Welding via bond exchange reactions has provided advances in obtaining high-quality joining performance. However, the reported welding method requires a relatively high press force, and challenges are still encountered in welding hard vitrimer. In this work, a facile surface depolymerization strategy was introduced to weld high-performance epoxy vitrimer. The vitrimers were firstly dissolved into ethylene glycol for depolymerization based on the solvent-assisted bond exchange reactions. Then, the depolymerized vitrimers were welded under heat and press force. The effect of the depolymerizing time, welding pressure, welding temperature and welding time on the welding strength were further investigated. It was found that there were optimal values for the depolymerizing time, welding pressure, and welding temperature, respectively, for the welding strength, while the welding strength increased with increasing welding time. Through facile surface degradation, the welding pressure was highly reduced, while the welding strength was increased. With surface depolymerization, the welding strength was 1.55-times higher, but the magnitude of press force was 1/1000-times than that with no surface depolymerization. It is elucidative that surface depolymerization can be used to weld hard vitrimer composites alongside reducing the press force effectively.  However2) . To dateEpoxy vitrimer is intrinsically composed of ester bonds and hydroxyl groups, rendering bond exchange reactions (BERs) attainable, which makes it possible for them to be welded from a bulk state or ground powder state. Yu et al. ,18,19 usAdditionally, attempts have been made to lower the press force for welding epoxy vitrimer, where BERs were triggered between hydroxyl groups in alcohol solvent  and esters in the epoxy network ,23,24. TIn this paper, solvent-assisted BERs, which have been used for recycling rather than welding, are used to weld hard epoxy vitrimer. Ethylene glycol (EG), which offers hydroxyl groups, reacts with ester bonds of epoxy vitrimer reversibly. The vitrimer is firstly depolymerized and then forms a gel layer, in which the depolymerized polymer can diffuse across the welding interface easily. This will highly reduce the welding pressure. On the other hand, the depolymerized polymer can be repolymerized in the welding region, resulting in strong covalent bonds at the welding interface. This paper is organized as follows. In The polymer network of vitrimer can be depolymerized into small segments, and further be repolymerized thanks to the reversibility of solvent-assisted BERs ,23,24. A2, Sigma Aldrich, St. Louis, MO, USA), and ethylene glycol .Diglycidyl ether of bisphenol A , glutaric anhydride , zinc acetylacetonate hydrate (Zn(ac)2 to the epoxy groups was added into melted DGEBA and stirred powerfully at 130 °C for 10 min. Then, the melted GA was poured into the mixture with a stoichiometric ratio of epoxy groups and acyl groups of 1:1. The above mixture was poured into home-made molds to cure at 140 °C for 12 h after undergoing vacuum degassing at 100 °C for 15 min. The mechanical and thermal properties of the epoxy vitrimer are presented in Hard epoxy vitrimer was prepared with the following synthesis procedures: DGEBA and GA in solid states were heated at 130 °C until melted. Then, 5 mol % Zn(ac)An appropriate depolymerization condition for welding was roughly obtained by a dissolving experiment for epoxy vitrimer. In our previous work , the disT1 for a depolymerizing time of t1 in a vacuum oven  with a tensile rate of 1 mm/min. The force and crosshead displacement were recorded. The strain was calculated as the displacement divided by the initial distance of the crosshead. The stress was calculated as the force divided by the welding area in the undeformed state. Thus, the welding strength of epoxy specimens was calculated by:The surface morphologies of the welded region were investigated by a 3D microscope system .The depolymerizing time for welding was determined by the dissolving experiment of cubic epoxy vitrimer into EG solvent at 180 °C, as shown in After partially dissolving, the depolymerized vitrimer was assumed to contain three parts: the fresh network, the swelled network, and the depolymerized network, as shown in To quantitatively provide criteria for optimizing the depolymerizing time, we welded the depolymerized vitrimer and compared their welding strength. At the beginning of the welding, two pieces of the depolymerized vitrimer specimens are connected to each other a. The deAfter being depolymerized for 20 min, 30 min, 40 min, and 50 min, respectively, the depolymerized vitrimer were welded at 180 °C under 20 kPa for 12 h , the welding strength using surface depolymerization is 35 MPa under 20 kPa, while that using hot-pressing welding is only 21.9 MPa. That is, the welding strength using surface depolymerization is 1.55-times higher, but the magnitude of press force is 1/1000. This work provides an example of using surface depolymerization to develop welding methods for vitrimer composites. Nevertheless, these are comparatively high welding temperatures determined by the boiling point of EG. Therefore, it is worth further exploring solvents with low boiling points.


As no reliable diagnostic methods are available, gallbladder cancer (GBC) is often diagnosed until advanced stages, resulting in a poor prognosis. In the present study, we assessed whether volatile organic compounds (VOCs) could be used as a diagnostic tool for GBC. The VOCs in bile samples collected from 32 GBC patients were detected by gas chromatography-ion mobility spectrometry (GC-IMS), and 54 patients with benign gallbladder diseases (BGD) were used as controls. Both principal component analysis and unsupervised hierarchical clustering analysis gave a clear separation of GBC and BGD based on the bile VOC data collected from GC-IMS. A total of 12 differentially expressed VOCs were identified, including four upregulated  and eight downregulated  in GBC compared with BGD. ROC analysis demonstrated a 12-VOC panel con-structed by four machine learning algorithms, which was superior to the traditional tumor marker, CA19-9. Among them, support vector machines and linear discriminant analysis provided the highest AUCs of 0.972, with a sensitivity of 100% and a specificity of 94.4% in the diagnosis of GBC. Collectively, VOCs might be used as a potential tool for the diagnosis of GBC. Gallbladder cancer (GBC) is one of the most common malignant tumors of the biliary system in Eastern Asia with high mortality . In 2020Metabolomics is one of the most promising approaches for identifying biomarkers of disease and increasing understanding of metabolic processes in cancer . As an iIn the present study, we performed GC-IMS to obtain the metabolomic profiling of VOCs in bile from patients with GBC and BGD. Moreover, we aimed to develop a volatile biomarker panel that could act as a minimally invasive approach for the early detection of GBC. To the best of our knowledge, we, for the first time, showed that VOCs could be used as bile biomarkers for the diagnosis of GBC.In the present study, patients who were older than 18 years and histologically diagnosed with GBC were recruited from 2018 to 2021 in Qilu Hospital of Shandong University. Inclusion criteria were set as follows: 1) patients without any history of other malignant tumor or anti-cancer therapy, 2) patients who were cooperative with supplying fresh bile sample and complete medical records, and 3) patients who underwent radical resection and reported GBC by pathological examination. Patients with BGD, such as cholecystitis and gallbladder polyps, who met the above-mentioned conditions except for the pathologically reported GBC, were included as controls. The experimental scheme was approved by the Ethics Committee of Qilu Hospital of Shandong University, and the informed consent were got from each patient.Bile samples were collected when the patient was first treated with endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiodrainage (PTCD). The collected specimens were centrifuged at 3,000 rpm for 10 min at 4°C. The supernatants were aliquoted and stored at -80°C immediately.VOC profiles from bile samples were detected using GC-IMS . All samples underwent the same procedure. Briefly, 0.5 mL bile was placed in each headspace bottle and incubated at 80°C for 10 min. Subsequently, 1 mL headspace gas was extracted for analysis. Nitrogen was used as the carrier air. The IMS drift gas was always maintained at 150 mL/min, while the initial flow rate of the carrier air was maintained at 2 mL/min for 2 min, and then it was linearly increased to 150 mL/min in 10 min. Other major experimental parameters were as follows: drift tube temperature: 45°C; gas chromatography column temperature: 60°C; inlet-chromatography column converter temperature: 60°C; column-migration tube converter temperature: 45°C; ion mode: positive ion mode. Each analysis was conducted in triplicate.The software R (x64 3.6.2) and the software package “ggord” were used for principal component analysis (PCA). The level of each VOC was compared with Mann–Whitney U test. The area under the curve (AUC) was calculated on the receiver operating characteristic (ROC) curve and compared using MedCalc 9.3.9.0. The analysis of machine learning was carried out using Matlab R2016a  based on the Statistics and Machine Learning Toolbox. Based on identified VOCs, decision tree (DT), support vector machines (SVM), linear discriminant analysis (LDA), gradient enhancement machines (GBMs), and K-nearest neighbor (KNN) were used for classification. Hierarchical 10-fold cross-validation was used to optimize the parameters of the training cohort.A cohort consisting of 86 patients with definite pathological diagnoses, including 32 GBC patients  and 54 BGD patients , were included in the present study. Moreover, 70% recruited subjects were randomly selected as a training cohort (n = 24 GBC and n = 36 BGD), while the remaining 26 samples (n = 8 GBC and n = 18 BGD) were set as a test cohort. There were no significant differences between the two cohorts in terms of age, sex, and some biochemical indexes. Room-temperature stability was assessed in the same samples that were measured in parallel every hour within 12 h, with a total of 12 injections. The VOC was characterized by the molecular gas chromatography preservation index, and the migration time of molecular ions was measured and quantified according to the signal peak strength. For each sample, we would generate the 3D data   with a GC-IMS library, a total of 45 VOC peaks were manually selected based on retention index and migration time in all patients. These species (peaks) included 19 defined substances and six unknown substances -2-octenal, (E)-hept-2-enal, (E)-2-hexenal, (E)-2-pentenal, cyclohexanone, and acetophenone were robust in distinguishing GBC patients from BGD patients, with AUCs>0.75  were used to construct diagnostic models. There are still numerous challenges for us to more accurately diagnose GBC. In the present study, we explored the potential role of GC-IMS in the detection of GBC and thus reported several meaningful findings. First, GC-IMS could discriminate between bile samples collected from patients with GBC and BGD. Second, we identified 12 specific VOCs, which might play a relevant role in assessing GBC. Third, the model based on the VOC profile allowed for accurate discrimination between GBC and BGD groups. However, this observation was limited to a small cohort of patients.There is increasing interest in the application of VOCs in exhaled breath for diagnosing a variety of cancers , 18. Sin-9 or