Considering the effect of regions, the MT and PM GlutoPeak indices are powerful parameters to discriminate whole wheat flour samples by their gluten strengths. In conclusion, the GlutoPeak test can be a powerful and reliable tool for prediction of refined and unrefined wheat quality without being time-consuming.The study aims to accurately detect gluten quality of whole wheat flour without a refining process by measuring gluten aggregation properties with a novel and non-destructive chemometric technique called GlutoPeak, coupled with principal component analyses (PCA) and hierarchical cluster analyses (HCA). For this purpose, whole wheat flour samples from 125 common bread wheat cultivars were analyzed for protein content (PC), wet gluten content (WGC), and Zeleny sedimentation value (SV). The correlations of GlutoPeak indices  with other conventional wheat quality parameters were evaluated. Results indicated that MT had high correlations with WGC ( Gluten quality is the most important criteria both to characterize the flour performance and to decide its end use in the cereal industry . The effWidescale innovative analytical techniques based on chemical, electrophoretic, immunological, and spectroscopic factors have been developed and applied to determine and evaluate the gluten quality of wheat cultivars . EmpiricGlutoPeak is a quick tool that can determine the gluten quality and fingerprint, and classify the wheat-based flours, measure the main mixing parameters , characterize the gluten components, and predict the rheological behavior of different wheat flours ,5,6,7. TIn the present study, many unrefined wheat flour samples were evaluated by both conventional chemical quality tests used in cereal chain and a recently introduced chemometric GlutoPeak test method. Correlation between results obtained from both conventional and GlutoPeak measurements were examined. It is strongly believed that the results of this study will provide valuable information about the general chemical composition of selected whole wheat flour samples. The results enable us to classify the whole grain flour samples by their end-product functional properties at raw material receiving stage. Moreover, this study demonstrates the GlutoPeak usability as a fast and reliable evaluation method, particularly for millers and wheat breeders.A total of 125 winter wheat varieties harvested during the 2018–2019 growing period in rainfed conditions were obtained from different experimental farms located in Turkey  . Wheat sn = 125) were determined according to the AACC method 44-01, 08-01, 46-12, 38-10, and 56-61A, respectively [Moisture content (MC), ash content (AC), protein content (PC), wet gluten content (WGC), and Zeleny sedimentation value (SV) of whole wheat flour (WWF) samples , maximum torque , torque 15 s before MT , and torque 15 s after MT .The gluten aggregation properties of whole wheat flour samples were determined using the Brabender GlutoPeak device  as described by  with som®19, 2020) at a significance level of 0.05. Tukey’s test was used to differentiate among the mean values (r) were determined to analyze the relationship between the GlutoPeak indices and the commercial gluten quality parameters. For evaluating the sample discrimination, principal component analysis (PCA) and hierarchical cluster analyses (HCA) were also employed.All measurements (mean ± standart deviation) were performed in duplicate. Statistical analyses were conducted using one-way ANOVA  are provided in Conventional quality parameters and GlutoPeak indices of WWF samples (p < 0.05). Among the chemical parameters, ash contents of WWF samples changed the values between 1.4% and 2.1%. Ash content, one of the most important chemical quality parameters, determines the technological properties, mineral content, extraction rate, and nutritional labelling of flour samples [It was discovered that region and variety had significant effects on both conventional and GlutoPeak quality parameters of WWF samples  between conventional quality parameters and GlutoPeak indices are provided in The correlation coefficients  was found between PMT and WGC among all samples, regardless of region. Since PMT provides information about gluten aggregation kinetics, the wheat varieties having stronger gluten with higher WGC indicated a high peak (MT) with a shorter peak time (PMT) [r = 0.589, p < 0.05) and a strong correlation with WGC . PMT and MT values are crucial indices to discriminate flour samples as weak and strong due to their gluten qualities [r = 0.511, p < 0.05) and with WGC , as listed in Generally, a negative moderate correlation (me (PMT) ,16. MT hualities . Resultsualities . Therefor = −0.782, r = −0.747, r = −0.687, r = 0.659, p < 0.05) between PMT and WGC values of flours, while Corum demonstrated a negative moderate correlation . The MT GlutoPeak indices of flour samples from Edirne produced a very strong positive correlation with both PC  and WGC , which are important conventional quality parameters. Additionally, MT of flour samples from Eskisehir, Erzurum, and Corum had positive strong correlations both with PC  and WGC . These very strong and strong correlations have been supported by previous findings, suggesting that the MT value is a critical parameter to differentiate protein quality of whole wheat flour samples [r = 0.626, p < 0.05) and WGC  in Corum, while it demonstrated a positive moderate correlation with PC  in Erzurum and with SV  in Edirne. Although PM value does not have as strong of a discrimination power as other GlutoPeak parameters, such as MT and PMT, the PM value has a strong correlation with the gluten strength of whole wheat flours [r = 0.908 for Edirne, p < 0.05), strong , and moderate correlations  with PC, except Adana. Only in the Urfa region, a negative very strong correlation  between PM and PC was observed. The flour samples from the Erzurum, Edirne, Eskisehir, and Corum regions produced very strong  and strong correlations  in descending order between GlutoPeak PM index and conventional WGC parameters. Overall, protein contents  of whole wheat flour samples have been significantly affected by environmental conditions . Therefore, correlations between GlutoPeak and conventional properties of whole wheat flour samples from different regions (in rainfed conditions at about 21–24 °C) demonstrated a range from moderate to very strong correlations due to their changing protein contents and gluten qualities.The effect of region on the correlation between GlutoPeak indices and conventional quality parameters was also portrayed in  samples ,17. Amon samples . In thist flours . PM valuThe results of PCA obtained from 9 variables and hierarchical clustering (HCA dendogram) of conventional and GlutoPeak gluten quality parameters were presented in In In the present study, GlutoPeak and conventional test parameters of whole wheat flours from different regions of Turkey were compared. The results prove that it is possible to accurately predict some conventional quality indices of whole wheat flour samples in the GlutoPeak test. Significant correlations obtained from results enabled us to discriminate the whole wheat flours with varying gluten qualities. GlutoPeak indices, particularly MT and PM values, demonstrated strong correlations with protein content, wet gluten content, and Zeleny-sedimentation values of whole wheat flour samples. The results of this study have strongly indicated that GlutoPeak is a promising instrument for testing the gluten quality of both refined and whole wheat flours without being time consuming. Therefore, gluten quality of different wheat flours can be determined accurately and rapidly using the GlutoPeak test method, benefitting both wheat breeding programs and the flour milling industry.


Polycomb repressive complex 2 (PRC2) is a multisubunit histone-modifying enzyme complex that mediates methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) is an epigenetic hallmark of gene silencing. PRC2 plays a crucial role in a plethora of fundamental biological processes, and PRC2 dysregulation has been repeatedly implicated in cancers and developmental disorders. Here, we review the current knowledge on mechanisms of cellular regulation of PRC2 function, particularly regarding H3K27 methylation and chromatin targeting. PRC2-related disease mechanisms are also discussed. The mode of action of PRC2 in gene regulation is summarized, which includes competition between H3K27 methylation and acetylation, crosstalk with transcription machinery, and formation of high-order chromatin structure. Recent progress in the structural biology of PRC2 is highlighted from the aspects of complex assembly, enzyme catalysis, and chromatin recruitment, which together provide valuable insights into PRC2 function in close-to-atomic detail. Future studies on the molecular function and structure of PRC2 in the context of native chromatin and in the presence of other regulators like RNAs will continue to deepen our understanding of the stability and plasticity of developmental transcriptional programs broadly impacted by PRC2. Drosophila melanogaster  is a multisubunit chromatin-binding complex, which also harbors intrinsic enzymatic activity responsible for methylation of histone H3 lysine 27 (H3K27) –5; trimenogaster . Reversinogaster –16. On tnogaster ; loss ofnogaster . This rePRC2 impacts gene expression at the transcriptional level in large part through H3K27 methylation , 19, 20.Drosophila, PRC2 is targeted to chromatin via DNA motifs known as Polycomb response elements (PREs) and cognate transcription factors; in comparison, how the chromatin recruitment of mammalian PRC2 is achieved remains incompletely understood, primarily due to the lack of conservation of PREs in mammals (PRC2 is preferably targeted to CpG islands (CGIs) in cells , 30. At  mammals –38. Unli mammals , 40. Acc mammals . PRC2 is mammals , 42. For mammals . In cont mammals , 44. ProA functional mammalian PRC2 holo complex consists of core and accessory subunits –48. OtheDrosophila PCL protein, and among them MTF2 is the most abundant in mESCs. In the absence of AEBP2, MTF2 and JARID2 can form an atypical hybrid holo complex with the core subunits  . Both gaIn line with its role in tumor suppression, loss-of-function of PRC2 caused by inactivating mutations or chromosomal translocation of essential components of PRC2, including EZH2, EED, and SUZ12, is frequently found in myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), T cell acute lymphoblastic leukemia (T-ALL), and malignant peripheral nerve sheath tumors (MPNSTs) , 68. Notvia cell signaling and metabolic pathways. For example, EZH2 phosphorylation by the AKT kinase suppresses PRC2 enzymatic activity and promotes hormone-refractory prostate cancer independent