element method, whereas the boundary condition on a frontal plane was assumed upon the basis of measurements by experimental means. The achieved outcomes of the experimental investigations were approximated with the application of the following analytical correlations in the function of temperature:Operating temperature at the cogging process for the investigated austenitic steel consists of heat exchange between anvils, workpiece, and environment, as well as the thermal effect of mechanical energy transferred into heat. Heat transfer is carried out by convective and radiative exchange with the environment from the free surface of a workpiece. A heat convection coefficient to the environment equal to 20 W/.Determining the boundary deformability of a material in the cogging process is a significant issue from the point of view of designing a technological process. As the outcome of investigations into this issue, numerous criteria of the formation of ductile fractures were developed. After a preliminary verification of different ductile fracture criteria for predicting crack formation in the course of plastic shaping of ingots made of the X5CrNi18-10 steel, three criteria: the normalized Cockcroft–Latham criterion, Oyane et al. criterion, and Brozzo et al. criterion were assumed. In the models, it was assumed that reaching the maximum possible level of plasticity and formation of a fracture in a material subjected to deformation will be initiated when the determined value for particular criteria exceeds a certain determined boundary value  to the effective stress Oyane et al.  proposeda is a parameter scaling the influence exerted by mean stress upon the boundary deformability of a metal. In Equation (10), σ1), and also by mean stress (mσ), upon the boundary strain of a material in the form of the correlation:In the model of Brozzo et al. , the modφ30.0 mm, and the height of which was varied. The state of stress and strain for compressed specimens was determined with the application of the finite element method, in the case of the application of the DEFORM-3D software. The calculation was conducted for specimens having the slenderness ratio of 0.50–1.50, for four temperatures: 1273, 1323, 1373, and 1423 K, and also variable values of friction factor (0.05–0.70). The values of friction factor assumed for the purpose of calculation were as well determined with the application of the ring compression method. The final height of compression meant the height of the specimen at the moment of observing cracks on a free surface (midway through the specimen height). Having at disposal detailed data on the state of stress and strain, achieved with the application of the finite element method, the following material constants C2 = 0.753 (Equation (10)) and C3 = 0.872 (Equation (11)) were determined.For determining the boundary values for the criteria developed by Oyane et al. and by Brozzo et al., it was attempted to conduct a uniaxial compression of cylindrical specimens made of the X5CrNi18-10 steel, the diameter of which was cε = 0.22, two more passes were conducted). The forging ratio is defined as A0/A, where 0A is the original forging cross-section area before cogging and A is the final forging cross-section area after the cogging process. The schedule of the cogging process, together with the dimensions of the cross-section after particular passes, is presented in In this article, the achieved distributions of the effective strain, the effective stress, mean stresses, and temperature in the cogging process of the X5CrNi18-10 stainless steel in hot forging conditions in three original convex anvils for the purpose of preliminary shaping were analyzed. The further stage of forging was conducted in flat or V-shaped anvils having the following angle: 140° × 140°. Geometric models of anvils and a material subjected to deformation is presented in The specific character of the forging process in convex anvils brings about the fact that local strains on the section of a material subjected to deformation after the first pass a were dicε = 0.22, are presented. The values of the effective strain throughout most of the cross-section reached the following values: T = 40 °C, was observed.In 38 135°, a, ε¯ = 000 150°, a and ε¯ 00 165°, a. In thecε = 0.51, are presented. A two-fold increase in the value of reduction ratio brought about a significant increase in the effective strain for the central part of the cross-section of a forging subjected to deformation in particular convex anvils, and it amounted to: T = 40 °C, was observed.In 25 135°, a, ε¯ = 152 150°, a, and ε¯80 165°, a. In the–114 MPa b. In the–114 MPa d. It wasxT in the function of the shape of convex anvils, and also reduction ratio, is presented in xT in the function of reduction ratio is presented for three investigations into convex anvils having the following angles: 135°, 150°, and 165°. A convex shape of working surfaces of convex anvils brought about the concentration of significant compression stresses in the central zone of a deformation valley in the case of negligibly small areas, not significant for forging technologies, and subjected to the impact of tensile stresses. Stress triaxiality, together with increase in reduction ratio, for particular applied convex anvils, reached larger and larger negative values, favorable for the stress state in a forging subjected to deformation. The