less without enrichment and concentration. Meanwhile, the detection time is shortened to 10 min, which significantly improves the simplicity of detection operation, detection time, and efficiency. In contrast, traditional methods usually need to concentrate the samples, and the detection process can last more than 1 h. It was worth noting that the advantage of this study was to explore the experimental conditions by using orthogonal experiments and to test the effects of room temperature and repeated freeze-thaw cycles on VOCs. The sample was relatively stable within 12 h at room temperature, which facilitated the detection.Although GC-IMS has been first used in the detection of bile VOCs, it has been successfully implemented feasibly into others. Maxine and her colleagues have observed a significant difference in fecal VOC profiles using GC-IMS between coeliac disease and refractory coeliac disease . Based oIn this study, 12 specific VOC molecules linked to GBC were identified. Some of them have also been suggested as potential biomarkers in breath or stool for other diseases. For instance, cyclohexanone is associated with colorectal cancer , breast At present, the diagnosis of GBC mainly depends on the clinical manifestations of the disease, CT, B-ultrasound, and other imaging examinations, while these approaches are too subjective, and there are too many external interference factors. Serum CA19-9 test is one of the few non-invasive markers for clinicians to make a preliminary diagnosis , 39. HowIn the present study, our sample size was small, so it was difficult to avoid bias. Therefore, a large sample size and multicenter study should be carried out to further demonstrate the existing data. Meanwhile, it is necessary to further explore the relevant mechanism between the production of endogenous VOCs and the occurrence and development of GBC or BGD. This study provided an experimental basis for the application of VOC analysis in GBC and made it possible to be used in the early diagnosis of GBC, which had an extremely broad application prospect.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Qilu Hospital of Shandong University. The patients/participants provided their written informed consent to participate in this study.Conceptualization, XZ, YiZ; methodology, XG, LZ; validation, XG, JG; investigation, YaZ, QL; data curation, JJ, JG; writing—original draft preparation, XZ; writing—review and editing, YaZ; visualization, LZ; supervision, YiZ; funding acquisition, XZ, JJ, YiZ. All authors have read and agreed to the published version of the manuscript.This research was funded by National Natural Science Foundation of China, 82172339 and 81972005, Natural Science Foundation of Shandong Province, ZR201910220159, Key Research and Development Program of Shandong Province, 2019GSF108064 and 2021CXGC011105, and Shandong Medical and Health Technology Development Project, 2018WS327.Authors LZ and JG were employed by Hanon Advanced Technology Group Co.,Ltd,.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Fluorescent fibers are capable of discoloration behavior under special light sources, showing great potential for applications in biomedicine, environmental monitoring, heavy-metal-ion detaction, and anti-counterfeiting. In the current paper, temperature-sensitive fluorescent poly-acrylamide (PAM) nanofiber (AuNCs@PAM NF) membranes are prepared by mixing red fluorescent gold nanoclusters (AuNCs) synthesized in-house with PAM using the electrospinning technique. The AuNCs@PAM nanofibers obtained using this method present excellent morphology, and the AuNCs are uniformly dispersed in the fibers. The average diameter of the AuNCs@PAM NFs is 298 nm, and the diameter of AuNCs doped in the fibers is approximately 2.1 nm. Furthermore, the AuNCs@PAM NF films present excellent fluorescence and temperature-sensitive performance between 15 and 65 degrees. While under the 365 nm UV light source, the fiber film changes from white to red; this discoloration behavior weakens with the increase in temperature, and changes from deep to light red. Therefore, the approximate temperature can be identified using the color change, and a visual temperature-sensing effect can be achieved. The dual functions of temperature-sensitivity and fluorescent properties improve the scientificity and safety of nanofibers in the use of anti-counterfeiting technology. The fiber can emit pure red light and has good luminous properties. Although melt-spinning is widely used due to its low cost and advanced technology, its disadvantage is the high temperature required for spinning, which restricts its application. In comparison to melt-spinning, the conditions required for wet-spinning are simple. Lu et al. [2 to coat the surface of Fe3O4 nanoparticles, and then, modified using a low-surface-energy fluorinated silane-coupling agent to prepare Fe3O4@SiO2@POTS. It was mixed with a PVDF solution in its preparation for spinning, and a dual-scale-structure nanofiber membrane was constructed, which improved the surface roughness of the composite membrane and presented the membrane with excellent superhydrophobicity, superparamagnetism, and tensile strength. In addition, electrospinning has been used to fabricate inorganic, organic, and hybrid (organic/inorganic) compound nanofibers with unlimited application potential [2+-sensing process. AuNCs have attracted considerable attention from researchers [Fluorescent fiber is commonly referred to as a “functional nanometer material”; its typical feature is that it can emit a special color under irradiation of an excitation light source of a specific wavelength. When the special light source disappears, the fiber can restore its own color. Thus, to date, fluorescent fibers have attracted widespread attention based on their potential application in different fields because of their unique photochromic properties ,2,3. Genu et al.  used theu et al. . With thu et al. ,12,13. Wu et al.  used SiOotential ,16. Amonotential ,17,18. Sotential  preparedotential  presenteearchers ,22 due tearchers ,24,25, aearchers  developeearchers  composedOn this basis, temperature-sensitive AuNCs were easily synthesized by using BSA as a reducing agent and stabilizer, and exhibited good fluorescence properties under a UV lamp. AuNCs@PAM NFs exhibiting fluorescence properties and temperature-sensitivity were prepared by being mixed with PAM via electrospinning, and their morphology and properties were characterized and tested. AuNCs were successfully doped into the nanofibers. AuNCs@PAM NFs presented consistent and excellent temperature-sensitive fluorescence performance with AuNCs, and the red fluorescence color gradually lightened with increasing temperature. The bifunctional nanofibers used in this study can improve the science and security of anti-counterfeiting technology, and can be broadly applied in fluorescent anti-counterfeiting technology.3+ to Au0.Firstly, 10 mL of 10 mM chloroauric acid  solution was added to 10 mL of 50 mg/mL BSA  solution, and stirred vigorously for 2 min in a water bath at 37 °C. Then, 1 mL of 1 M NaOH  solution was added and continuously stirred for 12 h at 37 °C to produce a brown-gold nanocluster solution ,29, whicA total of 5 g of AuNC solution was magnetically stirred in a beaker with 14.6 g of deionized water (σ ≤ 0.1 us/cm), and 0.4 g of PAM  particles were weighed and slowly added to the beaker, and stirred at room temperature for 14 h. AuNCs@PAM NFs were prepared using the electrospinning technique; the voltage was 13 Kv, the receiving distance was 27 cm, and the injection-pushing speed was 0.03 mm/min. The spinning was carried out at room temperature and the relative humidity was 35%.Scanning electron microscopy (SEM) and a fluorescence microscope BX53 were used to characterize the morphologies of the nanofibers. The inner lattice structure and morphologies of the AuNCs were characterized using transmission electron microscopy (TEM) . The diameters of the AuNCs and nanofibers were measured using Nano Measurer software. X-ray photoelectron spectroscopy  was performed to validate the elemental properties of the AuNCs. The crystal structure information of the AuNCs in the AuNCs@PAM NFs was measured via X-ray diffraction . A fluorescence spectrometer (RF-6000) was used to measure the fluorescence properties. The UV lamp model previously mentioned in this study was a tri-purpose ultraviolet analyzer (ZF-1), which uses light with a wavelength of 365 nm.7/2 of the AuNCs are deconvoluted into two distinct components centered at 83.28 and 84.88 eV, corresponding to Au0 and Au+, respectively; this indicates that Au is present in two chemical environments [0 constitutes the gold core of the AuNCs, while Au+ covers the surface of the AuNCs. Taken together, the above results indicate the successful preparation of the AuNCs.The photographs of the AuNC solution under visible light and UV lamp irradiation are presented in ronments . Among tThe fluorescence tests of the AuNC solution were performed at different temperatures, as illustrated in The successfully prepared AuNCs were doped in a PAM solution to prepare the electrospinning solution, and the AuNCs@PAM NFs were prepared via high-voltage electrospinning. Considering that the properties of the polymer-spinning solution play a crucial role in the morphology and performance of the nanofiber membranes, we conducted further studies on the spinning solution concentration. 3) [This may have been due to the fact that when the PAM concentration was low, the distance between the molecular bonds in the polymer PAM molecules was greater, and the intermolecular adhesion was lower. As the PAM concentration increased, the probability of the interpenetration and entanglement of the bonds between the PAM molecular chains increased, and the viscosity of the spinning solution gradually increased, too. At the same time, the conductivity of the spinning solution increased with the gradual increase in the PAM mass fraction, which was due to the fact that PAM particles are water-soluble solid materials; additionally, the conductivity in the aqueous solution increased with the increase in the concentration of the dissolved solids. The relationship between the conductivity and dissolved-solid concentration was approximated as μS/cm = 1 ppm or 2 μS/cm = 1 ppm (per million units of CaCO3) ,34. In aWith a fixed spinning voltage of 13 Kv and a receiving distance of 27 cm, the nanofibers formed by solutions of different concentrations are presented in the inset of The comparison photographs of AuNCs@PAM NFs prepared with a mass fraction of 2% under visible light and a UV lamp with a wavelength of 365 nm are presented in The letters “TGU” were removed from the prepared AuNCs@PAM NFs, placed on the surface of the PAM NFs, and observed under visible light and a UV lamp, as shown in In this paper, temperature-sensitive AuNCs with a diameter of about 2.1 nm were easily fabricated using BSA as a reducing agent and stabilizer, and presented excellent fluorescence properties under a UV lamp. The red fluorescence color of the AuNCs was deepest at 15 °C, and the color gradually decreased with increasing temperature. By doping AuNCs into PAM, AuNCs@PAM NFs were successfully prepared via the traditional electrospinning technique with an average diameter of 298 nm, and were characterized using TEM, SEM, XRD, fluorescence microscopy, and UV. AuNCs@PAM NFs presented excellent temperature-sensitivity properties and an obvious fluorescence effect under a UV lamp, indicating their potential for the application of fluorescent fibers in heating stickers, temperature-sensitive responses, and anti-counterfeiting materials.