of other PRC2 subunits  transcription machinery and to orchestration of high-order chromatin structure.Drosophila embryos and mESCs have shown that H3K27ac and H3K27me3 are antagonistic, suggesting a direct competition between PRC2 and p300/CBP in posttranslational modification of H3K27  of CBX proteins  bound to a minimal fragment of SU(Z)12 (homolog of SUZ12 in Drosophila) represent the initial attempts to tackle the puzzle of complex assembly  of EZH2 associates with the bottom face of the EED WD40 repeat, dominating the interaction of these two proteins  domain and the SBD (SANT1-binding domain) of EZH2 mediate intramolecular interactions (6.2 below).ractions , 139. Thractions . The TADractions . These tractions , 81, 146ractions . Intriguractions . Indeed,ractions –150. In C2 domain, a mediator of mutual exclusivity and PRC2 dimerization. In the accessory subunit-binding module of PRC2, SUZ12(N) contains a mobile non-canonical C2 domain, which becomes ordered only when bound to the accessory subunit PHF19 or AEBP2  domain of PHF19 (PHF19(RC)) mediates concurrent binding to the C2 domain of SUZ12 from one protomer and the SUZ12(N)–RBBP4 protein body from the other protomer  domain and ZnB helix of SUZ12 assemble into a binding platform for the TR domain of JARID2 (JARID2(TR)) (ID2(TR)) . An overID2(TR)) . The N-tID2(TR)) . In lineID2(TR)) , 159.EZH1 and EZH2 represent a distinct lysine methyltransferase family . The SETThe catalytic mechanism, cancer mutations, and SAM-competitive inhibitors. Histone substrate recognition and methyl transfer reaction are two key steps for PRC2-mediated H3K27 methylation. The A25-R26-K27-S28 core sequence within the histone H3 tail and in particular residue R26 are read by the SET domain directly; residue A25 also contributes to the substrate specificity by the mechanism of steric exclusion , 35 bp, or 40 bp linker DNA, are captured mostly engaged with the catalytic module of PRC2  and XL (2nd author) wrote the manuscript. All authors contributed to the article and approved the submitted version. XL (1The work in the Liu laboratory was supported by Welch Foundation research grant I-1790 and NIH grants GM121662 and GM136308. Xin Liu is a W. W. Caruth, Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


During the study, it was observed that the activity of enzymes such as lipase and polyphenol oxidase, was significantly reduced under certain conditions of scCO2 treatment, while the enzymes α-amylase and protease show better stability. In particular, polyphenol oxidase was effectively inactivated below the 60% of preserved activity at 200 bar and 3 h, whereas α-amylase under the same conditions retained its activity. Additionally, the moisture content of the scCO2-treated spelt flour was reduced by 5%, and the fat content was reduced by 58%, while the quality of scCO2-treated flour was maintained. In this regard, the sustainable scCO2 process could be a valuable tool for controlling the enzymatic activity of spelt flour since the use of scCO2 technology has a positive effect on the quality of flour, which was verified by the baking performance of spelt flour with the baked spelt bread as an indicator of quality.In this research, a new approach to enzyme inactivation in flour was presented by supercritical technology, considered a sustainable technology with lower energy consumption compared to other technologies that use ultra-high temperature processing. Total protein concentration and the activity of enzymes α-amylase, lipase, peroxidase, polyphenol oxidase, and protease were determined in flour pre-treated with scCO It is well known that supercritical fluids are substances above their critical temperature and pressure [2 under supercritical conditions is an approach that offers many suitable properties that are crucial in food processing [2 is available with a relatively low price, and it is approved as a solvent in the food industry [2 is not considered as an expensive gas and process temperatures are particularly low. Heat consumption is not required, which is important from an economic and ecological point of view. Certainly, the most expensive part is the process equipment for scCO2 technology. However, the most important fact is an important and positive environmental impact due to the high-pressure processing implementation, as a food production process is achieved because of substantial water and energy savings. The use of sustainable scCO2 technology, where CO2 is considered as a green solvent, provides processes which attract considerable interest. In this paper, some relevant advantages of using scCO2 to improve the quality of flour and, consequently, bakery products are presented. Based on these facts, it is highly recommended from an environmental, safety, and economical point of view to develop alternative routes for reducing the activity of undesirable enzymes. Furthermore, due to the extension of the shelf-life of processed products, this allows a significant reduction of food waste [2 treatment is an expensive investment, but the costs of serial production are expected to be lower, especially for CO2, which is considered a cheap gas.The food industry is currently facing several challenges, where the biggest concern is the consumer’s decision to select food for consumption. Today’s technologies are mainly aimed at providing the consumer with quality food, as minimally processed as possible, but, at the same time, with the possibility of maintaining the freshness of food for a long time. Therefore, researchers have recently been focusing on research that would achieve higher food quality and extend the shelf life of a particular food . For sevpressure . Their ipressure . High-processing . In addiindustry . The advod waste . On the 2 can be achieved at relatively low pressure and temperature (7.38 MPa and 31.2 °C) [2 is the most widely used supercritical fluid, with the very important possibility of achieving a non-thermal (35–55 °C) process. In relation to that, our research focused on enzyme inactivation in spelt flour, which was exposed to various scCO2 conditions. Additionally, the effects of the non-thermal scCO2 processing of the spelt flour on the nutritional compounds, physicochemical properties, sensory attributes, and bread quality, as well as its enzymatic inactivation, were discussed. The supercritical phase of CO31.2 °C) , so as n2 in certain foods are summarized in In the beginning, it is crucial to understand the importance of specific enzymes and their role in the food production. Therefore, the results of enzyme inactivation by scCO2 technology for inactivation of enzymes in fruit and vegetables, enzymatic browning and off-flavor formation is prevented. These changes lead to degradation and quality changes of juices during storage. Enzymatic browning is a major problem in the beverage industry (apple juice), as PPO oxidizes the o-diphenols to quinones, leading to polymerization, which produces brown pigment (melanin) [Furthermore, with the use of scCOmelanin) . Our res2 processing are usually below 50 °C. ScCO2 technology works with pressures between 8 and 60 MPa [2 technology for the inactivation of polyphenol oxidase (PPO). The optimal process conditions were obtained at 37 °C, 25 MPa, and 10 min, which are very similar to the conditions used in this study. Moreover, only 10% of PPO residual activity was maintained [2 processing of apple juice, aiming to inactivate PPO, both achieving total PPO inactivation, but Ferrentino and Spilimbergo used lower pressure conditions and shorter treatment time, which are more economical [2. The temperatures during scCOd 60 MPa . Zhang eintained . Murtazaonomical ,22. In aonomical . The cau2 conditions on the activity of specific enzymes in spelt flour. Obtained results were an important accomplishment for the further use of spelt flour in the production process. However, it must be known what reactions are catalyzed by specific enzymes in spelt flour and why additives in the form of enzymes are increasingly being used in the bakery industry, ultimately contributing to improved production. Previously published studies have focused mainly on the addition of certain enzymes to flour and the consequent monitoring of the rheological characteristics of the dough by the addition of different enzyme concentrations. In our research, the emphasis was on already existing enzymes in spelt flour, which, with the green approach using scCO2 technology, improve or reduce the enzyme activity according to the individual type of enzyme. Therefore, the novelty of this study was the approach by which the inactivation of enzymes in flour occurs by the use of scCO2 technology. So far, there has been a greater emphasis on maintaining quality in preserving fruit and vegetables and inhibiting the growth and reproduction of microorganisms [2 technology in flour, where the importance of enzymes has not been well researched so far. It is of the utmost importance that this process does not negatively affect the quality of flour as a raw material for bakery products. It is definitely necessary to have a good knowledge about the enzymes present in the flour and their function, especially in the production process of the final product and its quality.Our study included research on the influence of different scCOrganisms . UpgradiTriticum spelta) from the company Hlebček d.o.o., Pragersko, Slovenia. Ethanol (96%), phosphoric acid (≥85%), sodium chloride (≥99.5%), Coomassie brilliant blue G250 (1.15444.0025), and acetonitrile (99.9%) were supplied from Merck. Chicken egg albumin (≥98%), sodium acetate (≥99.0%), acetic acid , and p-nitrophenyl butyrate (≥98%) were supplied from Sigma. The enzymes α-amylase (~30 U/mg) from Aspergillus oryzae and protease (≥0.6 U/mg) from Aspergillus saitoi were obtained from Sigma, while lipase (~200 U/g) from Aspergillus niger was obtained from BioChemics. Peroxidase (232-668-6) from horseradish was purchased from BBI Enzymes . Polyphenol oxidase (LS003793) from Agaricus bisporus was supplied from Worthington Biochemical Corporation. All the chemicals were used as received without any further purification. All other chemicals used in the laboratory were of analytical grade.Spelt flour used in this study was obtained by grinding the cleaned whole grain of spelt wheat  from a cylinder was cooled to 3 °C by a heat exchanger and delivered by a CO2 pump. After treatment with CO2, the vessel was slowly depressurized, and the sample was collected and immediately used for further protein extract preparation. Treatments were performed in triplicate.In a typical experiment, 5 g of spelt flour was weighed into a filter bag, which was then placed into the pressure vessel, where the temperature was maintained at 35 °C. A treatment time was defined as that after the system reached the set pressure and temperature. COProteins were extracted from spelt flour in accordance with the established procedure , where 5The protein concentration was determined directly by measuring protein content in the extract of flour proteins with UV-spectrophotometer using the Bradford dye-binding method . MoreoveThe activity of a specific enzyme was determined based on specific enzymatic activity assays using a UV-spectrophotometer  at different wavelengths.The α-amylase activity was determined based on the increased glucose in the solution using the DNS reagent . 