most favorable conditions for reforging the axial zones of a forging were achieved in convex anvils having the following angle: 165°. The collation of the course of changes in the effective strain cα =150°, cε = 0.356) in flat anvils, are presented. Those confirmed the occurrence of significant qualitative and quantitative changes in the distribution of the effective strain and effective stress, and also mean stresses. The largest values of the effective strain were observed in the central part of a forging, and they amounted to T = 150–175 °C. In .95–5.47 a. The art anvils . Throughincrease b. The dicα = 150°, cε = 0.356) in V-shaped anvils having the following impression angle: 140° × 140°, in the case of maintaining the identical forging ratio like in flat anvils (4.2), is presented. The largest values of the effective strain were achieved in the axial zone of a forging  in V-shaped anvils having the following impression angle: 140° × 140°, in the case of maintaining the identical forging ratio like in flat anvils (4.2), is presented. The application of preliminary forging with the application of convex anvils having a larger angle and higher reduction ratio brought about a significant increase in the effective strain in the axial zone of a forging reaching the value of T = 100–130 °C. In .18–6.80 a, contriincrease b. The princrease c indicatincrease d. On thexT for particular passes in the case of forgings with selected concavities and subjected to deformations in flat anvils is presented in cε = 0.51 , assumed larger negative values, favorable for the stress state in a forging subjected to deformation. For those parameters of deformation conducted in flat anvils, the most favorable conditions for reforging the axial zones of a forging were achieved.The collation of the courses of changes in the effective strain c = 0.51 a. For thxT for particular passes in the case of forgings with selected concavities, subjected to deformation in V-shaped anvils, is presented in cε = 0.51 , and significantly smaller values of the effective strain were achieved in that case; in addition to that, the shape of concavity is determined. In xT for particular passes in the case of forgings with selected concavities having the following angle: 135° and 165°, and applied reduction ratio cε = 0.22 and cε = 0.51 forged in V-shaped anvils, is presented. Stress triaxiality, together with increase in reduction ratio and the angle of concavities (cε = 0.51 and cα = 165°), similarly to what occurs in flat anvils, assumed favorably negative values. A convex shape of the working surfaces of anvils having the following angle: 165° and a high value of reduction ratio brought about, at an initial stage of deformation, concentrating a significant compression of stresses in the central part of a deformation valley, in the case of negligibly small areas subjected to the impact of tensile stresses. The further forging process conducted in V-shaped anvils contributed to an increase in the concentration of compression stresses in the volume of a forging, and also to eliminating non-favorable tensile stresses situated in the lateral zones of a shaped material.The collation of the course of changes in the effective strain t anvils a. For thcα =150°, cε = 0.356), is presented. What was conducted, was the calculation of the value of selected criteria of cracking, and it rendered it possible to determine the areas exposed to the greatest danger of crack formation. In the axial zone of a forging, the smallest values of crack formation factor were achieved for Oyane et al. criterion (OΨ = 0.250–0.375), whereas the largest values for Brozzo et al. criterion (BΨ = 0.632–0.685). The intermediate values were achieved in the case of the application of the normalized Cockcroft and Latham criterion (C-LΨ = 0.456–0.513). In the case of assuming the normalized Cockcroft and Latham criterion as the basic one, the one by Oyane et al. decreases the values of crack formation factor in the axial zone of a forging by 35.5%, whereas for this zone the criterion of Brozzo et al. gives rise to expecting the values of the crack formation factor increased by 35.9%. Numerical calculation conducted in the case of the application of these criteria demonstrated the same areas where the values of crack formation factors reached or exceeded the determined boundary values for particular criteria. That means that, in a material subjected to deformation, the maximum possible level of plasticity was reached, and the formation of discontinuity is possible. The largest values of the crack formation factors were achieved in the corners of a forging subjected to deformation, and it is also there that positive values of mean stresses are observed  is presented. In the axial zone of a forging, the smallest values of the crack formation factor were achieved for the criterion of Oyane et al. (OΨ = 0.250–0.375), whereas the highest ones for Brozzo et al. criterion (BΨ = 0.550–0.625). The intermediate values were achieved in the case of the application of the normalized Cockcroft and Latham criterion (C-LΨ = 0.500). Assuming the normalized Cockcroft and Latham criterion as a basis, it is ascertained that Oyane et al. criterion decreases the values of the crack formation factor in the axial zone of a forging by 37.6%, whereas for the same zone, the criterion of Brozzo et al. gives rise to expecting the larger values