This review will focus on antifungal agents which contain at least one guanidine group, for the treatment of human-related fungal pathogens, described in the literature between 2004 and 2022. These compounds include small molecules, steroids, polymers, metal complexes, sesquiterpenes, natural products, and polypeptides. It shall be made clear that a diverse range of guanidine-containing derivatives have been published in the literature and have antifungal activity, including efficacy in  Fungal infections are a growing problem in healthcare, and are associated with high mortality rates ,2. The a1), the stomach acid reduction drug famotidine (Pepcid), (2) and the muscle relaxant tizanidine (Zanaflex) (3), The guanidine group is found in a broad range of drugs, including the anti-influenza drug zanamivir (Relenza) (anaflex) , 3]. TheGuanidine-containing compounds can also be isolated from natural sources ,7, and nMonanchora unguifera, [1.2.4.6] tetrazepine-3,7-dione ring system, . The gua27), Mucor, Aspergillus niger, and Fusarium solani strains, with activities of 53–76% inhibition. The non-complexed ligand of (27) showed lower activity against these strains (37–61% inhibition), indicating that the complexation was having a slightly beneficial effect on the antifungal activity. An LD50 of 779 μg/mL was found for (27) in brine shrimp. It is not clear that these complexes have a path forward towards greater activity and efficacy in vivo.A series of guanidine ligands, and the homoleptic copper (II)) complexes thereof were evaluated for their antifungal activity by Badshah et al. in 2012, . Eight c28) and (29), 28) and (29) showed IC50, MIC, and MFC values between one and ten-fold higher than those found for the positive control amphotericin B, versus three Candida strains, Cryptococcus neoformans, and Aspergillus fumigatus. Analogs (28) and (29) display good activity, particularly against Candida krusei, and Cryptocossus neoformans (~3 μM) and it would seem that the next step with these derivatives would be to test them in in vivo experiments against candidiasis and/or cryptococcosis.Tricyclic guanidine analogs of batzelladine K, a polycyclic marine alkaloid, were synthesized and evaluated for their antifungal activity by Bhutani and coworkers in 2013, . Fifty dXenorhabdus cabanillasii, named cabanillasin (30), 30) was shown to be highly active against strains of Candida albicans (IC50: 0.78 μg/mL), Candida lusitaniae (IC50: 1.56 μg/mL), Candida glabrata (IC50: 6.25 μg/mL), and Candida krusei (IC50: 6.25 μg/mL). At a 48 h timepoint, cabanillasin was found to have only low activity against the filamentous fungi Aspergillus fumigatus and Rhizopus oryzae. Cytotoxicity testing revealed that the LD50 of (30) in human prostatic carcinoma cells (PC-3) was 47 μg/mL, whilst in human normal mammary epithelial cells (hTERT HME-1) the LD50 was 25 μg/mL. With cabanillasin showing good in vitro potency and lower cytotoxicity, it would be valuable to have the compound tested in vivo, for example in a Candida albicans experiment.A metabolite isolated from Fusarium moniliforme, Aspergillus fumigatus, and Aspergillus flavus. Three of the compounds, 31), (32), and (33) of 65–96%. The other five complexes were found to be appreciably less effective. The authors speculate that the presence of electron-withdrawing groups on the aryl ring, as seen in (31), (32), and (33), causes a decrease in the basicity of the guanidine, and an increase in the lipophilicity of the compounds, which is beneficial to antifungal activity. It is noteworthy that the three analogs shown in Following on from their previously described work on the copper (II) complexes of guanidine-containing ligands , ferroce34), 34) was tested for its EC50 against human erythrocytes, which was found to be 175 μg/mL. The performance of compound (34) was analyzed in specific candidiasis and onychomycosis studies. In the candidiasis study, the compound was compared to terbinafine in an assay utilizing human skin. A 2% hydrogel formulation of (34) produced significant reduction in ATP levels against Candida albicans compared to untreated references. The compound also showed a significant increase in treatment efficacy compared to terbinafine. In the onychomycosis study, (34) was tested versus amorolfine as positive control and deionized water as placebo. The study, run over 48 h, demonstrated that compound (34) lowered recovery of ATP from nail samples, compared to the infection control and placebo. Four nail penetration enhancers were also investigated in conjunction with (34), and it was found that MedNail was the most effective, producing baseline recovery of ATP comparable to the non-infected controls. Given that (34) was selected as a development candidate by Lytix Biopharma, and is being investigated by Amicoat and Pharma Holdings, it would be expected that the compound has a good overall profile, and with its efficacy in both candidiasis and onychomycosis experiments, the compound is highly interesting.In 2016, Svenson et al. reported their studies on short cationic antimicrobial peptides, . The gro35), 35) was evaluated in a time-kill experiment, which showed that at 12 h of incubation there was a pronounced fungistatic effect on Candida albicans. The derivative was next tested for its effect on Candida albicans membranes using a concavaline/propidium iodide (PI)/4′,6-diamidino-2-phenylindole (DAPI) cell staining assay. The result of the assay showed that upon treatment with (35), membrane damage and cell death were observed in 20% of the cells, and that the treatment did not induce apoptosis. Compound (35) was also shown to disperse preformed Candida biofilms. The site of action of (35) was interrogated by the effect of the compound on sheep red blood cells, and it was determined that the compound did not act as a membrane disruptor as its primary mechanism. In vivo embryotoxicity was determined using zebrafish, which showed that the compound was less embryotoxic and teratogenic than the azole drug voriconazole. Overall compound (35) looks to be an interesting antifungal agent. The information that the compound is not a membrane disruptor is useful, but it would be more valuable to be able to understand how the compound exerts its effect.In 2016 Opsenica and coworkers described thiophene-based guanylhydrazones as antifungal agents, . The gro35), and tested them against four Candida strains. The results showed that compound (36), 36) showed the lowest cytotoxicity against healthy human fibroblasts (MRC5) of the three analogs (40 μg/mL). The potential of compound (36) to interact with chromosomal DNA from Candida albicans was investigated, with the result showing that the compound did appear to interact with DNA, for which additional evidence was presented though the use of circular dichroism spectral analysis, which showed that the compound significantly perturbs the conformation of double stranded template DNA (dsDNA). The DNA-specific probe DAPI was utilized to investigate the pro-apoptotic effect of (36). The result of this work showed that there was a significant decrease in the intensity of the blue color associated with the DNA-DAPI complex in the Candida albicans cells treated with the MIC of compound (36), indicating perturbation of the conformation of the dsDNA. The reactive oxygen species (ROS) generation and accumulation caused by (36) was found to be significantly lower than for amphotericin B. The synergism between (36) and known antifungal agents was investigated, which showed that (36) and amphotericin B acted synergistically against Candida albicans and Candida parapsilosis, but that (36) did not act synergistically with nystatin or itraconazole against these two Candida species. As only three additional derivatives were synthesized this only provides a small sample size to select from. It would be instructive to see more analogs made, preferably of (36) as it is the superior compound, to incorporate substituents on to the phenyl ring, incorporate substituents on to the furan ring, phenyl replacements such as pyridyl and thiophene, and furan replacements such as oxazole and oxadiazole.In 2018 the same group published on additional bis-guanylhydrazones as effective agents for the treatment of candida infections, . The groCandida albicans and Aspergillus niger, and the two most interesting compounds were (37) and (38), 37) and (38) produced zones of inhibition for Candida albicans of 40 and 41 mm, respectively, and for Aspergillus niger the zones of inhibition were lower, 21 and 25 mm respectively. The MIC values for Candida albicans and Aspergillus niger were also calculated- for Candida albicans the MIC values were 3.125 μg/mL for (37) and 1.56 μg/mL for (38), and for Aspergillus niger the MIC values were lower at 12.5 μg/mL for both (37) and (38). The MIC values calculated for Candida albicans and Aspergillus niger for (37) and (38) are modest, and suggest the need for additional SAR investigation- changes such as additonal SAR on the aniline phenyl ring, substitution on to the benzoyl group, replacement of the benzoyl group, and replacement of the benzothiazole all appear to be reasonable approaches.In 2016 a set of thirteen guanidine -containing benzothiazole analogs were synthesized and tested by Bhat and Belagali, . The com39), Candida albicans. The concentration of (39) needed to lyse 50% of rabbit red blood cells (HC50) was 48 μg/mL, showing selectivity for the fungal species. Compound (39) was further tested against four strains of Candida albicans, four strains of Fusarium solani, and five Aspergillus species. The results showed that (39) had MIC values of 0.78 μg/mL for the Candida albicans strains, 3.13 μg/mL for the Fusarium solani strains, and 3.13–6.25 μg/mL for the Aspergillus species. Compound (39) was tested in combination with six known antifungal agents, and there was synergy observed with only one, terbinafine. A time-kill kinetics experiment was carried out for (39), which showed that the compound had a rapid fungicidal effect on a strain of Candida albicans. The cytotoxicity of (39) was evaluated, and it was found that the IC50 was 64.1 μg/mL in human corneal fibroblasts. The mechanism of action of (39) was assessed using the DNA-binding dye Sytox Green, which readily penetrates compromised membranes of dead cells, but cannot cross the intact membranes of living cells. This experiment suggested that compound (39) was killing fungi by disrupting the fungal cell membrane. Finally, an in vivo experiment was carried out with (39), in a murine fungal keratitis experiment with a Candida albicans strain. The compound was dosed as a 0.2% solution, and was given over a six hour period. Relative to untreated controls, compound (39) produced a 93% reduction in fungal burden in the eye. With excellent MIC values against Candida albicans, synergism with terbinafine, low cytotoxicity, an implied mechanism of action, and in vivo efficacy, compound (39) has one of the most complete profiles of the antifungal agents presented. It would be interesting to have investigated if the derivative could also prove efficacious in other in vivo models, such as disseminated candidiasis.Semisynthetic analogs of α-mangostin were described as possessing antifungal activity by Liu et al. in 2017 . The gro40), Candida albicans, Candida auris, and Aspergillus fumigatus, the group found that the most active agent was alexidine dihydrochloride. The group also tested the top six compounds from the original screening for their ability to inhibit biofilm formation, and showed that only two compounds, alexidine dihydrochloride and the organomercury agent thimerosal , 57) was tested against strains of Aspergillus flavus, Candida albicans, and Candida glabrata. (57) was found to be fungicidal for all three strains, with MIC values of >80–150 mg/mL, comparable to the reference standard antifungal agents employed, amphotericin B and miconazole. The scientists carried out molecular docking studies with the four polypeptides for their interactions with lanosterol 14-α demethylase , for such cyclic polypeptides to be able to progress further it would seem that additonal analogs need to be synthesized and tested to discover more potent derivatives.Four cyclic polypeptides were described as antifungal agents by Jadeja et al. in 2022 . The anaMonanchora clathrata and Monanchora unguiculata, , polypeptides (34), and (54), and polymers (56) all display in vivo efficacy. Among these, perhaps the most interesting agents are macrocycle BM1 (11), abafungin-related analog (53), and polymer (56), due to the large amount of positive data collected for each compound, coupled with in vivo antifungal efficacy. Another agent of high interest is (48), a GlcN-6-P synthase inhibitor, as this agent is known to have a novel mechanism of action. It is hoped that in time there will be a guanidine-containing analog that progresses in to clinical evaluation for antifungal treatment, and that one day there will be an approved antifungal agent that contains a guanidine functionality which can benefit patients with fungal diseases, perhaps (11), (34), (53), or (56).As can readily be discerned, there have been a plethora of guanidine-containing compounds disclosed between 2004 and 2022 as having antifungal activity against human related fungal pathogens, both