1 g DNS was stirred with 20 mL of 2 M sodium hydroxide, and the mixture was diluted with 50 mL water. Afterwards, 30 g of sodium potassium tartrate was added and dissolved. The incubation mixture contained enzyme extract (0.5 mL) and 1% substrate (starch from wheat) in 0.02 M sodium phosphate buffer (0.5 mL). The incubation mixture was mixed with 1 mL DNS reagent and kept in a boiling water bath for 5 min. After cooling the incubated tubes, 10 mL reagent grade water was added. The absorbance was measured with a UV-Vis spectrophotometer at a wavelength of 540 nm.v/v) Triton by adding 0.10 mL of enzyme extract from spelt flour. The contents were mixed, and the tubes were incubated in the water bath at 37 °C for 3 min. Then, 10 µL of substrate solution (50 mM p-nitrophenyl butyrate (pNPB) diluted in acetonitrile and stored at −20 °C) was added. The activity of lipase was determined based on p-nitrophenol (pNP) reaction, which was monitored at 400 nm for 5 min. One unit of lipase activity released 1 nanomole of pNP per minute at pH 7.2 and 37 °C, using p-nitrophenyl butyrate (pNPB) as the substrate. Briefly, an incubation mixture was prepared by the addition of a solution of 100 mM sodium phosphate buffer with 150 mM sodium chloride and 0.5% , and hydrogen peroxide as substrates. Our previous research has published the method for peroxidase activity determination and detailed calculation procedure of peroxidase activity .PPO activity was determined spectrophotometrically at 265 nm by monitoring the reaction at 25 °C for 5 min. The final concentrations in the reaction mixture were 50 mM potassium phosphate, 0.17 mM L-3,4-dihydroxyphenylalanine, 0.072 mM ascorbic acid, 0.0022 mM ethylenediaminetetraacetic acid, and 50–100 units of PPO. One unit of enzyme activity was defined as the amount of enzyme causing a 0.001 change in the absorbency per minute at pH 6.5 at 25 °C in a 3 mL reaction mix .Protease activity was measured using casein as a substrate. A procedure for determining the protease activity was previously described in our research .All experiments were conducted in triplicate, and the error bar represents the percentage error (±3%) in each set of readings.p ≤ 0.05) by using IBM SPSS Amos 28.All determinations were conducted at least in triplicates. Analysis of variance (ANOVA) and Duncan’s multiple range test were employed to determine the statistical significance of the differences between the means (2-treated spelt flour.Scanning electron microscopy (SEM), attenuated total reflectance—Fourier transform infrared spectroscopy (ATR-FTIR), moisture content, and fat content were carried out for characterization of untreated spelt flour and scCOA scanning electron microscope (SEM) model Quanta FEI 200 3D microscope  was used to study the microstructure of the sample. The pressure chamber was 60 Pa, and the accelerating voltage for imaging was 10 kV. 2-treated spelt flour. To test repeatability, analyses were performed in triplicate and average spectra were used.ATR-FTIR  was used for functional group identification of untreated and scCO2 at a pressure of 300 bar and 35 °C in a high-pressure batch reactor for 24 h was determined using the Mettler Toledo HX204 Moisture Analyzer halogen moisture analyzer with a heating temperature of up to 130 °C.The moisture content in untreated spelt flour and spelt flour that was exposed to scCO2-treated spelt flour samples with petroleum ether as a solvent, according to Official Method AOAC 963.15, with minor modifications [Soxhlet apparatus was used to extract fats from untreated and SCCOications . After t2 was studied by the volumetric expansion of spelt flour saturated with CO2 on a magnetic suspension balance (MSB). The solubility of CO2 is the total amount of carbonate species that can be dissolved in the solution. Solubility of CO2 in spelt flour was measured by a gravimetric method involving magnetic suspension balance  at pressures ranging from 1 bar to 300 bar. The temperature of the cell was kept constant using a Lauda P5 thermostat. The software MessPro recorded the conditions . The duration of the measurement at each pressure was approximately 120 min, as after this time, equilibrium was reached. To define the volume of the sample, a photo of the sample was taken with a digital camera and analyzed using ImageJ software [Equilibria solubility of COsoftware .3/g) was calculated. Ten readings were taken from baked bread to determine the specific volume. Meanwhile, loaf height and crumb thickness were measured using ImageJ software, where loaves were vertically sliced in the centre.The specific volume of baked bread was determined by the water displacement method. Loaf weight was recorded, and specific volume  at 200 bar and 3 h. Oxidation of amino acids present in free and polypeptide chains is a primary mechanism of enzyme inactivation in scCO2, with additional occurrences of depolymerization of polypeptide chains and destruction of secondary structural elements of enzymes [In this study, a green approach using scCO enzymes .Pressures of 100, 200, and 300 bar were used in the experiments. The data showed that it was not always the highest pressure that caused the highest enzyme inactivation. An interesting finding was that a higher level of enzyme inactivation was achieved even with a shorter exposure time (3 h). It was shown that the α-amylase activity of flour directly affected the fermentability of flour and the quality of fermented foods, therefore the process parameters, 300 bar and 3 h, were set in continuation of the experiment, which, in most cases, represented the conditions where the highest level of inactivation of enzymes that negatively affect the further process of preparation of the bakery product was achieved.2 treatment was more effective in the inactivation of various enzymes with the prolongation of the exposure time. This technology physicochemically inactivates enzymes at a relatively low temperature (≤60 °C) and moderate pressures (8–40 MPa) without residual waste generation [2 solubilization, facilitating its contact with and penetration into food. This is the reason for higher enzyme inactivation efficiency. At the same time, higher pressure can generate the same inactivation efficiency at lower temperatures. Despite that, higher pressure can induce a decrease in pH, leading to enzyme inactivation [2 on enzyme inactivation is the formation of carbamates with lysine residues on the enzyme surface. In addition, carbonic acid is formed and pH decreases [In many studies, COneration . Howeverneration . Only thtivation . The dirssures 8– MPa with2 technology, was also achieved. In the process of scCO2, PPO in the spelt flour was inactivated to prevent the enzymatic oxidation of spelt flour. Generally, quinones in food are produced because of the reaction of naturally present phenolic compounds with PPO enzymes. In the case of flour, this can lead to the darkening of flour dough. For this reason, it was proven that the use of scCO2 successfully inactivated the enzyme PPO and thus reduced the occurrence of enzymatic browning reactions in spelt flour. As shown, the highest inactivation of PPO was reached at 200 bar and 3 h, whereas the inactivation of amylase at these conditions was around 10%.If amylases are applied the staling rate of bread, this may result in reduction, which results in the most successful enzymatic approach. Amylases catalyze the cleavage of 1,4-glycosidic bonds. The starch molecules are, therefore, degraded. Dextrin with lower molecular weight and other short-chain unbranched molecules are formed. Additionally, amylases are the reason for a greater loaf volume, improved crumb structure, and, at the same time, starch retrogradation is hindered . Therefo2, which would effectively extend the shelf-life of spelt flour. On the other hand, some flavor compounds are produced by lipid degradation and oxidation pathways. Therefore, understanding lipid degradation and oxidation pathways are important in the quality control of spelt flour and products. Lipase catalyzes the first step of lipid catabolism, which can cause deterioration of food quality . Moreove2-treated, on quality deterioration, characteristics of spelt flour and sensory analysis of a baked product, untreated and scCO2-treated spelt flour analyses were performed and evaluated below.To clarify the effects of enzymes in flour, which were previously scCO2-treated spelt flour were characterized using SEM analysis. The differences in microstructure of untreated and scCO2-treated spelt flour were obtained to contribute to the knowledge about the consequences of the use of scCO2-treated spelt flour in the bakery. In order to assess the role of scCO2 on the structure of starch grains of spelt flour, SEM and FTIR analyses were performed to confirm the differences between spelt flour before and after exposure to scCO2. The SEM images of spelt flour samples obtained before and after scCO2 treatment are shown in 2 treatment. The starch granule size of spelt flour was determined in the range of 16–35 µm. Certainly, the SEM analysis clearly showed the difference in the formation of aggregates in the size of 100 µm in untreated spelt flour, which can be explained by the fact that the moisture content in untreated spelt flour was minimally higher (4.7%) than in scCO2-treated spelt flour because of carbonic acid formation. Thus, these results were in accordance with the moisture and fat content measurements. In addition, it was observed that the scCO2-treated flour was more free-flowing, as it also contained less fat. SEM analysis confirmed that scCO2 did not adversely affect flour quality, and, in addition, changes in flour properties such as reduced fat and moisture content in scCO2-treated spelt flour were observed.The structural properties of untreated and scCO2-treated spelt flour shows the contribution of different functional groups belonging to carbohydrates, lipids, proteins, and others, according to the main chemical composition of flour. FTIR analysis was performed for untreated and scCO2-treated spelt flour using an ATR accessory. Samples’ spectra were obtained over the range of 400 cm−1 and 4000 cm−1 with a spectral resolution of 0.5 cm−1. 