of the crack formation factor (the difference amounts to 17.5%). Numerical calculations conducted in the case of the application of these criteria indicated, in the course of forging in V-shaped anvils, these same areas on the lateral surfaces of forgings, where the values of the crack formation factors reached, or to a non-significant degree, exceeded the determined boundary values for particular criteria. That means that, in a material subjected to deformation, the maximum possible level of plasticity was reached, and that initiating the formation of discontinuity is possible. The largest values of the crack formation factors were achieved in small areas situated on the lateral surfaces of forgings subjected to deformation, and it was there as well that positive values of mean stresses are observed  pass. For concavities, the achieved reduction ratio cε = 0.22, a required number of passes was increased to 6, in which significantly smaller values of the particular analyzed criteria crack formation and their slow increase were achieved. In the axial zone of a forging subjected to deformation in V-shaped anvils, none of the analyzed criteria reached the boundary values.The collation of the course of changes in the crack formation factors for the axial zone of a forging in accordance with three analyzed criteria, and also for particular passes with selected concavities having the following parameters: The comparison of calculated and measured temperature in selected points on the deformed surface of a forging after particular passes is presented in cα = 135°), V-shaped (α = 140° × 140°), and flat.In φ80 × 150 mm and made of the X5CrNi18-10 steel. In the division plane, a square coordination grid (3.0 × 3.0 mm2) was plotted with the precision of 0.01 mm. The situation of process units before and deforming was measured on a measuring microscope coupled with a computerized workstation. The specimens were subjected to deformation on a hydraulic press (pressure: 500 kN). For determining the components of the Cauchy strain tensors, the relocation field of a sixth-degree double-parameter polynomials was approximated, whereas coefficients were determined with the application of the method of least squares, taking under consideration the condition of a constant volume (Lagrange multiplier method). The comparative analysis of effective strain based upon numerical calculations, and obtained by means of experiments, demonstrated their being mutually commensurate to a relatively high degree.Experimental confirmation of the outcomes of investigations into numerical distribution of the effective strain after forging in convex, V-shaped, and flat anvils is presented in , rendered it possible to determine the local values typical in the case of the strain state, stress state, the distribution of temperature, and also the possibility of cracking a material in the form of three selected ductile fracture criteria: the normalized Cockcroft and Latham criterion, Oyane et al. criterion, and Brozzo et al. criterion. In the presented paper, the focus of attention was upon the analysis of a new, two-stage manner of cogging of a forging-type shaft consisting in preliminary shaping of an ingot in convex anvils having convex working surfaces, and also principal forging conducted in flat or shaped anvils. The new manner of forging brought about the formation of favorable conditions for achieving the maximum values of the effective strain in the axial zone of a forging, accompanied by a simultaneous absence of tensile stresses, which was improving reforging and quality of achieved forgings. What was determined, was the effective geometric shapes of convex anvils; the efficiency of different technological parameters in the case of intensity of reforging the axial zone of an ingot was also analyzed. It was recommended to conduct preliminary shaping in convex anvils having the following angle: 165°, and the depth of achieved concavities ought to be matching the reduction ratio of cε ≥ 0.356. Taking under consideration the achieved values of the effective strain in the axial zone of a forging and the distributions of mean stresses, the further forging process can be conducted in flat anvils, or in the V-shaped ones. Numerical investigations, confirmed by selected experimental researchcα, and also reduction ratio cε, demonstrated significant influence exerted upon the values of particular criteria in the axial zone of a forging. In the axial zone of a forging for the forging ratio amounting to 4.2, none of the analyzed criteria reached the boundary values.The course of changes in certain values of particular criteria in the course of forging in different investigated anvils, with different values reduction ratios, informed about the values of criteria in particular zones of plastic deformation, and rendered it possible to the situations and phases of deformation, in which the loss of cohesion of a material subjected to deformation is possible. In further passes, an increase in the total values of the particular crack formation factors was observed, whereas the largest values were achieved for Brozzo et al. criterion, and the lowest ones for Oyane et al. criterion. The intermediate values were achieved in the case of the application of the normalized Cockcroft and Latham criterion. The shape of concavity achieved by means of deformation in convex anvils and a certain angle