Phosphatase and tensin homolog (PTEN) is one of the highly susceptible genes to breast cancer (BC); however, the role of PTEN-related RNAs in BC remains poorly understood. Understanding the effect of PTEN-related RNAs and their mechanisms may be helpful to clinicians. We screened the differentially expressed RNAs (deRNAs) related to PTEN and established the competitive endogenous RNA (ceRNA) network by integrating several databases. After that, the RNA model, prolactin receptor (PRLR)/calcium voltage-gated channel auxiliary subunit alpha2delta 1 (CACNA2D1), was obtained by KM survival analysis and logistic regression analysis. Finally, mutation, methylation, functional enrichment, and immune correlation were analyzed to explore the roles of these RNAs. Our results showed that PRLR might be harmful to BC, while CACNA2D1 might be beneficial to BC. Furthermore, the abnormal expression of PRLR in BC might result from mutation and hypomethylation, while the aberrant expression of CACNA2D1 might be ascribed to methylation. Mechanistically, PRLR might affect the prognosis of BC by inhibiting the expression of immune checkpoints, while CACNA2D1 might improve the prognosis of BC by increasing the immune cells infiltrating into BC and up-regulating the expression of immune checkpoints. The abnormal expression of PRLR and CACNA2D1 in BC is closely related to the prognosis of BC, and they may serve as targets for the treatment of BC. In 2020, breast cancer (BC) surpassed lung cancer to become the highest incidence cancer world-wide . While mPublic databases, such as The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), provide clinical data and molecular information to facilitate the research of various cancers. Meanwhile, high-throughput RNA sequencing, gene mutation analysis, methylation analysis, and immune microenvironment analysis have been extensively employed in cancer research . In receAs a common tumor suppressor gene, phosphatase and tensin homolog (PTEN) inhibits the proliferation of cancer cells by down-regulating the PI3K/AKT/mTOR pathway ,7, and iOn the basis of prior studies, we constructed a competitive endogenous RNA (ceRNA) network related to PTEN in BC, and then analyzed a KM survival, functional enrichment, and PPI network on the RNAs in the network. Then, the obtained RNAs linked to BC prognosis were subjected to logistic regression, thereby establishing a BC prognosis model containing prolactin receptor (PRLR) and calcium voltage-gated channel auxiliary subunit alpha2delta 1 (CACNA2D1). Furthermore, the model was evaluated by an internal test set. Then, a correlation analysis showed that both PRLR and CACNA2D1 were related to PTEN, which might be involved in the pathogenesis of BC. Finally, the potential biological functions of PRLR and CACNA2D1 in BC were examined by functional enrichment analysis, and the possible roles of PRLR and CACNA2D1 in BC were studied by analyzing mutation, methylation, and immune correlation . The stuhttps://portal.gdc.cancer.gov/, accessed on 21 December 2021), and RNA and miRNA sequences were obtained from IlluminaHiSeq_RNASeq and IlluminaHiSeq_miRNASeq sequencing platforms. All data could be freely downloaded. In terms of the median PTEN expression, 1059 BC samples were divided into PTENhigh group (n = 529) and PTENlow group (n = 530). Against the requirements of data integrity, the samples with missing items were excluded. Finally, data of 1000 patients were obtained from BC samples, and the caret package was further randomly allocated to the training set and the test set at a ratio of 7: 3. In addition, the GSE21422 data set was downloaded from GEO database  to verify the expression of RNAs in the model.Data on RNA expressions and clinical features of BC samples and adjacent tissues were downloaded from TCGA database .The criteria for eligible deRNAs in BC samples and normal samples were as follows: mRNA with a http://jvenn.toulouse.inra.fr/app/example.html, accessed on 10 April 2022), and the targeted relationships of the identified RNAs were predicted by utilizing miRcode , TargetScan , and miRTarBase  databases. Then, the protein–protein interaction (PPI) network for the resulting mRNAs was constructed by employing the STRING database . Finally, Cytoscape software  was used to visualize the ceRNA network and PPI network.Venn diagram analysis was performed by using the online tools jvenn  to conduct gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the identified network and important RNA molecules (the top 200 genes related to PRLR and CACNA2D1 in BC). An adjusted p < 0.05 was deemed statistically significant.In order to understand the biological functions of the network and mRNA molecules, we used MetaScape . The survival package of R software was employed to assess the KM survival of deRNAs in ceRNA network, and the variables included in the prognosis model were first screened in terms of a p < 0.15. Then, the logistic regression analysis was performed to establish the model, and the model was then verified by chi-square analysis, Hosmer–Lemeshow Test, calibration curve, and ROC curve. In addition, the survival analysis of PRLR and CACNA2D1 was re-verified by using the GEPIA2 database .The survival analysis of PTEN in BC was carried out by using Kaplan–Meier plotter . Then, UALCAN  and DeatherMeth 2.0  were used to evaluate the methylation levels of PRLR and CACNA2D1 in BC tissues and normal tissues, and MEXPRESS  was employed for visualization. Finally, a heatmap was drawn by using MethSurv . Meanwhile, the expression of RNAs in BC tissues and normal tissues was analyzed by using the UALCAN database and GEPIA 2 database, and the expression of RNA in BC cell lines and at protein level was verified by the Human Protein Atlas (HPA) database  [The mutation analysis of PTEN, PRLR, and CACNA2D1 was carried out by utilizing cBioPortal for Cancer Genomics (ry 2022) ,14,15.https://cistrome.shinyapps.io/timer/, accessed on 28 May 2022) was used to assess the relationship between the expression of PRLR and CACNA2D1 in BC and the abundance of tumor immune infiltrating cells, the relationship between tumor immune infiltrating cells and prognosis, and the correlation between PRLR and CACNA2D1 and immune checkpoints.The TIMER database  and PTENlow group (530). First, BC samples and normal samples were screened for deRNAs, with p < 0.05 and |log FC| < 0.7 as eligibility criteria of mRNA, p < 0.05 and |log FC| < 0.5 as the criteria for lncRNA, and p < 0.05 and |log FC| < 0.3 as the criteria for miRNA. In total, 4346 demRNAs, 446 delncRNAs, and 332 demiRNAs were identified from BC samples and normal samples. Second, the deRNAs of PTENhigh group and PTENlow group were screened, with p < 0.05 and |log FC| < 0.5 as the mRNA cutoff value, p < 0.05 and |log FC| < 0.5 as the lncRNA cutoff value, and p < 0.05 and |log FC| < 0.3 as the miRNA threshold. Finally, 1329 demRNAs, 109 delncRNAs, and 109 demiRNAs were found from the PTENhigh group and PTENlow group , the samples were divided into PTENow group .high and PTENlow groups with those of BC and normal tissues, and finally identified 700 demRNAs, 47 delncRNAs, and 84 demiRNAs, which were displayed by Venn diagrams , potassium calcium-activated channel subfamily N member 4 (KCNN4), PRLR, protein kinase cGMP-dependent 1 (PRKG1), has-miR-429, CACNA2D1, NADPH oxidase 4 (NOX4), EPH receptor A3 (EPHA3), glypican 6 (GPC6), HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1), and HOTAIR were preliminarily eligible for being included in the prognostic model. Logistic regression analysis of these variables and clinical characteristics , tumor stage, and prior malignancies) revealed that only distant metastasis, lymph node metastasis, CACNA2D1, and PRLR registered a significant correlation with the prognosis of BC. Then, the four significant variables were used to construct a model, in which the p values of all variables were found to be < 0.05 , indicating that the current data had been fully extracted and the goodness-of-fit of the model was high. Furthermore, the goodness-of-fit of the model was further demonstrated by the calibration curve   . These dp < 0.001), which was consistent with the results of the bioinformatic analysis   A. DNA me< 0.001) B, while < 0.001) C. Finall< 0.001) A, while < 0.001) B.To further understand the biological functions of PRLR and CACNA2D1 in BC, the top 200 genes related to PRLR and CACNA2D1 in BC were retrieved from GEPIA2, and the GO and KEGG pathway enrichment analyses of these genes were performed by using Metascape. It was found that PRLR was mainly implicated in cytoskeleton-dependent intracellular transport, protein catabolism, and the Hedgehog signaling pathway A, while As a part of the tumor immune response, immune cell infiltration is related to the development, progression, and outcome of tumors. We used TIMER to evaluate the effect of immune cell infiltration on the clinical prognosis of BC patients, and found that lower levels of B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells were related to a worse prognosis of BC patients, while the expression of macrophages was not significantly correlated to the prognosis of BC A. Then, The expression of immune checkpoints was also found to be intimately related to the prognosis of a tumor . ProgramGlobally, BC is the highest-incidence cancer world-wide and the leading cause of cancer-related deaths in women . While vPTEN can regulate the PI3K–AKT–mTOR signaling pathway to control a wide array of biological processes, including cell survival, migration, proliferation, and metabolism, and its abnormal expression may lead to cancer susceptibility and tumor progression . In thisFirst, a PTEN-related ceRNA network composed of eighty-two mRNAs, two lncRNAs, and five miRNAs was constructed in this study based on TCGA database. Enrichment analysis showed that the biological functions of this network mainly involved the cell morphogenesis and cell adhesion. Then, PPI network analysis, KM survival analysis, and logistic regression analysis exhibited that PRLR and CACNA2D1 were related to the prognosis of BC. Furthermore, we conducted several analyses on PRLR and CACNA2D1, and found that PRLR may be overexpressed in BC tissues through mutation and hypomethylation, thus affecting the prognosis of BC patients. Meanwhile, the expression of CACNA2D1 may decrease due to aberrant methylation, which is related to the poor prognosis of BC patients.PRLR is the receptor of prolactin that promotes the growth of breast tissues, and it belongs to the transmembrane receptor of cytokine receptor family . PreviouThis study found that PRLR was significantly enriched in cytoskeleton-dependent intracellular transport. Prior studies have shown that cytoskeleton-dependent intracellular transport was related to tumor immunity. ShiYongNeo et al., showed that NK cells could efficiently express and secrete CD73 through vesicle transport, and promote tumor immune escape . MeanwhiWe used TIMER to evaluate the influence of immune cell infiltration on the clinical prognosis of BC patients, and found