2-treated spelt flour, as the characteristic peaks occur at the same values in both spectra. The results demonstrated the band ~1078 cm−1 was related to the crystalline structure of starch, and the absorption band at ~995cm−1 in the starch region was linked to hydrated crystalline domains. A similar observation was reported previously by Zhou et al. in potato starch [−1), lipids , and proteins (1650–1500 cm−1) in both spelt flour samples, where minor differences, quantitative or structural, were difficult to detect [A FTIR spectrum of untreated and scCOo starch . Moreoveo detect .2 does not affect major functional groups of spelt flour.Taken together, these data demonstrate that there was no change in the intensity of the characteristic peaks of FTIR spectrum, which means that scCO2 extraction, where it was confirmed by SEM that native full-fatted quinoa starch granules were presented as aggregations. Comparable to the results of this study, it was found that scCO2 defatted quinoa samples was more disordered, presenting less aggregation between the components [2-treated spelt flour-less aggregates, and greater lightness of the sample were detected.Solaesa et al. reported structural, physicochemical, pasting, and thermal properties of quinoa defatted by scCOmponents , as was In comparison, Ma et al. treated wheat flour with superheated steam, where partial gelatinization of starch granules and protein denaturation induced starch–starch and starch–protein aggregate formation, which changed the particle size distribution and resulted in the increase in flour particle size. Specifically, the FTIR spectrum of superheated steam-treated wheat flour had lower values than native wheat flour, indicating the lower short-range order of starch molecules. This result could be due to the destruction of hydrogen bonds during superheated steam treatment, which uncoiled the double helices in starch molecules .2 medium, it was demonstrated that the fat content was reduced to 0.6%, which is 58% less fat in our scCO2-treated spelt flour sample. The lower fat content of flour also has some permeability, as this reduces the possibility of flour rancidity and, also, from a dietary point of view, flour is deficient in calories and, consequently, healthier.Chemical compositions may affect the flour properties of dough kneading (water absorption rate), gluten network formation, dough properties  and cooking characteristics , which are especially important in bakery production . The res2 technology has also reduced the fat content of spelt flour, which consequently reduced the possibility of flour rancidity. An excellent result is also that the flour maintained nutritional value and sensory quality after scCO2 treatment. That is undoubtedly an important factor influencing the durability of flour. The moisture content of the flour is important since the higher moisture content causes a lower amount of dry solids in the flour. Additionally, moisture values inform us indirectly about flour storage conditions. Flour specifications usually limit the flour moisture to 14% or less because flour with greater than 14% moisture is not stable at room temperature. Excessive moisture facilitates mould growth , since o2 used at an adequately high concentration. The freshness of foods and their shelf life is therefore extended. Moreover, the high solubility of CO2 in foods enables antimicrobial effect, whereby CO2 gas is readily soluble in aqueous and fatty foods and, at lower temperature, a higher level of solubility occures. Investigating the effect of CO2 solubility on spelt flour, the amount of CO2 (or measured solubility of CO2) in spelt flour was measured using MSB at a temperature of 35 °C. CO2 solubility was determined under the same conditions as scCO2 treatment of spelt flour in a high-pressure batch reactor to determine the connection between CO2 solubility and enzyme activity in spelt flour. As seen in 2 solubility increased as saturation pressure increased, indicating that CO2 gas dissolves, resulting in a sorption behaviour that follows Henry’s law [2 solubility of 0.77 g/g was determined at 200 bar and 35 °C, and 0.86 g/g at 300 bar and 35 °C. It has been reported that the higher level of CO2 solubility at lower temperatures favours chilled storage to allow a reasonable level of CO2 accumulation during the required shelf life of the product [Microbial growth on foods is inhibited with COry’s law . For the product .2 treatment on flour properties and the quality of baked bread. 2-treated spelt flour. It can be seen that bread made from scCO2-treated spelt flour was higher and, consequently, had a higher specific surface volume, which indicated that scCO2 has a positive effect on exposed spelt flour, where inactivation of certain enzymes and hyperactivation of α-amylase enzyme gave bread with better characteristics compared to control bread, where untreated spelt flour was used. Additionally, our findings from the determination of the activity of individual enzymes are consistent with the final product, as the presence of the enzyme α-amylase had a positive effect on the rising process, whereby it improved the internal texture of bread and increased the volume of bread. The average height of bread calculated using the ImageJ program for bread baked from untreated (control) spelt flour in three different samples was 10.2 ± 0.25 cm, while the average height of bread beaked from scCO2-treated spelt flour in three different samples was 10.4 ± 0.17 cm. In accordance with these measurements, measurements of the specific surface volume of the bread were also carried out, and it was found that the bread from untreated spelt flour has a specific surface volume 2.74 ± 0.1 cm3/g, which is slightly lower than bread baked from scCO2-treated spelt flour with specific volume 2.81 ± 0.08 cm3/g.In many studies it was found out that enzymes application leads to the improved sensory quality of bread, which is the consequence of the improved flour quality. In addition, a dough that has more machine tolerance was created, and bread structure was enhanced . The focUsually the quality of the final product changes with different processing techniques of flour or other food products. Our study shows that the quality of flour has improved, which is clear from the final product and its specific properties.2 treatment of spelt flour. This was reflected in the results of the α-amylase, lipase, POD, PPO, and protease enzyme activity measurements and other characteristic differences in the final product—bread with better properties compared to the control product from flour that was not exposed to scCO2 conditions. Fat content in scCO2-treated spelt flour significantly decreased due to scCO2 exposure, which is essential in reducing the rancidity of flour. In addition, it can be said that, by inactivating unwanted enzymes such as POD and PPO, scCO2 treatment affected the prolongation of spelt flour shelf life. In addition, high-pressure processing is more sustainable than conventional processes, which, besides the benefits in terms of non-thermal inactivation of enzymes, and the minimal effects on nutritional and organoleptic properties of food products, is considered to be environmentally friendly. A novelty of this study is the approach in which enzymes are inactivated in the flour using scCO2 technology. This study demonstrates that using scCO2 technology in flour regulates the activity of individual enzymes in flour. It is extremely important that this process does not negatively affect the quality of flour as a raw material for bakery products. The results of the present study could be used as the basis for the future optimization of spelt flour characteristics and to extend the shelf life, with further studies to be carried out. Considering the obtained results, scCO2 exposure of spelt flour can improve some rheological properties of dough and positively affect specific activities of flour enzymes that are important in the production of the final product.Consumers are becoming more aware of the benefits of living a healthier lifestyle and maintaining fat-free and other diets. During this study, indirect baking quality parameters were significantly influenced by scCO


Immunotherapy significantly improves the prognosis of advanced lung cancer. It has become an important treatment option for advanced lung cancer. However, there remain many limitations in clinical treatment, and only a small portion of patients can benefit from immunotherapy. Our study aimed to identify markers that can precisely forecast the efficacy of immunotherapy in patients. We analyzed a non-small-cell lung cancer (NSCLC) immune checkpoint inhibitor (ICI) cohort (n=240). We used this discovery cohort to identify CNVs in genes associated with immunotherapy. We further analyzed immune biomarkers and immune infiltration in The Cancer Genome Atlas (TCGA)-NSCLC cohort and the Gene Expression Omnibus (GEO) cohorts. By analyzing an ICI dataset from MSKCC, we found that progression-free survival (PFS) was improved after UBE3A deletion (UBE3A-del). The analysis results showed that UBE3A-del had higher immunocyte infiltration levels and higher expression levels of immune checkpoint biomarkers and affected the enrichment levels of immune signaling pathways. Our results suggest that UBE3A-del can be used as a predictive biomarker of NSCLC to screen for NSCLC patients who may benefit from ICI therapy. Abbreviations: NSCLC: Non-small cell lung cancer; CNV: Copy number variation; ICIs: Immune checkpoint inhibitors; TCGA: The cancer genome atlas; GEO: Gene expression omnibus; GSEA: Gene set enrichment; PFS: Progression-free survival; OS: Overall survival; TMB: Tumor mutational burden; CTLA-4: Cytotoxic T lymphocyte antigen 4; PD-(L)1: Programmed cell death (ligand) 1; MSI: Microsatellite instability; dMMR: DNA mismatch repair; SCNAs: Somatic copy number alterations; TME: Tumor microenvironment; MSK-IMPACT: The Memorial Sloan Kettering-Integrated Mutation Profilng of Actionable; Cancer Targets; FDA: Food and Drug Administration; WES: Whole-exome sequencing; SNP: Single Nucleotide Polymorphisms; FDR: False discovery rate; DCR: Disease control rate; DDR: DNA damage response and repair; MDSCs: Myeloid-derived suppressor cells; FAO: Fatty acid oxidation  Our topic is interesting and investigates a current and concrete unmet clinical need, given the lack of reliable predictors of benefit for immunotherapy in NSCLC. We first identified UBE3A-del as a biomarker of immunotherapy response from an ICI-treated cohort(the MSKCC dataset).UBE3A deletion in ICI-treated cohort.To investigate the relationship of UBE3A-del and immunotherapy, we verified it in the non ICI-treated cohorts, a TCGA dataset and two GEO datasets. After analysis, we determined that UBE3A-del has a close relationship to