that low levels of B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells were all related to the poor prognosis of BC patients. However, immune infiltration analysis showed that PRLR expression was feebly correlated with the infiltration level of CD8+ T cells, but not with that of CD4+ T cells, neutrophils, and dendritic cells, indicating that PRLR might affect the prognosis of BC patients but not by regulating the abundance of tumor immune cells. Therefore, immune cell infiltration may not be the cause of poor prognosis in BC patients with high PRLR expression.The abundance of infiltrating immune cells can exert a substantial impact on the prognosis of BC patients and tumor cells can also evade the identification and clearance by the immune system through immune checkpoints, both contributing to the prognosis of BC. Furthermore, this study evaluated the correlation between some common immune checkpoints and the prognosis of BC patients, and the relationship between PRLR and these immune checkpoints. The results showed that BC patients with a high expression of PDCD1, CTLA4, TIGIT, and BTLA had a better clinical prognosis, while the expression of PRLR was negatively correlated with the expression of these immune checkpoints, indicating that PRLR might participate in the immune process of BC by inhibiting the expression of the above immune checkpoints, and targeting PRLR might improve the efficacy of BC immunotherapy. To sum up, this study suggested that PRLR might affect the prognosis of BC patients by regulating the expression of immune checkpoints.CACNA2D1 is a gene encoding a T-type calcium channel subunit α2δ1. CACNA2D1 is involved in the regulation of calcium current density and dynamics of calcium channels, and plays an important role in excitation–contraction coupling. It was found to be profoundly involved in the metabolism, immune system, cardiovascular system, musculature, and other phenotypes in vivo . In lineFunctionally, CACNA2D1 was found to be mainly involved in myofibrillar composition, adhesive spots, and muscular structure development, and cytoskeleton-dependent intracellular transport and the cytoskeleton were all related to tumor immunity. Therefore, we were led to assume that CACNA2D1 might be related to tumor immunity. However, so far, no studies have previously examined the relationship between CACNA2D1 and BC immunity. In this study, analyses on immune infiltration and immune checkpoints of CACNA2D1 found that CACNA2D1 was positively correlated with immune cells such as B cells, CD8+ T cells, CD4+ T cells, neutrophils, and dendritic cells, and the low CACNA2D1 expression in the immune cells was related to the poor prognosis of BC, suggesting that CACNA2D1 might impact the prognosis of BC patients by regulating the abundance of tumor immune cells. In addition, CACNA2D1 was positively correlated with the expression of immune checkpoints, such as CTLA4, TIGIT, and BTLA, which, in turn, are positively correlated with the prognosis of BC patients. These findings indicated that CACNA2D1 might influence the prognosis of BC patients by regulating the expression of immune checkpoints, and targeting CACNA2D1 may improve the efficacy of immunotherapy in BC.This study has some limitations. First, BC types were not taken into consideration in the grouping of the samples. Second, only TCGA database was used in the model construction and verification, and no external data sets were used for verification. In addition, the expression and functions of the selected molecules were not experimentally confirmed. Further studies are warranted to verify the reliability and accuracy of the model. Moreover, in vitro and in vivo experiments are needed to substantiate the roles of PRLR and CACNA2D1 in the pathogenesis of BC and their effects on the prognosis of BC.To sum up, this study established a model that evaluated BC prognosis on the basis of the PTEN-related ceRNA network, and found that PRLR and CACNA2D1 might be involved in BC by regulating the abundance of tumor immune infiltrating cells and the expression of immune checkpoints, thus affecting the prognosis of BC patients. PRLR and CACNA2D1 hold promise to serve as therapeutic targets for BC.


TP53 mutation statuses, revealed an altered expression of 49 genes to affect the prognosis and/or treatment response. Next, these genes were investigated in the validation cohort, to confirm the clinical significance of their expression alterations, and to identify genetic variants with an expected high or moderate impact on their products. The expression changes of five genes, PROM1, CXCL8, RUNX1, NAV1, TP73, were found to predict prognosis or response to treatment in both cohorts, depending on the TP53 mutation status. In addition, we revealed novel and confirmed known SNPs in these genes, and showed that SNPs in the PROM1 gene correlated with its elevated expression.Considering the vast biological diversity and high mortality rate in high-grade ovarian cancers, identification of novel biomarkers, enabling precise diagnosis and effective, less aggravating treatment, is of paramount importance. Based on scientific literature data, we selected 80 cancer-related genes and evaluated their mRNA expression in 70 high-grade serous ovarian cancer (HGSOC) samples by Real-Time qPCR. The results were validated in an independent Northern American cohort of 85 HGSOC patients with publicly available NGS RNA-seq data. Detailed statistical analyses of our cohort with multivariate Cox and logistic regression models considering clinico-pathological data and different  CEBPA [EMSY [TP53 gene result in the p53 protein accumulation which creates a permissive environment for the activity of oncogenes. In the present study, we found five genes, the changed expression of which concordantly affected patient outcomes in both cohorts. Furthermore, by using bioinformatic and statistical tools, we identified novel and known sequence variants in TP53 and in the other analyzed genes, and correlated them with expression changes. This comprehensive study, summarizes the results obtained with two different techniques, i.e., Real-Time qPCR and NGS RNA-seq, and reveals significant relationships between genetic alterations, aberrant expression of the studied genes, and the clinical outcomes in two independent cohorts of HGSOC patients.Ovarian carcinoma (OvCa) is the leading cause of death from gynecological malignancies worldwide. The mortality in this disease is exceptionally high, because the early-stage disease is usually asymptomatic while the symptoms in the late-stages are often nonspecific and there are no efficient screening methods. Thus, the majority of patients are diagnosed at late stages, which are characterized by poor prognosis. Despite nearly 70% remission rate after the first line of treatment, the disease recurs in about 50% of the patients, mostly due to chemoresistance . TherefoCEBPA  and EMSYPA [EMSY  genes, bPA [EMSY . Thus, iPA [EMSY , 7. MissTP53 gene [TP53 mutations revealed regularities undetectable when the entire group of patients was analyzed as a whole. Furthermore, the expression of three genes  exerted opposing impact on DFS in the subgroups with different TP53 mutation statuses  had a number of reads too low to properly assess their mRNA expression. For each of these genes, the number of normalized NGS RNA-seq reads equaled 0 in over 90% of samples, which made the validation impossible  >2 cm as an independent factor worsening drug response, while overexpression of the NAV1 gene was associated with the lower chance of CR in the same group of patients. Consistently, NAV1 overexpression also increased the risk of death in the subgroup with missense mutations in TP53, though this regularity was not confirmed in the validation cohort of HGSOCs.As to the potential factors predictive of treatment response, elevated expression of the No time trends were found for the frequencies of death, recurrence, complete remission or sensitivity to chemotherapy in the experimental cohort . InformaMUC16 and TP53, all of a high impact. Two unique, previously unreported variants were identified in this subset, both in the TP53 gene (chr17:g.7676061dup and chr17:g.7675205del). As to the five genes characterized by concordant expression profiles in both cohorts, PROM1, CXCL8, RUNX1, NAV1, and TP73, in total, they harbored 24 SNP changes ; chr1:g.201780560G>C ; chr1:g.201809166C>A ; chr1:g.201793831A>G, ), and 0 non-SNP alterations. The detailed results of the VEP analysis are presented on heatmaps . The Pearson’s Chi-squared test with Yates’ continuity correction p-values equaled 0.0006 and 5.361e-15, respectively.All the genes, the altered expression of which correlated with either cancer prognosis or treatment response prediction in the experimental cohort, were also subjected to the analysis of genetic variants with the Ensembl VEP app in the validation cohort. In summary, 716 high or moderate SNP changes were identified in 31 of 49 genes analyzed, 186 of which were of a high impact. Twenty eight of all these SNPs were unique, novel variants, previously not reported in the Ensembl database. Considering non-SNP alterations in the same cohort and gene set, six genetic changes were found in only two genes, heatmaps , in TablPROM1 gene only, revealing a positive correlation between the occurrence of genetic variants and mRNA overexpression . Remarkably, this genetic change leads, in fact, to alternative splicing of the PROM1 gene, resulting in the formation of an abnormal transcript, detectable in the NGS RNA-seq analysis  but also proved that SNPs in the PROM1 gene correlated with its elevated expression which, in turn, was a negative prognostic factor in patients with HGSOCs harboring missense mutations in TP53.Based on the data available in the scientific literature, 80 genes were nominated for evaluation of their mRNA expression in pre-treatment tumors from an experimental cohort of the TP-treated HGSOC patients. This step involved the Real-Time qPCR analysis and detailed statistical inference with multivariate Cox and logistic regression models in subgroups with different The first question that needs to be discussed is the relatively low fraction of genes the altered expression of which was successfully confirmed in the validation cohort (5/49 = 10.2%). This might relate to several factors. First, the experimental and validation cohorts came from completely distinct populations (European vs Northern American). The so-called genetic background, being specific for each human population, is a well-known factor affecting susceptibility to ovarian cancer and other neoplasms , 13, as TP53 mutations. The fraction of tumors harboring TP53 mutations in the experimental and validation cohorts equaled 92.9% and 69.4%, respectively, and the difference was statistically significant. The lower frequency of the TP53 mutations in the validation cohort may reflect the phenomenon known as a nonsense-mediated mRNA decay, leading to low concentrations of mRNAs transcribed from alleles carrying nonsense mutations [TP53 and in the other genes in the latter group can be underestimated. p53 is called the guardian of the genome for its imperative role in cancer prevention. The normal p53 protein partakes in the regulation of proliferation, apoptosis and DNA repair. It is also associated with the control of cell metabolism, autophagy and cell senescence [TP53 mutations, especially of the missense type, considerably speeds up tumorigenesis [CD44, MKI67 and RUNX2, exerted opposed effects on patient DFS depending on the TP53 mutation status. Interestingly, the three genes revealed their adverse prognostic value in tumors harboring the TP53 missense mutations, thus supporting the aforementioned superior role of p53 in carcinogenesis. By contrast, the positive impact of CD44, MKI67 and RUNX2 overexpression on HGSOC patient survival is likely caused by their proliferation-promoting function which, in turn, sensitizes cancer cells with the normal p53 protein to cisplatin and other DNA-damaging agents [MKI67 mRNA and Ki-67 protein expression were also found to be downregulated by the p53/p21 pathway upon DNA damage [Runx2 gene was demonstrated to be indirectly repressed by p53, leading to an inhibition of the osteogenic differentiation of bone marrow stromal cells. Consistently, the p53 loss sped up the differentiation process [CD44, MKI67 and RUNX2 has previously been demonstrated to depend on the p53 protein function. This seems to provide a reasonable explanation of the apparent discrepancies in the prognostic value of these genes in subgroups with different TP53 mutation statuses, found herein in the experimental HGSOC cohort. However, the prognostic meaning of all three oncogenes discussed in this paragraph was not confirmed in the validation cohort. The inconsistency could relate to the fact that, unlike the experimental group of HGSOC tumors, the validation series of samples comprised tumors obtained from patients cured in three different hospitals. This might negatively affect the uniformity of the validation cohort, and introduce some additional, hard-to-define variables, that ultimately could significantly interfere with the statistical results. For example, the differences in patient outcomes are likely to be influenced not only by the treating institution and the clinicians involved, but also by the date of patient admittance to the hospital. As stated above, the time trend analysis could not be performed for the validation cohort. Thereby, we were unable to determine whether and how the date of tumor excision affected the risk of patient death and tumor recurrence or the chance for a positive response to chemotherapy in this cohort. Considering the above, the findings related to the superior role of p53 in carcinogenesis, demonstrated herein for the CD44, MKI67 and RUNX2 genes, although supported by data available in the scientific literature, should be interpreted with caution.There is, however, another interesting variation between the two cohorts investigated herein, the prevalence of the nescence . On the nescence , and whenescence .The occuigenesis . In lineg agents . In factg agents . SimilarA damage . The exp process . In summTP73, belongs to the evolutionarily oldest family of tumor suppressors, comprising also TP53 and TP63. Considering over 70% similarity of the DNA-binding domains in p53, p63 and p73 proteins, many known p53 target genes , PUMA, NOXA, BAX and MDM2) have been demonstrated to be regulated by the other members of this protein family, too. Nevertheless, the full repertoire of common and private target genes for each suppressor still needs to be unraveled [TP53, the TP63 and TP73 genes are rarely mutated in neoplastic cells [TP73 (SNP with a moderate impact), no alterations in TP63 (data not shown) and as much as 59 mutations in the TP53 gene  accumulation [TP73 gene overexpression as a predictive marker of worse HGSOC response to the TP chemotherapy, independent of the TP53 mutation status. The tumor-promoting role of TP73 in ovarian cancer was also reported in two other studies [TP73 in chemotherapy response remains unclear, too. In some studies, TAp73 has been demonstrated to be an anti-apoptotic agent, inhibiting drug- and p53-induced apoptosis in ovarian and small-cell lung carcinomas [The most prominent and interesting discovery of this study concerns five genes the altered expression of which was found to be associated with the clinical outcome, what was successfully validated in an independent cohort of HGSOC patients. The first of these genes, nraveled . Interesnraveled . Furthernraveled . Unlike ic cells . AccordiP53 gene  and S4. syndrome , TP73 hamulation . The mecmulation . On the mulation . Our res studies , 31, andrcinomas , 33. Howrcinomas , 35. TheNAV1 (neuron navigator 1) was another gene found in our research to adversely affect HGSOC treatment response when overexpressed. Similarly to TP73, this impact was also independent of the TP53 mutation status. Importantly, NAV1 must not be confused with Nav 1.1–1.9 sodium channels proteins, which are encoded by SCN1A-SCN11A genes. So far, no scientific reports on the NAV1 role in ovarian cancer have emerged. As to other cancer types, the NAV1 promoter hypomethylation was observed in hormone receptor positive  breast cancers [NAV2 and NAV3) showed their opposed roles in colon cancer development [NAV1 gene seems to exert pleiotropic effects. In mice, it was shown to be involved in the directional neuron migration process [NAV1 associations with cancer, we referred to the Pan-Cancer Analysis of Whole Genomes (PCAWG) study, based on an international collaboration to identify common patterns of mutations in more than 2,600 whole cancer genomes from the International Cancer Genome Consortium [NAV1 expression was elevated in ovarian carcinomas (N = 110) compared to normal ovaries (N = 39) (medians of transcripts per million (TPM) equaled 7 and 4, respectively). Thus, this outcome seems to be concordant with the negative predictive value of NAV1 overexpression demonstrated herein. The NAV1 protein’s association with microtubules, reported by Martínez-López et al. [NAV1 gene expression levels. cancers  which seelopment , 38. The process . In huma process , and dia process . Given snsortium . This stries N =  , a well-known chemotactic factor, a key mediator protein associated with inflammation, where it plays a pivotal role in neutrophil recruitment and degranulation [CXCL8 overexpression in cells with no TP53 missense mutations by showing its negative impact on overall survival of HGSOC patients. Other studies involving clinical material also corroborated our findings, by presenting a negative correlation between the survival time of ovarian cancer patients and the expression levels of IL-8 and IL-10 [The next validated gene, nulation . Despitenulation . By contnulation –47. In anulation . Concordnd IL-10 . Moreovend IL-10 .RUNX1 gene, found herein to present significantly altered expression in both the experimental and validation cohorts, is involved in the generation of hematopoietic stem cells [RUNX1 also occur in many solid tumors. In gastric [RUNX1 in breast cancer appears to be more ambiguous and hormone-dependent. On the one hand, tumor suppressor capabilities of RUNX1 have been observed at the mRNA and protein levels in ER+ and/or PR+ breast cancers [RUNX1 gene in breast cancer, intriguingly associated with the ER negative and triple negative  subtypes [RUNX1 mRNA upregulation in the TN breast carcinomas [in vitro studies showed that RUNX1 inhibition in cell lines promoted cisplatin-induced apoptosis [RUNX1 knockdown significantly attenuated proliferative, migratory and invasive abilities of SKOV3 cells [RUNX1 promoter was observed in HGSOC patients with chemoresistant tumors, suggesting its adverse, oncogenic role in cancer progression [RUNX1 in HGSOCs, but only in the subgroup without missense mutations in TP53, where its elevated expression correlated with the decreased risk of tumor recurrence. The reasons for these seemingly antithetical outcomes can relate to hormone-dependence of ovarian neoplasms, just like in breast cancers. In line with this assumption, RUNX1 alterations reported in the cBioPortal database  for ovarian cancers comprised both amplifications and deletions, thus highlighting the ambiguous and context-dependent role of the gene in this neoplasm [RUNX1 changes identified herein in the VEP analysis of the HGSOC validation cohort was classified as a high-impact SNP (rs200431130), resulting in the loss of the RUNX1 start codon in the recently predicted RUNX1 transcript (XM_017028487.1). This discovery provides another supporting evidence for RUNX1 to conceivably play a tumor suppressor role in ovarian cancers.The em cells . It has em cells . However gastric , hepatoc gastric , and eso gastric  carcinom cancers –58. On tsubtypes . Accordircinomas , 61. Thercinomas , and in rcinomas . In ovarpoptosis . MoreoveV3 cells . In linegression . In addigression . Interesneoplasm . One of PROM1, encodes Prominin-1 . CD133 is a glycoprotein frequently expressed in cancer stem cells [PROM1 gene is driven by five alternative promoters, three of which are located on CpG islands and are partially regulated by methylation. The occurrence of several promoter regions leads to alternative splicing, resulting in CD133 structural variants with potentially unique roles [PROM1 gene overexpression on patient DFS in the subgroup of tumors with missense TP53 mutations, found in both the experimental and validation cohorts. We also discovered a positive correlation between the elevated expression of PROM1 and its SNPs. It is worth to note that nine out of ten samples with genetic changes within the PROM1 gene harbored the same high-impact alteration: chr4:g.15980539T>A (ENST00000447510.7:c.2374-2A>T), being a novel, splice acceptor variant, resulting in the formation of an abnormal PROM1 mRNA transcript. Splice site mutations have been extensively studied in recent years in various human disorders, including cancers. Abnormalities in alternative splicing were linked to tumorigenesis, as they induced a plethora of physiological alterations in neoplastic cells, including the imbalance between proliferation and apoptosis, increased invasiveness, angiogenic and metastatic potential as well as an elevated rate of metabolism, all of which belong to the well-known cancer hallmarks [EMID1 and IL19, respectively, by Mucaki et al. [PROM1 gene.The last of the validated genes, em cells . The expem cells . Transcrue roles . In ovarue roles –72. Addiue roles . Our resPROM1, CXCL8, RUNX1, NAV1 and TP73, as potential biomarkers to predict prognosis or response to treatment. This was achieved by combining the results of Real-Time qPCR analyses performed in our Polish cohort and the NGS RNA-seq studies carried out by Ducie et al. [TP53 mutation status for ovarian cancer biology and biomarker discoveries.In summary, in two independent cohorts of HGSOC patients, we identified five genes, e et al.  in the APROM1, CXCL8, RUNX1, NAV1 and TP73, affects patient prognosis or predicts treatment response. Remarkably, the context of the TP53 missense mutations in the tumors is crucial to unravel these associations, which confirms our previous results, pointing to p53 as an important confounding determinant in the biomarker discovery. In addition, hundreds of genetic alterations with the expected high or moderate impact on the encoded proteins’ sequence and function were identified, including thirty unique changes so far unknown. Noteworthy, some of these variants are likely to affect the expression of the altered genes, as shown herein for the PROM1 oncogene. Our results add to a better understanding of ovarian cancer-driving mechanisms, thus provide the grounds for the development of novel, targeted, less aggravating and, hopefully, more effective methods of treatment.The altered expression of five genes, The study was conducted according to the guidelines of the Declaration of Helsinki, and approved in writing by the Institutional Review Board of the Maria Sklodowska-Curie National Research Institute of Oncology (nos. 49/2003 and 39/2007). Informed consent in writing was obtained from all subjects involved in the study.TP53 mutations. Other mutations do not cause the p53 protein accumulation, just like in case of the wild-type TP53 gene [TP53 missense mutations and the p53 protein accumulation. Out of 70 specimens that we examined, 46 (65.7%) exhibited the p53 accumulation, while the TP53 missense mutations were found in 43 tumors (61.4%). For the evaluation of clinical endpoints in the experimental group, CR was defined as disappearance of all clinical and biochemical symptoms of ovarian cancer assessed after completion of the first-line chemotherapy and confirmed four weeks later [The Real-Time qPCR analysis was performed in a uniform series of 70 high-grade, serous, ovarian cancer samples from previously untreated patients . All patients were treated at a single institution, the Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland in the years 1995–2006 and received TP chemotherapy after surgery. Medical records of all the patients were critically reviewed by at least two clinicians. The material was carefully selected to meet the following criteria: no chemotherapy before staging laparotomy, adequate staging procedure according to