the efficacy of immunotherapy. UBE3A-del showed higher TMB in the ICI dataset and TCGA-NSCLC dataset. UBE3A-del showed a better disease control rate (DCR).We found that PFS of patients was improved after UBE3A-del mRNA expression was lower and that the CNV fraction was positively correlated with RNA expression, UBE3A-del methylation degree was higher, expression of some proteins was higher after UBE3A-del.Difference analysis of TCGA transcriptome data showed that UBE3A-del enhances immune infiltration. Our results showed that immune genes, immune cells, chemokines and immune checkpoint markers were significantly upregulated in UBE3A-del. Our results show that UBE3A-del enhances antitumour immunity and immunogenicity.UBE3A-del is enriched in the immune pathway. Our results showed that UBE3A-del activated the DNA damage repair mismatch complex pathway and immune pathway.The rise of tumor immunotherapy is setting off a new revolution in tumor treatment. Compared with the traditional curative effect, the lasting curative effect is an important advantage of immunotherapy. Immunotherapy has made rapid progress in lung cancer, bringing new hope for advanced lung cancer and becoming an important treatment for advanced lung cancer. However, only a few people show sustained responses, and we need to search for effective biomarkers that can identify potential beneficiaries .Previous studies have shown that PD-L1 expression, tumor mutational burden (TMB), microsatellite instability (MSI), deficient DNA mismatch repair (dMMR), immune microenvironment (CD8 + T cell and immune factors), and neoantigen load can predict the response of various tumor types to ICIs . HoweverPrevious studies have mostly followed the effects of somatic mutations on immunotherapy . In the UBE3A gene deletion (UBE3A-del) showed higher TMB and better PFS, and CNV level of UBE3A was correlated with mRNA, methylation and proteomics. After UBE3A gene deletion, a better curative effect was achieved by increasing immunocyte infiltration and activating immune pathways [In this study, CNV and clinical information from multiple databases were collected to identify gene CNVs associated with ICI efficacy. We found that pathways .Figure https://www.cbioportal.org), a dataset of 240 non-small-cell lung cancer patients was used. MSK-impact sequencing was used in our study, an entire genomic analysis panel authorized for use by the Food and Drug Administration (FDA) [To assess the influence of SCNA on the curative effect of ICIs, we analyzed a discovery cohort. From HIRA Rizvi et al. (on (FDA) .https://www.cbioportal.org) [We further analyzed the immune biomarkers and immune infiltration in TCGA-NSCLC, and 586 patients with NSCLC were collected from the TCGA dataset  , mainly We used GISTIC 2.0 to analyze the arm-level SCNA of copy number . For TCGUBE3A-del and UBE3A-wt were compared, and the outcomes were visualized by the R package ggpubr.The CIBERSORTx deconvolution method was used to estimate the levels of tumor immunocyte infiltration ; 22 typeWe used edger to analyze the transcriptome data of the TCGA-NSCLC and GEO cohorts. Then, we used clusterProfiler to analyze the correlation of CNVs and the activation levels of pathways ,26. The UBE3A-del. Kaplan–Meier survival analysis and Cox regression analysis were used to analyze the PFS and OS of UBE3A-del and UBE3A-wt. We used the R package ggstatplot to estimate the relationship between CNV status and mRNA and methylation in the TCGA dataset. The limma R package was used to analyze the protein chip expression of UBE3A-del and UBE3A-wt in the TCGA dataset [We used the Mann–Whitney U test to analyze TMB discrepancies, mRNA expression, methylation values, immunocyte infiltration levels and immune checkpoint biomarkers from  dataset . We usedWe performed GISTIC analysis on 586 NSCLC samples from the TCGA cohort . The resABCG1, and 662 genes were significantly deleted (P < 0.05), such as UBE3A. Then, the genes with significant amplification and deletion were verified in the TCGA cohort. According to the CNV amplification or deletion score and RNA expression in the TCGA cohort, a total of 36 genes with correlation coefficients > 0.6 were all significantly deleted genes, such as UBE3A and SPG11. After analysis, we determined that UBE3A-del has a close relationship to the efficacy of immunotherapy. UBE3A-del showed higher TMB in the ICI dataset and TCGA-NSCLC dataset   and immune checkpoint markers  were significantly upregulated in UBE3A-del  and immune checkpoint markers  was significantly upregulated in UBE3A-low cells  and hinder the immune response of natural hosts, thus affecting the efficacy of ICIs [UBE3A-del patients had higher TMB and longer PFS related to immunotherapy, and the CNV of UBE3A was positively correlated with mRNA expression and negatively correlated with methylation degree, which was consistent with previous research results [UBE3A-del exceeded that in UBE3A-wt.Although a growing number of studies have demonstrated that chromosome CNV is relevant to ICIs ,19, therhibitors . Yuchen  results ,27,39. T results . Hassan  results . In our BRAF, KRAS, and MET was higher after UBE3A-del. J Mazieres et al. reported a multicentre study on immunotherapy in 551 NSCLC patients in 2019. The study found that the KRAS gene-driven lung cancer ICI treatment response rate was the highest at 26%, followed by BRAF, with a response rate of 24%, and the MET-driven lung cancer ICI response rate was 16%, which were relatively higher than those of other driver genes [UBE3A-del was highly infiltrated in immunocytes, highly expressed immune genes, and affected immune-, autophagy-, and DNA damage repairable pathways. These sequencing data analyzed based on various databases increased the evidence of the close relationship between UBE3A-del and NSCLC ICI efficacy, which supported epigenetics and immunotherapy.We analyzed the common driver genes of NSCLC and discovered that the mRNA expression of er genes . In addiUBE3A-del, which is closely related to NSCLC immunotherapy. UBE3A is also known as ubiquitin protein ligase E3A. The ubiquitin proteasome pathway participates in the regulation of the cell cycle, which is the principal protein degradation pathway, signal transduction and DNA damage repair. We summarized the possible mechanism of UBE3A in the treatment of ICIs  promote tumor growth by increasing fatty acid uptake and activating fatty acid oxidation (FAO) to inhibit T cell immunity, thus promoting the proliferation and migration of malignant cells. The inhibition of FAO alone could significantly delay the growth of T cell-dependent tumors and increase adoptive T cells and the antitumour effect of treatment [UBE3A-del resulted in the upregulation of the chemokines CXCL9 and CXCL10, the enrichment of the DDR pathway and the inhibition of the metabolic pathway, which were consistent with the findings of previous reports.Previous studies have revealed that the Th1 chemokines blockade . Min Yueblockade ,41. N Aublockade . Previoublockade . In our reatment . Mauro Dreatment . In our UBE3A-del in only the immunotherapy of NSCLC, which is unknown for other tumors, and lacked verification in experiments and clinics. Further validation results are still needed to verify the predictive value of UBE3A-del in the immunotherapy of NSCLC, and it is necessary to expand to other tumors to further analyze its predictive value for immunotherapy.Although UBE3A-del has significant predictive value for NSCLC, the limitation of this study is that we only used bioinformatics to analyze the predictive value of UBE3A-del patients have better PFS with ICIs. Consequently, UBE3A-del can be used as a new marker for the ICIs of NSCLC. We still need to verify this result in the clinic and expand the predicted tumor range in clinical practice.Our study provides solid evidence that Click here for additional data file.


In order to improve the water solubility and stability of lutein, soy protein isolates (SPI) and their hydrolysates via pepsin (PSPI) and alcalase (ASPI) were used as nanocarriers for lutein to fabricate the lutein-loaded nanoparticles (LNPS) of SPI, PSPI, and ASPI. The encapsulation properties, light, and in vitro digestive stability of lutein in nanoparticles, and protein–lutein interactions were investigated. Compared with SPI-LNPS and ASPI-LNPS, PSPI-LNPS was characterized by uniform morphology (approximately 115 nm) with a lower polydispersity index (approximately 0.11) and higher lutein loading capacity (17.96 μg/mg protein). In addition, PSPI-LNPS presented the higher lutein retention rate after light exposure (85.05%) and simulated digestion (77.73%) than the unencapsulated lutein and SPI-LNPS. Fluorescence spectroscopy revealed that PSPI had stronger hydrophobic interaction with lutein than SPI, which positively correlated with their beneficial effects on the light and digestive stability of lutein. This study demonstrated that PSPI possessed significant potential for lutein delivery. Lutein, a type of natural carotenoid found in vegetables, fruits, and eggs , is beneEmulsion systems were widely used to encapsulate carotenoids. However, fabricating lutein-loaded nanoemulsions caused a low encapsulation efficiency due to the homogenization process . FabricaSPI has a much higher molecular weight than animal protein, and the hydrophobic groups are mostly embedded. Due to their high molecular weight and limited hydrophobic binding sites on the surface, hydrophobic nutrients have a lower loading capacity in nanoparticles than animal proteins . ThroughThis study sought to increase lutein’s encapsulation effect via enzymatic hydrolysis of SPI, thereby increasing lutein’s light and in vitro digestive stability. First, SPI hydrolysates were prepared using pepsin (PSPI) and alcalase (ASPI), and their structural properties and lutein encapsulation properties were analyzed. The lutein-loaded SPI hydrolysates nanoparticles with the best encapsulation properties and particle characteristics were chosen for comparison to unencapsulated lutein and lutein-loaded SPI nanoparticles in terms of stability against light and in vitro digestion. Meanwhile, the surface hydrophobicity, fluorescence spectroscopy, circular dichroism spectrum, Fourier transform infrared spectrum, and X-ray diffraction analysis were used to investigate the potential mechanism leading to the different encapsulation effects and lutein stability when SPI and its hydrolysates nanoparticles were used. This work will aid in the improvement of lutein’s aqueous solubility, stability, and bioavailability, and the development of lutein-enriched nutraceuticals.Lutein (purity > 90%), alcalase (enzyme activity > 200 U/mg), bile salts, and molecular weight standards including bacitracin, aprotinin, myohemoglobin, bovine serum albumin, and