the recommendations by the International Federation of Gynecologists and Obstetricians (FIGO) , tumor tP53 gene . In the ks later . DFS wasIn order to validate the gene expression results in an independent cohort of 85 HGSOC samples, we have examined the publicly available NGS RNA-seq data set (id: PRJNA396544) from the European Nucleotide Archive (ENA), deposited there by Ducie et al. [TP53 mutation status. A detailed clinico-pathological characteristics of the patients and tumors in both the experimental and validation cohorts is presented in Remarkably, the clinico-pathological data available for the validation cohort differ, with respect to some variables, from the data we gathered for the experimental cohort, e.g., in the experimental cohort treatment response prediction was assessed based on two parameters: CR and PS, whereas for the validation cohort the information on complete tumor remission was missing. To overcome this limitation, the PS and CR variables in the experimental group were both compared with the PS in the validation cohort. Additionally, in the experimental cohort, OS and DFS times were calculated for each patient. By contrast, in the validation cohort OS was available but the DFS variable was missing, and the authors provided Progression-Free-Survival (PFS) data instead. As DFS and PFS variables cannot be treated interchangeably, we decided to compare the DFS in the experimental cohort with the recurrence status in the validation group. As to the validation procedure, in either prognosis or treatment response prediction, the results were considered as confirmed when HR/OR values in the experimental group and Fold Change (FC) values in the validation cohort were both either above or below 1 for the same gene, the same dependent variable  and the same RNA was isolated from frozen tumor sections with over 85% of cancer cells, using the RNeasy Plus Mini Kit . RNA quantity was measured with NanoDrop spectrophotometer , and its quality was assessed on Agilent Bioanalyzer . RNA integrity numbers (RINs) of the samples ranged from 6.5 to 9.4.HPRT1 (hypoxanthine phosphoribosyltransferase 1), PPIA (peptidylprolyl isomerase A), and GUSB (glucuronidase beta)) [PLUS app , utilizing an improved version of the geNorm algorithm [Based on data available in the scientific literature, 80 genes were nominated for their expression verification by Real-Time qPCR. The majority of the genes was analyzed in the entire experimental series of 70 samples, but some genes were assessed in a subset of the experimental series (51 samples or 50 samples). For the list of genes, their descriptions and series sizes, see the e beta)) . These tlgorithm , 83. In lgorithm . The calThe quality of the FASTQ files acquired for 85 HGSOC samples from the validation cohort was analyzed with the Fastqc app (version: 0.11.9) followed by adapter clipping and removal of poor-quality fragments of reads with the Trimmomatic app (version: 0.39). NGS reads were then mapped to a reference sequence of the human genome (assembly: GRCh38 (hg38)) using the HISAT2 sequence aligner (version: 2.2.1). After mapping, the PCR and optical duplicates were removed with the MarkDuplicates app, a part of the Genome Analysis Toolkit . The assessment of mapping quality was carried out using Samtools (version: 1.12) and Qualimap (version: 2.2.2-dev) apps, while Integrative Genomics Viewer  served for visualization of the mapping results. The DESeq2 package for R (version: 1.26.0) was employed for identification of differentially expressed genes. Importantly, due to the relatively low depth of sequencing coverage in the acquired NGS RNA-Seq data , the DESeq2 analysis had to be performed on unfiltered data. Otherwise, about two thirds of the genes  would be filtered out and thus excluded from further analyses.https://github.com/lukszafron/VAF.checker). Then, the vcf-annotate app from the VCFtools package (version: 0.1.16) was employed to filter out the variants that do not meet the following criteria: all filters with default values applied except for: MinAB = 2 , Qual = 20 , and MinMQ = 20 . Subsequently, the obtained VCF files were divided with bcftools into two subsets , containing snp variants vs all other sequence alterations, i.e., indels (insertions or deletions), mnps (multi-nucleotide polymorphisms), bnd (breakpoints), and others, respectively. Next, the variant identification and effect prediction analysis was carried out using the Ensembl Variant Effect Predictor (VEP) app  and the merged Ensembl and RefSeq databases [https://github.com/lukszafron. Ensembl VEP divides sequence variants into four categories: high, moderate, low and modifier, based on their expected impact on the transcript and protein sequences. For details, please refer to the web page [In the present study, the aforementioned NGS RNAseq data set generated for the validation cohort was used not only to find significant alterations in gene expression, but also to determine sequence variants. The Variant Call Format (VCF) files were generated  using the bcftools (version: 1.12) and the GRCh38 (hg38) assembly of the human genome. Next, the variants were subjected to a two-step filtering. First, the variants less frequent than 10% were filtered out based on the AD tag, using the VAF checker app (version: 1.0)  involved in the development of hereditary ovarian cancer . In those libraries, the gene set enrichment was carried out using the sequence capture technology and the SeqCap EZ Prime Choice Probes, both offered by Roche . The obtained NGS data were subsequently analyzed using the methods described above to find genetic variants with a high or moderate expected impact on the encoded proteins’ sequence and function.To assess the TP53 gene, and were adjusted for the FIGO stage and RT. Noteworthy, for all the analyzed genes, the expression was treated as a continuous variable to avoid arbitrary categorization of data, which could potentially lead to unreliable statistical results. In case of continuous variables, contrary to categorical variables, HRs/ORs cannot be treated as the ratio of the hazard/odds rates, corresponding to the conditions described by two sets of explanatory variables. For continuous variables, the same interpretation applies to a unit difference [TP53 mutations or patient deaths in the experimental and validation cohorts. All p-values shown herein were considered significant at the statistical significance level  of 0.05. Given the use of two independent HGSOC cohorts to study the impact of altered gene expression on ovarian cancer prognosis and prediction of treatment response, we decided not to apply the Benjamini-Hochberg correction controlling the false discovery rate. Otherwise, the rate of the statistical type II error (beta) would increase with concurrent, unintended decrease of the statistical power (1-beta) [All statistical analyses were carried in the R environment (version: 3.6.1). The survival analysis in the experimental group of patients was performed using the multivariate Cox proportional hazards models . All Cox models were also checked with respect to proportionality of hazards for each variable used. The prediction of treatment response in the experimental group of patients was carried by generating multivariate logistic regression models (R packages: stats (version: 3.6.1) and rms (version: 6.2–0)). In order to verify the discriminating capabilities of the Cox and logistic regression models, we performed their cross-validation in new data sets, generated from the original data by bootstrapping (with replacement) and subsequent comparison of areas under curves (AUCs) between the original and bootstrapped data sets, using the riskRegression package for R (version: 2020.12.8) . All thefference . As ment(1-beta) .S1 FigThe patients underwent their first surgical treatment in the years 1995–2006. Time trends concerned overall survival (OS) ; disease-free survival (DFS) ; sensitivity to chemotherapy (PS)  and complete remission (CR) . The trends are shown as a trend line of death, relapse, PS and CR frequencies, respectively, supplemented with the results of the Mann-Kendall homogeneity test, and supported with autocorrelation function (ACF) plots.(TIF)Click here for additional data file.S2 FigThe results of the VEP analysis in the validation cohort of HGSOCs for 49 genes with significantly changed mRNA expression in the experimental cohort . Abbreviations used: VEP–Variant Effect Prediction; HGSOCs–high-grade serous ovarian cancers.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


The role of oxidative stress, as well as inflammation in the pathogenesis of methotrexate (MTX)-induced oral mucositis, is a known fact. The anti-inflammatory, antitumor, antimicrobial, and antioxidant properties of taxifolin—the effect we tested against MTX-induced oral mucosal damage—are well known.  Evaluating biochemically and histopathologically the effects of taxifolin on methotrexate-induced oral mucosal damage in rats. In the taxifolin+MTX (TMTX) group, 50 mg/kg taxifolin was orally administered to rats by gavage. In the MTX and healthy (HG) groups, normal saline was applied to rats as solvent by the same method. One hour after administration of taxifolin and solvent, 5 mg/kg MTX was orally administered to rats in the MTX and TMTX groups. Taxifolin and methotrexate were administered once a day for 30 days. Macroscopic, biochemical, and histopathological evaluations were performed on the inner cheek and tongue tissues of rats. These parts were removed after rats were killed with a high-dose anesthesia. Taxifolin with MTX prevented the increase in oxidant and pro-inflammatory parameters, such as malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), on the inner cheek and tongue tissues of rats. Moreover, taxifolin antagonized the decrease in total glutathione (tGSH). Taxifolin decreased MTX-induced histopathological damage. These findings suggest that taxifolin may be useful to treat MTX-associated oral mucositis. Moreover, MTX is a gold-standard antirheumatic agent in the treatment of rheumatoid arthritis, psoriasis, systemic scleroderma, and other autoimmune disorders.2 The use of MTX is preferred in low doses in the treatment of inflammatory diseases while it is preferred in high doses in the treatment of malignant diseases.1 As well as high-dose MTX is safely applied to most patients during treatment, serious side effects, such as nephrotoxicity, myelosuppression, mucositis, dermatologic toxicity, and hepatotoxicity, can be observed.3 Moreover, various side effects may occur during the treatment of inflammatory diseases, ranging from mild to severe side effects and even leading to treatment discontinuation.4 One of the serious side effects of MTX is oral mucositis.5 Oral toxicities, such as mucositis and stomatitis, are some of the most serious and indispensable side effects related to cancer therapy.6 Mucositis is a pathological process that causes ulceration and inflammation of the mucosal tissue in the region from the mouth to the anus.7 Oral mucositis makes feeding difficult and may also increase the risk of infection, limit the dose of the medication, delay the treatment, and extend hospital stay.8 The role of reactive oxygen species (ROS) and various pro-inflammatory cytokines in the pathogenesis of mucositis have been shown.9 It has also been showed that MTX increases the levels of malondialdehyde (MDA), an oxidant parameter in the oral mucosa, interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), an indicator of inflammatory response.10 The literature shows that effective antioxidant, anti-inflammatory, antiulcer, and antimicrobial medication may be useful to treat MTX-associated oral mucositis.Methotrexate (MTX) is a folic acid antagonist—an antiproliferative drug, as it is known.11 The anti-inflammatory, antitumor, antimicrobial, and antioxidant properties of taxifolin are well known.12 It has been shown that taxifolin has an antioxidant effect by inhibiting ROS production.13 The antioxidant, anti-inflammatory, and antimicrobial properties of taxifolin suggest that it may be useful to treat MTX-induced oral mucosal damage. In the literature, the oxidative and inflammatory effects