thyroglobulin were purchased from Shanghai Yuanye Biotechnology Co., Ltd. . Soybeans were bought from Fengyuan Zhongye Co., Ltd. . Pepsin (enzyme activity > 2500 U/mg), trypsin (enzyme activity > 2500 U/mg), 8-aniline-1-naphthalensulfonic acid (ANS), and trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma-Aldrich Co., . All other reagents and solvents were of at least analytical grade.v/v). After drying the organic solvent in the air for 12 h, the defatted soy flour was blended with deionized water at a 1:10 (w/v) ratio with the pH adjusted to 8.0 using 2 M NaOH. The dispersion was stirred for 2 h at a constant pH of 8.0 and centrifuged for 20 min at 10,000× g, and the supernatant was adjusted to pH 4.5 with 2 M HCl and centrifuged at 3300× g for 20 min  The precipitate was redissolved in deionized water at a ratio of 1:4 (w/v) for at least 3 h while maintaining a pH of 7.0. The solution was lyophilized to yield SPI with a protein content of 90.23%, as determined by the Kjeldahl method. The SPI was stored at −80°C prior to use.SPI was prepared from soybeans with reference to a previous publication . First, w/v) after overnight hydration. Enzymatic hydrolysis both took 45 min using pepsin  and alcalase , respectively. Enzyme activity was 2500 U/g protein for pepsin and 2000 U/g protein for alcalase. After adjusting pH to 7.0, the solution was boiled for 10 min at 100 °C in a water bath to deactivate enzymes and then rapidly cooled. After centrifuging the solution at 8000× g for 15 min, the supernatant was collected and lyophilized to yield PSPI and ASPI. PSPI and ASPI were stored at −80°C prior to use.PSPI and ASPI were prepared in a manner similar to that used by other researchers ,19. SPI tot is the total concentration of broken peptide bonds after thorough hydrolysis, which is 7.8 mmol/g for soy protein.The DH of PSPI and ASPI was determined using the TNBS assay described by Adler-Nissen . SpecifiThe molecular weight distribution of SPI and its hydrolysates was determined using an HPLC system  equipped with a size exclusion chromatography column according to the method described by Jiang et al. . The tesChromatography column: protein KW-804 ; flow rate: 1 mL/min; wavelength: 220 nm; column temperature: 25 °C; mobile phase: 50 mM phosphate buffer (pH 7.0) containing 30 mM sodium chloride; injection volume: 10 μL; sample concentration: 10 mg/mL. Molecular mass standards include bacitracin, aprotinin, myohemoglobin, bovine serum albumin, and thyroglobulin. The molecular weight of the protein was determined using the regression equation for molecular weight. Furthermore, the standard curve was plotted with the retention time of the standard as the abscissa, and log (MW) as the ordinate to obtain the regression equation.β-mercaptoethanol to a concentration of 2 mg/mL, and the mixtures were boiled for 5 min at 100 °C. A standard medium molecular weight protein was used as a molecular weight marker. Each lane was loaded with 15 μL of the sample, and the initial voltage was set to 80 V. The voltage was increased to 120 V once the samples reached the separating gel. Staining with BeyoBlue™ Coomassie Blue Super Fast Staining Solution and destaining with distilled water was performed prior to scanning the gel image with Bio-Rad’s Image Capture System.SDS-PAGE images of SPI, PSPI, and ASPI were analyzed by SDS-PAGE according to the method of Jiang et al. . The sta0) was determined using the ANS probe assay  is the final lutein concentration (μM); Ksv denotes Stern-Volmer quenching; kq denotes the quenching rate constant; and τ0 denotes the fluorescence lifetime of the fluorophore in the absence of quenching, which is typically 10−8 s.The Stern-Volmer equation was used to investigate the fluorescence quenching process of protein, in order to clarify fluorescence quenching mechanisms.q confirms the quenching type. The binding constant (Ka) and the number of binding sites (n) for static quenching can be determined using the Stern-Volmer logarithmic equation as follows:The value of kSecondary structures of SPI, PSPI, and their mixtures with lutein were determined using the method of Shen et al.  on a Chiw/w) with freshly dried KBr powder and compressed to a pellet for measurement. The scanning wavelength was adjusted to be between 500 and 4000 cm−1.The FT-IR and XRD spectra were obtained using the method described by Li et al. . The FTIA D2 Phaser diffractometer was used to obtain XRD spectra (Bruker). The 2θ angle was adjustable between 5° and 50° with a step size of 0.05°.p < 0.05, a statistical difference existed.The experiments were repeated three times, and data were presented as the mean ± standard deviation (SD). Statistix 9.0 software was used to conduct the statistical analysis . Significant differences were determined by the least significance difference (LSD) method. When Enzymatic hydrolysis of food-derived protein for bioactive compound nanoencapsulation has been widely reported previously ,17. DiffPSPI and ASPI had a DH of 6.00% and 14.96%, respectively , indicatα, α′, and β subunits of β-conglycinin remained in PSPI, whereas the A and B subunits of glycinin were mostly hydrolyzed, which was consistent with the findings of Chen et al. [β-conglycinin was found to be greater than that of glycinin [0 of PSPI, with a higher proportion of β-conglycinin subunits, was greater than that of ASPI with no glycinin or β-conglycinin subunits remaining . When lutein was encapsulated with PSPI, the retention rate increased by 5.52% (p < 0.05), when compared with SPI-LNPS. After intestinal digestion, SPI-LNPS retained lutein at a non-significantly higher rate (p > 0.05) than unencapsulated lutein, whereas PSPI-LNPS retained lutein at a significantly higher rate with an improvement of approximately 8.40% (p < 0.05). Jiao et al. [As illustrated in In order to understand why PSPI-LNPS had a better encapsulation performance and stability against light and in vitro digestion, fluorescence emission spectroscopy was used to analyze the interaction between protein and lutein. Due to the presence of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues in proteins, endogenous fluorescence can occur . The fluIn The fluorescence intensity of SPI and PSPI decreased as lutein concentration was increased a,b, indi0/F and lutein concentration was observed in both SPI and PSPI. The Stern-Volmer equation was used to calculate the quenching constant Ksv and the quenching rate constant kq. As the values of kq in SPI-LNPS and PSPI-LNPS were significantly greater than 2.0 × 1010 M−1 s−1, the quenching mechanism was static [a) and the number of binding sites (n).As illustrated in s static . In ordea and n for lutein-PSPI were both greater than those for lutein-SPI, indicating that lutein had a greater binding capacity for PSPI than SPI. The stronger interaction between lutein and PSPI was positively associated with lutein’s increased loading capacity and increased stability against light exposure and a simulated digestive environment in PSPI-LNPS. This result was also consistent with a previous work of Ma et al. [0 than SPI  were used to encapsulate lutein. Compared to SPI-LNPS, PSPI-LNPS demonstrated increased lutein loading capacity, uniform particle size distribution, and improved lutein stability against light exposure and simulated gastric environment, whereas ASPI-LNPS demonstrated the poorest encapsulation properties and particle characteristics, which were positively related to the


For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of Moco involves the consecutive action of numerous enzymatic reaction steps, whereby molybdenum insertases (Mo-insertases) catalyze the final maturation step, i.e., the metal insertion reaction yielding Moco. This final maturation step is subdivided into two partial reactions, each catalyzed by a distinctive Mo-insertase domain. Initially, MPT is adenylylated by the Mo-insertase G-domain, yielding MPT-AMP which is used as substrate by the E-domain. This domain catalyzes the insertion of molybdate into the MPT dithiolene moiety, leading to the formation of Moco-AMP. Finally, the Moco-AMP phosphoanhydride bond is cleaved by the E-domain to liberate Moco from its synthesizing enzyme. Thus formed, Moco is physiologically active and may be incorporated into the different Mo-enzymes or bind to carrier proteins instead. Aspergillus nidulans five genes are essentially required for both, a functional nitrate reductase (NR) and xanthine dehydrogenase (XDH). As both depend on molybdenum, their simultaneous loss of function was explained by the assumption that these two enzymes depend on a common molybdenum cofactor  revealed for the first time the pterin three-ring structure found in Moco .,23.E. co2+ . Howeve-domain, . Given tnal ends . Next tonal ends . Work denal ends  indeed rnal ends . In subsnal ends . Somewhanal ends . Notablynal ends . Furthernal ends .R. norvegicus [E. coli Mo-insertase MoeA focused on the identification of the MPT-binding site and structure guided mutagenesis identified several residues putatively involved in MoeA functionality [A of the molybdate insertion reaction [2+ coordination and (ii) resides into a flexible loop, close-by the active site which was found to be disordered in the structure of the respective Cnx1 E-domain exchange variant [The first eukaryotic Mo-insertase E-domain structure to have been reported was that of the gephyrin E-domain from rvegicus . Structurvegicus  revealedrvegicus . This firvegicus . Notablyrvegicus . At thatrvegicus  and molyrvegicus ,27. Subsionality . As a maionality . This fiionality  coming aionality . Contempionality  binding ionality . Consideionality . This idionality ,27 implareaction . Furtherreaction . The mol variant . These f variant . Essenti variant  are full variant ,37,47 bu variant ,27 does Based on the molecular and structural characterization of the molybdenum insertase E- and G-domains, over time, a variety of reaction models for molybdate insertion into the MPT dithiolene moiety were suggested see , each co2+binding site exists which promotes hydrolysis [Mo-insertases are required to make molybdenum available for the molybdenum dependent reactions within the cell. Functionalization of molybdenum, however, requires the concerted action of the two Mo-insertase domains  which catalyze the formation of MPT-AMP  and Mocdrolysis . Subsequ


The Pd2+-modified COFs TpPa-1 electrode (Pd2+/COFs) showed high electrocatalytic activity for both hydrazine oxidation reaction and nitrophenol reduction reaction. In addition, TpPa-1-derived nitrogen-doped carbon presented high activity for the electro-oxidation of reduced glutathione (GSH), and sensitive electrochemical detection of GSH was achieved. The presented COFs TpPa-1 can be utilized as a precursor as well as support for anchoring electro-active molecules and nanoparticles, which will be useful for electrochemical sensing and electrocatalysis.Covalent organic frameworks (COFs) are a new type of metal-free porous architecture with a well-designed pore structure and high stability. Here an efficient electrochemical sensing platform was demonstrated based on COFs TpPa-1 constructed by 1,3,5-triformylphloroglucinol (Tp) with p-phenylenediamine (Pa-1), which possesses abundant nitrogen and oxo-functionalities. COFs TpPa-1 exhibited good water dispersibility and strong adsorption affinities for Pd The synthesis and construction of efficient electrode materials to gain high sensitivity and selectivity is crucial for electroanalysis. The successful development of nonenzymatic electrochemical sensors depends critically on electrode materials with excellent electrocatalytic properties because many analytes are electrochemically inactive when using traditional electrodes. Up to now, different categories of materials, including noble metals, metal oxides, and carbons (such as CNT and graphene), have been applied in electrochemical sensing . Recentl2 crystal on the GA matrix. The interface showed high sensitivity and selectivity for the electrochemical sensing of multiple heavy-metal ions. GA was employed as the backbone for UiO-66-NH2, which also improved the electrical conductivity of the composites by accelerating the electron transfer in the matrix. UiO-66-NH2 provided a binding site for heavy-metal ions due to the interaction between hydrophilic species and metal cations [2-CNTs via a simple and rapid one-pot strategy at room temperature. The prepared COF@NH2-CNT composites exhibited a high surface area and excellent conductivity, thus achieving high sensitive detection of furazolidone. However, most MOFs are not chemically stable in water. If the MOF is unstable in the aqueous buffer used for electroanalysis, the MOF coating modified on the electrode surface may suffer from dissolving or converting into MOF-derived metal hydroxides or oxides [2 carbon networks with a high degree of p-conjunction and thus lead to at least a modest electronic conductivity, which facilitates the electron transfer rate. Furthermore, the presence of electronegative atoms (N and O) and π bonds within COFs can allow more specific target analyte recognition through the formation of hydrogen bonds or π-π bonds, thus improving the sensitivity and selectivity of COF-based electrochemical sensors [Metal–organic frameworks (MOFs) and covalent organic frameworks (COFs), as two emerging families of crystalline porous materials, have been explored in electroanalysis and electrocatalysis ,4. Compa cations . In thisr oxides . Unlike  sensors . These u2+ immobilization and fabricating nitrogen-doped porous carbon. The presented electrocatalysts showed high responses for hydrazine oxidation, nitrophenol reduction, and glutathione (GSH) oxidation.COFs-derived porous carbons are also considered ideal platforms for electrochemical sensing due to their high conductivity, porosity, chemical and thermal stability, high electrocatalytic activity, and powerful supporting performances for anchoring molecules and nanomaterials. Most intrinsic electrocatalytic activities are contributed to the oxo-functionalities and edge-plane-like sites (defects) present on the surface of carbon materials . COFs ar2), hydrazine (wt. 80%), graphite powder, liquid paraffin, p-nitrophenol (PNP), and o-nitrophenol (ONP) were purchased from Sigma-Aldrich . COFs-TpPa-1 was purchased from Nanjing XFNano Materials Tech Co., Ltd.  The pore size of COFs-TpPa-1 is in the range of 1.5–1.8 nm with a BET specific surface area of ~1360 m2/g. All of the other chemicals were of analytical reagent grade and used without further purification.GSH, palladium chloride (PdCl63−/4− in the frequency range of 1 MHz to 0.1 Hz at 0.25 V.The morphologies were characterized by scanning electron microscopy (SEM)  and transmission electron microscopy (TEM) . The X-ray photoelectron spectroscopy (XPS) was recorded on a Thermo ESCALAB 250 Xi spectrometer  fitted with a monochromatic Al Kα X-ray source. X-ray diffraction patterns were collected by a Bruker D8  advance powder X-ray Cu Ka radiation diffractometer. All of the electrochemical experiments were conducted on a CHI 750E electrochemical workstation with a conventional three-electrode system consisting of a modified working electrode, platinum coil auxiliary electrode, and Ag/AgCl (saturated KCl) reference electrode. Electrochemical impedance spectra (EIS) were carried out in 0.1 M KCl containing 5.0 mM Fe (CN)2) was successively polished with 1, 0.3, and 0.05 µm alumina paste to a mirror finish and then rinsed with deionized water followed by ultrasonic treatment in water and ethanol, respectively. The COFs-modified GCE (COFs/GCE) was prepared by dropping 10.0 µL COFs aqueous suspension (2.0 mg/mL) on a cleaned GCE and dried at room temperature. In order to anchor Pd2+, the prepared COFs/GCE was dipped in 10 mM PdCl2 solution for 30 min, which was then washed with water and left to dry at room temperature (Pd2+/COFs/GCE). Pd2+/GCE was prepared by soaking the GCE in Pd2+ solution by electroless deposition. The electroactive surface areas of the different electrodes could be calculated by the Randles–Sevcik equation using the CV responses of the corresponding electrodes in 0.1 M KCl solution in the presence of 5.0 mM K3Fe (CN)6 at different scan rates at 298 K   for 80 min under nitrogen flow in the tubular furnace, and black carbonized materials were obtained after cooling to room temperature naturally. To prepare the PCs modified electrode, 3.0 mg PCs were ultrasonically dispersed in 1.0 mL water for 1 h. The carbon paste electrode (CPE) was prepared by thoroughly mixing 1 mg of the graphite powder and 300 µL of liquid paraffin using an agate mortar and pestle for 40 min. Then, a suitable amount of the above mixture was filled firmly into one glass tube. The electrical contact was made by inserting a copper wire into the paste in the inner hole of the tube. Finally, 5.0 µL of 3.0 mg/mL PC suspension was dropped onto the freshly prepared CPE surface and dried at room temperature (PCs/CPE).The nitrogen-doped porous carbon materials (PCs) were prepared by calcination. A number of COFs were calcined at 800 °C  and high energy band (Pd3d 5/2), respectively. The Pd2+ species might be attached to COFs via N coordination [2+ is successfully loaded on the COFs. The XPS data showed that the atomic amount of Pd is 0.16%. In order to investigate the stability of COFs, the XRD patterns of COFs, Pd2+/COFs, and Pd2+/COFs soaked in 0.1 M NaOH were performed  of −1 cm−2 and a detection limit of 0.2 μM (S/N = 3). 2+/COFs/GCE and other modified electrodes presented in the literature. The results show that Pd2+/COFs/GCE demonstrated an excellent analytical performance for hydrazine, owing to the high electrocatalytic activity.2+/COFs/GCE, the interference test was also examined at Pd2+/COFs/GCE with 50.0 μM hydrazine in 0.1 M NaOH containing 0.5 mM of the possible interference substances such as KCl, NaCl, Na2SO4, NaBr, CaCl2, NaAc, glucose, (NH4)2SO4, NaNO3, and NaNO2  value of 4.5%. A reproducible experiment was also evaluated by using five parallel electrodes for the same solution, and the RSD of the current response was found to be 6.4%, suggesting an acceptable reproducibility.2+/COFs/GCE for the determination of hydrazine spiked in real samples was tested by a standard addition method  by CV at a scan rate of 50 mV s−1 in 0.1 M PBS (pH = 7.0) (1) and a pair of reversible redox peaks centered at about 0.08 V (R2) and 0.14 V (O1) at bare GCE and COFs/GCE. Pd2+/COFs/GCE also exhibited the same redox peaks, but the peak of R1 shifted to −0.56 V. However, at bare GCE and COFs/GCE, upon the addition of ONP, four redox peaks, including the three peaks and an additional oxidation peak (O2) appeared at −0.68 (R1), −0.30 (R2), −0.25 (O1), and −0.23 V (O2), respectively. Pd2+/COFs/GCE demonstrated the same redox peaks but a much lower R1 peak (−0.54 V). This indicated that Pd2+/COFs/GCE presented superior electrocatalytic activity towards the R1 reduction process of ONP and PNP, which was ascribed to the combination of the high catalytic performance of Pd2+ and efficient supporting effect of COFs.The electrochemical behaviors of ONP and PNP were investigated at bare GCE (a), COFs/GCE (b), and PdH = 7.0) . The CVs1). Reversible transformation (O1/R2) between hydroxylaminophenol and nitrosophenol then occurred with the potential scanning back. In addition, the phenolic group of ONP gave further oxidation through a proton and an electron transfer process (O2). The detailed reaction mechanism was proposed as When the potential was scanned negatively, both ONP and PNP could be reduced to hydroxylaminophenol through a four-electron and four-proton transfer process  was studied (1/2), which suggested that the reactions of ONP and PNP on the surface of Pd2+/COFs/GCE were mixed controlled processes (surface-controlled at lower scan rates but diffusion-controlled at higher scan rates).To further investigate the electrochemical performance of Pd studied . The res1) of ONP and PNP (500 μM) was studied by DPV on the Pd2+/COFs/GCE in 0.1 M PBS  was investigated, respectively , and the balance of GSH and GSSG acts as an indicator of many human illnesses such as several cancers, Parkinson’s disease, Alzheimer’s, and HIV . Metal an of GSH . Furthern of GSH . FurtherThe pH value is a crucial factor for the electrochemical oxidation of GSH, which was examined in 0.1 M PBS with various pH values containing 5.0 mM GSH on the PCs/CPE . The anoUnder the optimal conditions, the amperometric responses of the PCs/CPE were conducted by successive injections of various concentrations of GSH into the stirred 0.1 M PBS (pH = 4.0) solution . Upon eaThe current responses of the PCs/CPE to various concentrations of GSH in the static 0.1 M PBS (pH = 4.0) were also conducted using the amperometric technique. The inset of To further assess the practical applications of the proposed PCs/CPE biosensor, the validity of PCs/CPE for the determination of GSH in spiked ample was investigated by both successive and non-successive addition methods . The recIn this work, COFs TpPa-1 was explored as an efficient supporting material and precursor to construct electrocatalytic interfaces for electrochemical sensing, and environmental pollutants and biological thiol were sensitively detected. COFs TpPa-1 possesses abundant oxo-functionalities and nitrogen elements, which endow good water dispersibility, powerful adsorption ability, and enhanced electrical conductivity. The results show that COFs TpPa-1 will be a potential material in synthesizing functional materials for electrocatalysis and electroanalysis.