of taxifolin associated with MTX have been reported. There are no studies in the literature evaluating the protective effect of taxifolin against MTX-induced oxidative and inflammatory mucosal damage. Thus, our study aimed to evaluate biochemically and histopathologically the effects of taxifolin on MTX-induced oxidative and inflammatory oral mucosal damage in rats.Taxifolin , which we tested against MTX-induced oral mucosal damage in this study, can be found in plants such as thistle, onion, French maritime, and tamarind.Male albino Wistar rats were obtained from Erzincan Binali Yildirim University Experimental Animals Application and Research Center. In the study, 18 healthy rats weighing from 280 to 295 g were used. Rats were fed and separated in groups (n=6) in the suitable laboratory environment at normal room temperature (22°C) before the experiment. Procedures and protocols were approved by the Erzincan Binali Yildirim University Ethics Committee for Animal Experimentation on January 27, 2022 (Decision no. 2).The chemicals used were methotrexate, thiopental sodium, and taxifolin and they were obtained from Med-İlaç, Turkey, I.E. Ulagay, Turkey, and Evalar, Russia, respectively.The rats used in the experiment were divided into three groups: MTX group , taxifolin+MTX group (TMTX), and healthy group (HG).14 was administered orally to the stomach of rats by gavage in the TMTX group (n=6). Normal saline (0.9% NaCl) was applied to rats in MTX (n=6) and healthy (n=6) groups as solvent by the same method. One hour after taxifolin and solvent administration, animals in the MTX and TMTX groups orally received 5 mg/kg MTXo.15 Taxifolin and methotrexate were administered once a day for 30 days. At the end of this period, all rats were killed with high-dose anesthesia . The inner cheek and tongue tissues were removed and macroscopically evaluated, and then the levels of MDA, tGSH, NF-kB, TNF-α, IL-1β, and IL-6 were determined from the tissues. Tissues were also analyzed histopathologically. The results of the experiments in all groups were compared between groups and evaluated.A total of 50 mg/kg taxifolin16 (1979). On the other hand, tGSH (nmol/g protein) was measured by the method described by Sedlak J and Lindsay RH.17Tissue MDA (µmol/g protein) was measured by the method used by Ohkawa, Ohishi and YagiThe samples were weighed and then the tissues were cut, frozen with liquid nitrogen, and homogenized with a pestle and mortar. The samples were maintained at 2°C to 8°C after melting. Phosphate buffered saline (PBS)  was added and, after vortex for 10 seconds, centrifuged at 10000 xg for 20 minutes, the supernatants were carefully collected. The levels of TNF-α (ng/L), IL-1β (pg/L), and IL-6 (ng/L) were measured according to a commercial kit supplied by ELISA kit, Eastbiopharm Co., Ltd., China.The inner cheek and tongue tissues were fixed in 10% neutral formalin solution. The tissues were subjected to routine alcohol-xylol series after fixation. Later, 5 µm sections of the sample were placed in paraffin blocks and six random areas were stained with hematoxylin and eosin. Sections were stained with hematoxylin and eosin. The evaluation was made according to For biochemical analysisFor biochemical analysis, the Shapiro-Wilk test was used to determine whether data were normally distributed. Since data were normally distributed, the evaluation was made by one-way ANOVA. The Tukey or Games-Howell test was performed according to the results of the Levene test as a post hoc test. Biochemical results are presented as mean±standard deviation. Since histopathological data are ordinal data, the evaluation was made with the Mann-Whitney U test. Histopathological results are presented as mean±standard deviation and median (minimum–maximum). All statistical procedures were performed with IBM SPSS 22.0  and p<0.05 was considered significant.In the MTX group, the inner cheek tissue was quite swollen and presented severe hemorrhage, hyperemia, and ulcerated areas. In the TMTX group, the inner cheek tissue was less swollen and there was moderate swelling and hyperemia. When the tongue tissue was macroscopically evaluated, hyperemic muscle—due to the loss of the full-thickness epithelial layer on the tip of the tongues of the MTX group—complete loss of papillae, significant swelling of the entire tongue, hemorrhage, and hyperemia were observed. In the tongues of the TMTX group, moderate hyperemia was observed in the body and tip of the tongues, with a decrease in the papillary prominence .The amount of MDA in the inner cheek and tongue tissues of rats treated with MTX increased significantly when compared to the HG, as it can be observed in As it can be seen in As it can be seen in MTX increased the amount of IL-1β in the inner cheek and tongue tissues of rats (p<0.001). However, taxifolin significantly inhibited the increase in IL-1β in rats receiving MTX (p<0.001) .The amount of IL-6 in the inner cheek and tongue tissues of rats treated with MTX increased significantly when compared to the HG, as it can be observed in The histopathological findings were higher in the inner cheek and tongue tissues of rats in the MTX group than in the TMTX group. Statistically significant differences were observed between groups in terms of histopathological findings ; p<0.05.The tongue tissue presented a normal histological appearance in rats in the HG . Keratinized papillae were slightly shaped in the TMTX group while no keratinized papillae were observed in the tongues of the MTX group. The reduction in epithelial thickness was moderate in the MTX group and mild in the TMTX group. Swelling was moderate in the MTX and TMTX groups .. Interstitial inflammatory cell infiltration and hemorrhage were severe in the MTX group while these histopathological findings were mild in the TMTX group. Similarly, muscle fiber degeneration was severe in the MTX group and mild in the TMTX group .The inner cheek tissue presented a normal histological appearance in rats in the HG The effects of taxifolin on MTX-induced oxidative and inflammatory oral mucosal damage in rats were evaluated macroscopically, biochemically, and histopathologically in this study. Our macroscopic findings showed that mucositis developed in the inner cheek and tongue tissues of rats treated with MTX. Our biochemical findings showed that MTX increased MDA, TNF-α, IL-1β, and IL-6, and decreased tGSH in the inner cheek and tongue tissues. MTX also caused histopathological damage. Taxifolin significantly prevented macroscopic, biochemical, and histopathological changes.5 Mucositis is a pathological process characterized by ulceration and inflammation of the mucosal tissue.18 Another symptom of mucositis is bleeding of the mucosa.19 In our study, symptoms such as swelling, hyperemia, ulcer, and hemorrhage were observed in the inner cheek and tongue tissues of rats treated with MTX, which is in accordance with the literature.Oral mucositis is one of the serious side effects of MTX, as aforementioned.9 The levels of MDA, tGSH, TNF-α, IL-1β, and IL-6 were measured in the inner cheek and tongue tissues in order to evaluate MTX-induced mucositis. Our biochemical findings showed that MTX increased MDA and decreased tGSH in the inner cheek and tongue tissues. These findings show that oxidative stress developed in the inner cheek and tongue tissues of rats treated with MTX. Oxidant increase and antioxidant decrease in living tissues are also considered oxidative damage in the literature.20 Our biochemical findings are in accordance with studies by Kuduban, et al.10 (2016), which showed that MTX increases MDA and decreases GSH in the oral mucosa. It is known that MTX changes the balance between MDA and tGSH in favor of MDA in other tissues besides the oral mucosa.21 MDA is the cytotoxic product of the lipid peroxidation (LPO) reaction initiated by ROS,23 as it is known. Thus, MDA became one of the most popular and reliable markers of oxidative stress nowadays.24The role of ROS and various pro-inflammatory cytokines in the pathogenesis of mucositis have been shown in the literature.25 Taxifolin has also been reported to have a scavenging effect on other ROS, especially hydroxyl radical (HO).26 The fact that taxifolin has an inhibitory effect on ROS27 can be considered as its mechanism to prevent MTX from decreasing tGSH in the oral mucosa. These findings suggest that tGSH was insufficient to neutralize ROS in the MTX group. GSH is a tripeptide consisting of L-glutamate, L-cysteine, and glycine that found in many cells, as it is known.28 GSH, when catalyzed by selenium-glutathione peroxidase, reacts with hydrogen peroxide (H2O2) and chemically detoxifies H2O2 and protects cells from ROS damage.29In our study, the amount of MDA in the TMTX group was close to that of the HG. These findings suggest that taxifolin is supported by studies that show its inhibitory effect on LPO and ROS production.10 Our experimental results and this information obtained from the literature point to the relationship between oxidative stress and inflammation. It has been reported in the literature that ROS, which cause oxidative stress, play an important role in the regulation of inflammation.30 Moreover, it has been stated that mitochondrial ROS are signaling molecules that trigger pro-inflammatory cytokine production.31 It was reported that GSH and enzymatic antioxidant levels decreased in cells with increased levels of TNF-α, IL-1β, IL-6, and LPO in a study evaluating the relationship between oxidant and pro-inflammatory cytokine.32 It was shown that antioxidants excessively produced ROS and reduced inflammatory responses in another study.30 Taxifolin with MTX significantly prevented the increase in TNF-α, IL-1β, and IL-6 in our study, which overlaps is in accordance with the literature. There were no studies evaluating the effects of taxifolin on TNF-α, IL-1β, and IL-6 in the inner cheek and tongue tissues of rats treated with MTX. However, the nephroprotective effect of taxifolin of inhibiting the increase in MDA, TNF-α, and IL-1β in the kidneys with acrylamide is a known fact.33 Inhibition of IL-6 is also involved in the pathogenesis of the anti-inflammatory effect of taxifolin.34Our biochemical findings showed that MDA, TNF-α, IL-1β, and IL-6 increased and tGSH decreased in the MTX group. In a study that support our findings, it was reported that MTX increased the levels of MDA, IL-1β and TNF-α in the oral mucosa.35 Moreover, MTX caused inflammatory cell infiltration, hemorrhage, and muscle fiber degeneration in the inner cheek tissue. Inflammatory cell infiltration was observed in the inner cheek of rats treated with MTX in a study by Bilginaylar, et al.36 (2022). Furthermore, it has been reported that MTX produces degenerative changes in the epithelial cells of the inner cheek and tongue surface.37 The fact that taxifolin alleviated histopathological damage in the inner cheek and tongue tissues due to MTX may be due to its antioxidant and anti-inflammatory effects. The role of ROS and various pro-inflammatory cytokines in the pathogenesis of mucositis has been shown in the literature.9Our biochemical findings, obtained from inner cheek and tongue tissue samples, were in accordance with the histopathological findings in our study. Papillary structures disappeared, epithelial thickness decreased, and there was swelling in the tongues of rats treated with MTX. These histopathological findings are also in accordance with previous studies.The development of oxidative and inflammatory damage in the inner cheek and tongue tissues of rats treated with MTX was evaluated biochemically and histopathologically. Taxifolin antagonized MTX-induced oxidant and pro-inflammatory cytokines increased and the antioxidant property decreased in the inner cheek and tongue tissues. Taxifolin also reduced the histopathological damage associated with MTX administration in the inner cheek and tongue tissues. Our biochemical and histopathological findings suggest that taxifolin may be useful in the treatment of MTX-induced oral mucositis. More detailed studies are needed in the future to clarify the protective effect of taxifolin against MTX-induced oxidative and inflammatory oral mucositis.