It was found that all the flow stress curves were characterized by a single peak, indicating the occurrence of dynamic recrystallization (DRX), and flow stress will increase with increasing strain rate and decreasing deformation temperature. Based on the experimental data, the constitutive equations and thermal activation energy were obtained (R-grains). Additionally, the calculated flow stress values fit in well with the experimental ones in general, which indicates that the established CA model has a certain ability to predict the deformation behavior of metal materials at elevated temperatures.The hot deformation behavior of T15MN high-speed steel during thermal compression was studied by experiment and simulation. Specifically, the hot compression test was carried out on a Gleeble-1500 thermal-mechanical simulator at temperatures from 1273 to1423 K and strain rates from 0.01 to 10 s High-speed steel (HSS) is widely used for cutting tools due to the advantages of high hardness, high wear resistance, good toughness, and particularly excellent elevated-temperature properties <600 °C) ,2,3. T1500 °C ,2,So far, the conventional means of performing experimental research on the microstructures of materials at different scales has mainly consisted of the use of optical microscope (OM), scanning electron microscopy (SEM), electron backscattered electron diffraction (EBSD), transmission electron microscopy (TEM), etc. These detection methods provide great assistance in studying the microstructure characteristics of materials, whereas microstructural evolution during DRX is discontinuous, which is difficult to track during experiments. Fortunately, the multiscale simulation of the microstructure and properties of materials has gradually become more mature due to the continuous development of computer technology. The common simulation methods for predicting the microstructure and properties of materials consist of finite element (FE), Monte Carlo (MC), phase field (PF), cellular automata (CA). Among these, FM method has advantages in the simulation of macroscopic physical fields such as flow field, temperature field, and electromagnetic field. While the others are regularly used for studying the microstructural evolution of materials on the mesoscopic scale. Compared with the PF method and the MC method, which have more complicated models, substantial computation, and slower calculation speeds, the CA method possesses the unique advantage of building spatial topological relations evolving over time, and can simulate the spatiotemporal evolution of complex systems more simply and efficiently . BecauseR-grain structure could be obtained under suitable hot working conditions. However, little information about the computational simulation of DRX for high-speed steel materials is available, and there is a lack of relevant literature on predicting its high-temperature deformability and microstructural evolution. To optimize TMP, especially for steel grades with a wide variety of elements and high alloy content, it seems to be an extremely rewarding attempt to develop an efficient computing model of DRX behavior.For HSS materials, a handful of studies on hot deformation behavior have also been carried out. For example, Ardakani et al.  studied −1 to 10 s−1. Based on the stress–strain curves, the material constants of the alloy steel are determined through a reverse analysis method, and then the constitutive equations and thermal activation energy of the material can be obtained. Additionally, a two-dimensional DRX-CA model is established for a visual simulation of the microstructural evolution during hot deformation. The effect of thermal deformation parameters  on the DRX behaviors during hot deformation are comprehensively analyzed. The results of the numerical simulation are well consistent with the experimental ones, in general. The main purpose of this research is to provide a certain reference for the prediction and regulation of microstructure.In this study, a series of hot compression tests were performed on the T15MN HSS in the temperature range of 1273 K to 1423 K and the strain rate range of 0.01 s−1 to 10 s−1 and the deformation temperature ranged from 1273 K to 1423 K. The compression deformation degree of the samples was 60% of the initial height, approximately corresponding to the true strain of 0.9. −1, 0.1 s−1, 1 s−1 and 10 s−1. Upon completion of compression, water quenching was adopted immediately to retain the high-temperature deformed morphology of samples. The deformed samples were cut along the compression axis, mechanically polished, and chemically etched for the observation of deformed microstructure  by a Leica optical microscope (OM). The thin graphite and tantalum were placed between the machine head and samples to reduce the friction between them during compression and ensure the samples undergo a relatively uniform deformation. After completing the compression experiment, the flow stress values were automatically stored as a function of each deformation temperature and strain rate. Additionally, the equilibrium phase diagram of T15MN HSS was also calculated using thermodynamic software JMatPro, as shown in 6C-type and MC-type carbides.The studied material in this experiment is one of the high-speed steel materials, and its specific chemical composition is given in The CA method is a grid mathematical model that is discrete in time, space, and state . By pre-For Von Neumann neighborhood type:f denotes the transformation rules of the cellular automata.For Moore neighborhood type:During hot compression of the samples, the evolution of dislocation density inside grains is controlled by the combined action of work hardening and dynamic softening. The softening mechanism consists mainly of DRV and DRX. The Kocks-Mecking (K-M) model is dedicated to describing the variation of dislocation density of cell siting  in a two-dimensional plane, which is a function of strain and can be expressed as :(3)dρ/dεHere, Here, lated by :(8)ρ¯=1nl can be calculated by [Before reaching the peak stress, unbalanced competition between work hardening and dynamic recovery leads to an increase in dislocation density. When the dislocation density reaches a certain critical value, the nucleation of DRX on the pre-existing grain boundary will be induced. In comprehensive consideration of free energy changes, Roberts and Ahlblom proposed a critical dislocation density, given by ,27:(9)ρclated by :(10)l=KGIn which a is usually a constant; δ is the characteristic grain boundary thickness; Many different models have been established to describe the nucleation process of DRX. For example, Ding and Guo  proposedR-grains is essentially the migration of high-angle grain boundaries. Since the dislocation density inside the newly formed R-grains is extremely low and approximately equal to zero, a larger dislocation density difference will be generated between the R-grains and the strained original grains in the matrix. The stored energy provides a driving force for grain boundary migration through its own reduction, and the changed energy can be composed of two parts, namely the volume energy and the surface energy. Assuming spherical grains, the changed energy can be defined as follows:R-grain; R-grain; R-grain; The growth of The migration rate of grain boundary essed as :(17)νi=Mmined as :(20)γm=GR-grains. In addition, the influence of carbides on the CA model is not considered in this study.During simulation, there are four state variables for each cell in the CA model, which are the grain orientation variable; dislocation density variable; grain boundary recognition variable and recrystallization recognition variable, respectively. The grain orientation variable is used to distinguish different grains and grain boundary energy. Each cell is randomly assigned an orientation angle within the range of 0–180°, and the adjacent cells with the same orientation angle are classified as one grain; the dislocation density variable may record the varication of dislocation density in grains with strain and the deformation stored energy; the grain boundary variable can recognize whether cells are located at grain boundaries; the recrystallization variable can recognize the formation and proportion of (1)Before the deformation of the samples, the initial dislocation density (2)R-grains is assumed to be zero, and its value also increases with the continuous increase of strain.The initial dislocation density of the newly formed (3)R-grains boundaries.The nucleation of DRX occurs only at the original grain boundaries and the During the simulation process, three assumptions are made as follows :(1)BefoAdditionally, The true stress–strain (σ–ε) curves of T15MN HSS were obtained based on the hot compression tests at different deformation conditions, as depicted in ardening . When thR-grains growth, thereby reducing the work hardening effect. Therefore, it can be concluded that both low strain rate and high temperature are beneficial in reducing the flow stress level.Given a strain rate, the flow stress gradually decreases markedly as deformation temperature rises. In addition, especially for lower strain rates, the effect of temperature on rheological behavior seems to be more notable, as shown in The hot deformation process of metal materials is dependent on thermodynamic parameters, and thus is regarded as a thermally activated process . The hypOther forms of expressions are also applied for depicting the relationship between flow stress and deformation parameters, such as a power-law form (Equation (23)), mainly used to describe low stress (ασ < 0.8), whereas an exponential form (Equation (24)) is preferred for relatively high stress (ασ > 1.2), given by :(23)ε˙=ATo calculate the parameter values of the constitutive equation, take the natural logarithm of the two sides of Equations (22)–(24) and rearrange the following formulas:According to Equations (25)–(27), the values of Here, it is assumed that the activation energy of hot deformation  is usually utilized to characterize the effects of temperature and strain rate on flow stress behavior, which is defined by:Therefore, the constitutive equation containing the Z parameter can be expressed as follows:Based on the above expressions and the solved parameters, the dynamic equation of T15MN HSS at elevated temperature during hot deformation can be obtained, as shown below:Additionally, the constitutive equations with the Z parameter are as follows:−1 during hot deformation. The white regions and colorful regions represent original grain and R-grains in a deformed matrix, respectively. Following the pre-set nucleation principle, the R-grains are mainly formed along the grain boundaries. The simulation results make it clear that the fraction and the average size of R-grains generally increase with the increase of strain. Furthermore, it is noticed that when the strain is 0.05 (corresponding to Point a), a sporadic newly formed crystal nucleus has already appeared at the grain boundaries, implying that the strain in the local region reaches a higher strain value than the critical strain R-grains dominate compared with the original grains. For a strain value of 0.9 (corresponding to Point d), the complete DRX process is basically achieved, and the average size of R-grains increases appreciably.The visual prediction of experimental results is very meaningful in further revealing the mechanism of DRX. Based on a mesoscopic CA model, the dynamic evolution process of the deformed microstructure of the samples under compression can be monitored more intuitively through the graphical form, which may be divided into three main procedures: the nucleation, spread and growth of recrystallization grains . Figure −1, where the z-axis is represented by the logarithm of the dislocation density.Dislocation density is a crucial criterion in terms of determining whether the DRX process occurs, so it is an important factor usually involved in the study of the thermal deformation of metal materials. The microstructural evolution of materials is dependent on the variation of dislocation density inside deformed grains, and because only when the dislocation density reaches and exceeds the critical dislocation density R-grains begin to nucleate at local grain boundaries. However, the mean dislocation density at this time is still very low, because the dislocation density ρ reaches and exceeds the critical value R-grains and the original grains in the matrix, which provides a driving force for the growth of the R-grains and consequently promotes the gradual growth of the grains. While at the subsequent stage of thermoplastic deformation, the average dislocation density varies little and approximates to a stable state of nucleating easily, attributing to the joint action of multiple mechanisms, as shown in It can be found from −1 and the deformation temperature range of 1273–1423 K, with a true strain of 0.9, were captured using the CA method, as presented in R-grains increases gradually as the deformation temperature rises. The growth of R-grains is attributed to the high atomic diffusion rate and the mobility of the grain boundaries at elevated temperatures [R-grains at the particles or defects of the grain boundaries. Of course, too high a temperature also leads to the coarsening of R-grains. For example, when the deformation temperature is increased from 1273 K to 1423 K at an interval of 50 K, the corresponding values of the average R-grain size based on CA simulation are 7.16 μm, 11.57 μm, 15.73 μm, and 21.83 μm, respectively. This characteristic is consistent with the corresponding optical microstructures of T15MN HSS under the same conditions  is to eliminate microstructure defects and refine grains. As noted above, a higher temperature or lower strain rate causes grain coarsening, while a lower temperature or higher strain rate may lead to an inadequate recrystallization process. However, a uniform microstructure and appropriate grain size are desirable. As far as the above image results are concerned, the microstructure obtained under the deformation conditions of 1373 K and 1.0 s−1 and 0.01–10 s−1 and 1373 K are depicted and compared with the corresponding experimental results, as shown in To further validate the accuracy of the proposed DRX-CA model, simulation work was also carried out on the flow stress behavior of the deformed material. The prediction values of the flow stress obtained by CA simulation at 1273–1423 K and 0.1 s(1)All the flow stress curves are characterized by a single peak, which indicates the occurrence of DRX behavior. Additionally, flow stress is very sensitive to deformation temperatures, strain rates, and strain. True stress will decrease with decreasing strain rate and increasing deformation temperature. Based on experimental analysis, the constitutive equation and thermal activation energy are obtained. In this regard, the Arrhenius equations containing the Z parameter can be expressed as:(2)The concepts and components of the cellular automata model were introduced, mainly including component cell, cell space, neighbor types, and transformation rules. In addition, the models of dislocation density evolution, nucleation, and growth of DRX grains were illustrated and integrated into the model. Then, a 2D CA model was established in this study to describe the DRX behavior of T15 HSS, which was implemented on the MATLAB platform. This method provided a mesoscopic model to bridge the macroscopic hot-working process with the microscopic DRX behavior, enabling real-time visualization of the microstructural evolution of metal materials during hot deformation.(3)R-grains, as well as the dislocation density. The good agreement between the experimental and simulation results indicates that the established DRX-CA model can provide a certain theoretical reference for the prediction of microstructure and regulation of properties for the material.Based on the proposed DRX-CA model, an investigation was performed on the effect of deformation parameters on the evolution and prediction of microstructure, variation of dislocation density, and flow stress behavior. Both the increased deformation temperature and the decreased strain rate can promote an increase in the average size and fraction of The hot deformation behavior of T15MN HSS during isothermal compression was evaluated through the method of combining experiments with CA simulation. Some of the main conclusions of this research can be summarized as follows: