Enzyme inactivation is important, due to the undesirability of certain flour enzymes that cause adverse reactions during storage as unpleasant rancidity of flour, and, at the same time, reduce the shelf life of flour. Therefore, crude enzymes and flour were initially exposed to scCO2 to determine the effect on specific enzyme activity under appropriate conditions. The activity of the unwanted enzymes lipase and peroxidase decreased under optimal process conditions of scCO2 exposure, lipase by 30%, and peroxidase by 12%, respectively. It was discovered that the inactivation of enzymes in wheat flour occurred, where, at the same time, this sustainable method allows the regulation of enzyme activity in the baking process. Afterwards, the effect of scCO2 on the physicochemical properties of flour, morphological changes on starch granules, and content of total lipids was studied. In scCO2-treated white wheat flour, the fat content decreased by 46.15 ± 0.5%, the grain structure was not damaged, and the bread as the final product had a lower specific surface volume. Therefore, this could be a promising technology for flour pretreatment, potentially impacting the prolonging of its shelf-life.The objectives of this study were to inactivate the enzymes α-amylase, lipase, protease, and peroxidase in flour with supercritical carbon dioxide (scCO The numerous enzymes present in the bran and germ fractions of a wheat kernel initiate many chemical changes that affect the compositional and functional properties of whole wheat flour. In general, wheat flour is composed mainly of macronutrients such as starch, water, proteins, and non-starch polysaccharides, such as lipids and ashes , and alsOne of the challenges faced by the food industry is the stability of whole wheat flour during storage . The devPrevious studies have used different thermal processing methods, including steaming, microwave heating, and passing through infrared and gamma radiation, to decrease enzyme activity ,8,9. The2 treatment on the compositional and functional properties of whole wheat flour, as CO2 is a thermodynamically stable molecule with low reactivity [2 treatment, as a green, waterless, more energy-efficient, cleaner, and safer production method, has attracted considerable interest in different industries in recent years [2 is used commonly, and has become the solvent of choice for the food, cosmetic and pharmaceutical industries as a result of its non-toxicity and moderate critical properties  [2, the most widely used supercritical fluid, enzyme activity may be tuned with different pressure/temperature/time combinations, and, at certain conditions, it may be reduced [2 treatment of flour may reduce enzymatic activities efficiently without thermal treatment, ensuring the same quality of flour after this process is finished. Due to the undesirable effects of some enzymes, a series of experiments were performed to achieve the inactivation of these enzymes in wheat flour.This paper is focused on the enzyme activities and compositional properties of different flour types for different applications. Optimization of the protein extraction process was performed, in order to describe the differences in the protein concentrations in various flour types. Furthermore, another aim of this work was to observe the effects of scCOactivity . In prinnt years . ScCO2 i04.35 K) . It is w reduced ,16. This reduced . Under c reduced . Subsequ2’s solubility, which is needed for understanding the fermentation of bread dough modeling. Fan et al. researched CO2 solubility in aqueous dough during baking, and found that CO2 solubility is responsible for the early oven rise of the dough, since vapor makes a significant contribution to the later expansion [2 were also measured in different flour types.Certainly, an important parameter is scCOxpansion . Therefo2, with the aim to inactivate the enzymes in the flour. Thus, after exposure of the flour to scCO2, the proteins were extracted, and the residual activities of the enzymes α-amylase, lipase, protease, and peroxidase were determined in the supernatant. Moreover, a comparison was conducted between the enzyme activity in unexposed flour to the residual enzyme activity in scCO2 treated flour and the activities of free enzymes treated in scCO2. To understand the mechanism of enzyme inactivation using scCO2 it is essential to know the interactions between biological molecules like amino acids, proteins, and CO2. Amino acids are the structural constituents of the proteins, containing amine (NH2) and carboxyl (COOH) functional groups with a side chain  specific to each amino acid. Moreover, an aqueous solution of amino acids and amino acid salts is advantageous in capturing CO2 where dipole-quadrupole interactions become significant in the investigation of such complicated thermodynamic systems (water + amino acid + carbon dioxide) [2 are oxidation, sulfonation, hydroxylation, ring-opening, and amidation [2 in the food industry and food science.In addition white wheat flour was exposed to scCOdioxide) . The funmidation , whereinmidation . The knoAspergillus oryzae and protease (≥0.6 U/mg) from Aspergillus saitoi were obtained from Sigma, while the lipase (~200 U/g) from Aspergillus niger was obtained from BioChemics. Peroxidase from horseradish (232-668-6) was purchased from BBI Enzymes (UK). The petroleum ether (32247) used for lipid extraction was supplied by Honeywell, Riedel-de Haen. All other chemicals used in the laboratory were of analytical grade.White wheat flour type 500, wheat flour type 850, rye flour type 1250, wholegrain rye flour, graham flour, and wholegrain spelt flour were kindly supplied by the bakery Hlebček, d.o.o. . Experiments were conducted using carbon dioxide (99.5% purity) produced by Messer, Ruše. Ethanol (96%), phosphoric acid (≥85%), sodium chloride (≥99.5%), Coomassie-Brilliant Blue G250 (1.15444.0025) and acetonitrile (99.9%) were supplied from Merck, while the bovine serum albumin (≥96%), sodium acetate (≥99.0%), acetic acid , p-nitrophenyl butyrate (≥98%) were supplied from Sigma. The enzymes α-amylase (~30 U/mg) from The extraction of proteins was performed in 0.1 M acetate buffer pH 5.3 with 5 g of wheat flour in an Erlenmeyer flask, followed by incubation at room temperature for a specific time with horizontal shaking at 300 rpm. Afterwards, centrifugation at 8000 rpm was performed for five minutes. To optimize the extraction process, three different combinations of centrifugation and filtration were performed, to obtain the highest value of protein concentration. The first sample was, thus, filtered through a filter with a pore size of 0.45 µm. For the second sample, additional centrifugation was performed under the same conditions . In the third sample, the supernatant was decanted after the first centrifugation. For all three combinations, the supernatant was separated and considered as a crude protein extract. The protein concentration was determined in the supernatant according to the Bradford method . Each sa2 technology was implemented to determine the effects of scCO2 on enzymes’ activities. First, the native enzymes were exposed to scCO2 under different conditions in a high-pressure batch reactor, and then white wheat flour was exposed to the scCO2 medium under the same conditions. A high-pressure batch reactor with a volume of 60 mL with a CO2 gas supply was used in the experimental research. When the temperature in the high-pressure batch reactor reached 35 °C, the pressure was raised to the desired value of 200 and 300 bar with cooled CO2. Afterwards, the system was maintained at a constant temperature and pressure for a preestablished exposure time. After the scCO2 medium treatment, the reactor was depressurized rapidly (0.37 ± 0.08 bar/min). Later, enzymes were extracted from the scCO2 treated wheat flour, where the supernatant was used to determine the enzyme activity and protein concentration. Residual enzyme activities in the treated wheat flour were correlated to the enzyme activities in untreated wheat flour, taken as 100%. The flour samples were subjected to the same conditions of scCO2 treatment for three times to ensure repeatability, whereby the percentage error was ±2.0%.An application of scCOThe moisture was determined from the weight loss of a sample dried by heating to 130 °C. Moisture determination in the flour samples was repeated three times, to obtain reliable results within an experimental error of about 0.5%.Previous studies have shown that the particle size distribution of flours from all wheat types could be measured more precisely by laser diffraction than by sieve analysis . Therefo2 high-pressure treatment ESEM. The pressure chamber was 60 Pa, and the accelerating voltage for imaging was 10 kV.A Quanta FEI 200 3D, Oregon, United States microscope was used in environmental mode to observe the morphological changes in the starch granules in the flour that occurred during the scCO2’s solubility and the volumetric expansion of flour saturated with CO2. The solubility of CO2 is the total amount of carbonate species that can be dissolved in the solution. The solubilities of CO2 in different flour types were measured by a gravimetric method involving magnetic suspension balance  at pressures ranging from 1 to 350 bar  using an ATR accessory was performed for the untreated flour and flour samples after scCO2 treated flour samples into petroleum ether. A total of 10 g of flour sample was used to extract the lipids with 180 mL of petroleum ether, boiling at 60 °C for 5 h [Lipids are a group of substances that are soluble in organic solvents . In gene for 5 h . In the extractm is the mass of lipids weighed after evaporating the petroleum ether, and flourm sample is the mass of flour used for lipid extraction.Moreover, the percent of lipid content in the sample was calculated using Equation (1),The activity of a specific enzyme was determined based on enzymatic activity assays using a UV-spectrophotometer  at different wavelengths.The α-amylase activity was determined based on the increase in the amount of glucose in the solution by using the DNS reagent . The absorbance was measured with a UV-Vis spectrophotometer at a wavelength of 540 nm.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 p-nitrophenol per minute at pH 7.2 and 37 °C using p-nitrophenyl butyrate (PNPB) as the substrate.The protease activity was measured using casein as a substrate. The procedure for the determination of the protease activity was described previously in research .The peroxidase activity was measured using phenol, 4-aminoantipyrine (4-AAP), and hydrogen peroxide as substrates. The method for peroxidase activity determination and a detailed calculation procedure of peroxidase activity has been published in our previous research .The residual activities of different enzymes were calculated using the following Equation (2).All experiments were carried out in triplicate, and the mean values express the average ± standard deviation.2-treated flour after the performance of a two-way analysis of variance (ANOVA). The data were distributed normally and were presented with mean and standard deviation. A post hoc Tuckey honest significant difference test was performed to define the inactivation of enzymes in different groups.R version 4.1.1 and Rstudio, version 1.4.1717 were used to perform the statistical analysis and evaluate the differences between the residual enzyme activity within enzyme types in native form and in scCO2-treated white wheat flour in the bread making process. A bread-making machine, an SBB 850 F2, 850 W  was used for dough baking, according to the recipe from the bakery Hlebček d.o.o, Pragersko, Slovenia. The dough was prepared from 540 g of white wheat flour with 1.8% yeast, 2% salt, 58% water, and 2.5% of professional dough improver, which contains wheat flour, wheat gluten, sugar, wheat malt flour, flour treatment agent (ascorbic acid) and enzymes . In general, bakeries add dough improver to improve the baking functionality in their bread production. For this purpose, a dough improver was added in the same quantity in baking bread with both types of flour, untreated and scCO2-treated white wheat flour. The ingredients were first preheated for 10 min, and the dough was mixed for 12 min and fermented for 20 min, whereas stirring and fermenting were then repeated for 7 min and 35 min. The bread loaf was baked for 60 min at 220 °C. The dough baking test was repeated with untreated white wheat flour and scCO2-treated white wheat flour under the same conditions and bread-making program. The loaves of bread were weighed after cooling, and their volume (cm3) was determined by the water displacement method. The specific volume (cm3/g) was calculated as loaf volume/bread weight. Values obtained were the mean of three replicates. A sensory analysis was carried out, involving evaluation of the sensory attributes of appearance, aroma, taste, and texture. Ten non-trained testers  made a hedonic evaluation of the acceptability of bread baked from untreated and scCO2-treated white wheat flour. In the test, participants were asked about their preferences concerning the differences in the experimental bread. The average height of the baked bread was measured, and determined using the ImageJ program.The baking test was developed to establish the suitability of the flour for making bread, and to determine the difference between untreated and scCOThe conceptual design of the entire study started with the optimization of the protein extraction process and determination of the protein concentration, which were carried out with regard to six different flour types: Wheat flour type 500, wheat flour type 850, rye flour type 1250, wholegrain rye flour, graham flour, and wholegrain spelt flour. Since white wheat flour is the most commonly used, we decided to perform further experiments on this type of flour. Optimization of the protein extraction process was carried out to ensure the proper determination of the concentration of proteins. Optimal shaking time  and a combination of the centrifugation and filtration procedures of the sample were modified experimentally using three different protocols, where, in the first procedure, the supernatant was obtained by centrifugation at 8000 rpm for 5 min followed by additional filtration through a 0.45 µm pore filter. In the second procedure, double centrifugation at 8000 rpm for 5 min was performed to obtain clear supernatant, and in the third procedure, single centrifugation at 8000 rpm for 5 min was performed to obtain clear supernatant. After the extraction of proteins from the wheat flour, protein concentration was determined by the Bradford method using a UV-Vis spectrophotometer at 595 nm.The highest protein concentration was obtained after 90 min of shaking and single centrifugation in the Graham flour , followeThe protein content of wheat varies widely, depending on the type or class of wheat, growing conditions, and fertilizer inputs, particularly nitrogen, whereas wheat flour proteins are related directly to bread volume. Therefore, protein quantity and quality have received considerable attention in bread-making processes. The behavior of the dough in the bread-making process depends primarily on the properties of the flour and its composition, resulting in the dough being able to retain gas bubbles during fermentation and baking, and leading to baked products with a high loaf volume and more regular crumb structure.Flour composition and quality are of vital importance in flour milling. The moisture content of wheat is important, and should not exceed 15%, being low enough to prevent spoilage. 2, a different effect on their stability and activity may be observed, depending on the amount of water present, pressure and temperature of the treatment. Certainly, the type of enzyme is also crucial, as there are different mechanisms of enzyme inactivation in scCO2. The mechanism of enzyme inactivation in high-pressure treatments refers to the disruption of interactions that affect primarily the tertiary and quaternary enzyme structures, and result in the loss of the catalytic enzyme function. The high pressure causes protein denaturation, which occurs due to the penetration of water into the protein structure. The penetration of water into the protein matrix leads to conformational transitions that cause its unfolding [2 treatment is the pH value. In principle, during the supercritical treatment, CO2 forms carbonic acid (H2CO3), which dissociates into bicarbonate (HCO3−), carbonate (CO32−), and hydrogen (H+) ions, thereby reducing the pH inside the high-pressure reactor [2 treatment, and caused the inactivation of α-amylase activity. Therefore, it was considered that α-amylase was inactivated predominantly by the pH-lowering effect of the microbubbles of scCO2 [When enzymes are exposed to scCOnfolding . Another reactor . Yoshimuof scCO2 . Furtherof scCO2 .2 medium under certain conditions, in order to determine the suitability of the scCO2 method for the inactivation of enzymes.Based on these premises, the activities of the pure enzymes α-amylase, lipase, protease, and peroxidase were determined initially before and after the exposure to a scCO2 treatment under different conditions. The results in 2 exposure. At 300 bar the decrease in its activity was even more significant. Similarly, a decrease in native peroxidase activity was observed to 12% after three hours of treatment at 300 bar.2, and are very often used as biocatalysts in this medium. In this regard, Bauer et al. performed a treatment of esterase from porcine liver and lipase from Candida rugosa with scCO2 at different pressures with different amounts of water, at a temperature used commonly in biocatalysis with hydrolases. Lipase from C. rugosa was treated at 300 bar and 40 °C for 22 h, where the residual activity of the lipase was 91.6%. Moreover, the esterase from the porcine liver was treated at 200 bar and 65 °C for 24 h, where the resulting residual activity was 71.3% of that of the untreated enzyme [Candida antarctica at scCO2 conditions of 80 bar and 40 °C for 60 min. Their findings suggest that an increase in temperature and density led to an enhancement of the enzyme activity losses, while the decompression rates had a weak influence on enzyme inactivation [In our study, the activities of lipase and protease decreased only after a longer exposure time of 24 h, as shown in d enzyme . On the tivation .2 process as well  . Tedjo e2. It is known that CO2 induces transient acidification during treatment, and, later, during degassing (CO2 expansion). In this regard, consistent with our results, some proteins could be subjected to conformational changes during acidification, including changes in surface hydrophobicity. For instance, Bednarski et al., reported that amino acid residue TRP retained its shape after exposure to scCO2 for thee hours. In our case, TRP is located in the alpha-amylase [Based on our results and previous assumptions about the inactivation of enzymes in association with amino acid residues of the enzyme, it can be emphasized that specific amino acid residues are important in the mechanism of inactivation with CO-amylase .2, unfolding occurred, but, overall, no deleterious effect could be detected on amino acid composition [2 treatment targets primarily the enzyme proteins’ secondary structure (α-helix and β-pleated sheets). The enzyme inactivation mechanism most probably includes disruptive changes in the active site, unfavorable substrate desolvation, blockage of substrate access, and effects of transition state destabilization and restriction of conformational mobility.position . Therefop = 0.02) and enzyme inactivation in wheat flour (p < 0.01). There was no statistically significant interaction between the inactivation of enzymes in native form and enzymes in flour  = 1.83, p = 0.17). Tukey multiple comparisons of means confirmed that enzyme inactivation was statistically different between enzyme groups (p = 0.03), and that α-amylase inactivation was statistically different (p < 0.05) from all other enzyme groups.The two-way ANOVA confirmed statistical differences between the enzyme types  wheat flour granules was 70.7 µm. The increase in the average diameter of the wheat flour particle size can be attributed to the formation of aggregates, which is clearly evident when looking at 2 during exposure. The particle size measurements were repeated after a certain time (one week). The results demonstrated that the average particle size of wheat flour treated with scCO2 decreased to 61.2 µm, and after two weeks, it became equal to the initial value of the average particle size of untreated wheat flour (60.3 µm).A laser diffraction particle size analyzer was used for the granulometric analysis of the wheat flour T500. A comparison of the particle sizes of untreated wheat flour and scCO2 medium does not affect the morphological properties of the wheat flour, and does not result in any damage to the flour components, which are important in the further use of flour in the production of bakery products.Regarding these analyses, we confirm that exposure to the scCO2-treated wheat flour particles appear to be larger in comparison to those of untreated wheat flour. After the scCO2 exposure significant particle size enlargement occurred, and microscopic observations observed spherical and ovoid granules of different sizes. Due to the influence of pressure and the addition of CO2 after exposure, some of the particles of flour aggregated, and this increased the average size of the scCO2 treated wheat flour. The most important observation with regard to the scCO2 treated wheat particles is that no mechanical damage on starch grains was caused due to the treatment. With ESEM studies, the effect of scCO2 on flour particles was observed. It has been proven that starch granules were not damaged by flour exposure to scCO2 and remains intact, which is a significant property for further use of flour in final bakery products.ESEM studies were used to determine the morphological characteristics of the starch grains. The microstructures of the flour samples were observed using ESEM , with ir2 solubility in different flour types was determined with a magnetic suspension balance (MSB), where the temperature and pressure range considered for these experiments were the same as the conditions of the scCO2 treatment used for enzyme inactivation. The solubility of CO2 in different flour types was measured at 35 °C. The obtained results are presented in 2 increased with pressure. The solubility of CO2 at 300 bar was the highest in wheat flour type 850 and rye flour type 1250, with a value of 0.85 g/g. Afterwards, the solubility of CO2 was a little lower in wheat flour type 500, continued with wholegrain rye flour and wholegrain spelt flour. The lowest solubility of CO2 was found in Graham flour (0.6 g/g). In addition, when the flour is exposed to a dense gas, penetration of the gas molecules into the flour occurs and starts changing the density of the flour–gas mixture. The number of penetrating molecules rises when using gas with higher permeability and more porous material. CO2 is recognized as a highly permeable gas, which can increase the density of a sample [The CO2 in different flour types is that the solubility of CO2 goes hand-in-hand with the protein concentration in the flour. 2. This may be proof that a lower protein concentration is somehow connected with the higher solubility of CO2. The same observation was found for rye flour. Namely, rye flour type 1250 has a lower protein content (9.0 g/100 g flour) than wholegrain rye flour (13.0 g/100 g flour), and, at the same time, higher solubility of CO2. On the other hand, Graham flour had the highest protein value and the lowest solubility of CO2.An important finding regarding the solubility of CO2 medium had a significant influence on enzymatic activity. In further research, the influence of scCO2 treatment on the primary structure and conformation of the non-treated wheat flour and the scCO2-treated wheat flour were investigated by employing an FTIR instrument. The results shown in 2 medium were observed in comparison with the untreated wheat flour. Nevertheless, the infrared spectrum of wheat flour T500 after scCO2 treatment shows stronger adsorption intensities for all characteristic peaks between the samples. The absorption spectrum of wheat flour shows a strong peak for water with a stretching vibration of O-H bonds at 3260 cm−1, which represents the presence of moisture in the flour [−1 to 2800 cm−1 correspond to stretching vibrations of the C-H bond, which could be attributed to the presence of polysaccharides, unsaturated lipids, and carbohydrates.According to the above investigations, the treatment of wheat flour with scCOhe flour . Further2 treatment on the nutritional value of wheat flour, it was important to focus on the protein regions. According to the results from the FTIR analysis, the intensity of the absorption of the amide I band (centered at 1645 cm−1) increased due to the scCO2 treatment. Moreover, the characteristically strong peak at 1645 cm−1 is also attributed to the stretching vibrations of N-H, which presents mainly the bands of protein, and can be attributed to amide I and amine II [Glycine max L.) seeds and white corn (Zea mays), where high temperature extrusion caused changes in the secondary, tertiary, and quaternary protein structures [To evaluate the effects of scCOamine II . A similructures .2 may have an influence on the lipid content in the sample. Therefore, the total lipid content in white wheat flour was determined after exposure to the scCO2. Moreover, the presence of lipids is important with regard to bakery product production. The percentage of lipids obtained by extraction from untreated white wheat flour was 1.3%, while the percentage of lipids in white wheat flour treated with scCO2 at 300 bar and 24 h was 0.7%. The total lipid content differed significantly between the untreated and scCO2-treated white wheat flour. Therefore, based on the obtained results, the percentage of lipid loss in white wheat flour after scCO2 treatment was recalculated to a 46.15% loss. This is due to the lipids being soluble in CO2, and being released from the high-pressure batch reactor, together with the CO2 gas. A similar trend was noted by Shin et al., who reported that scCO2 extraction  reduced the fat content of soy flour significantly from 19.5% to 7.1% [2 technology holds the key to prolonging a product’s shelf life while reducing enzyme activity. By inactivating certain unwanted enzymes, it is assumed that this affects extending the shelf life of flour, as adverse reactions that affect the quality of the product occur more slowly. During the research, we performed experiments where we put the scCO2-treated flour and untreated flour for storage in the warehouse in a bakery, with which we cooperated during this research. Thus, we took a flour sample every few months and determined the activity of the enzymes in the stored flour. We also found that in scCO2-treated flour, bugs did not develop, as we know that scCO2 also affects the inactivation of microorganisms in food. Moreover, lipids play an essential role in food products’ shelf life and physical and sensory properties, as they present different structures, which affect their behavior, stability, plasticity, and texture. Certainly, due to the lower lipid content, the energy value of scCO2-treated wheat flour is lower than that of untreated white wheat flour, and, thus, pre-treated flour has a longer shelf life.ScCO to 7.1% . Many fa2-treated white wheat flour were evaluated for a sensory analysis. The aroma, taste, and texture were evaluated during the sensory analysis, which was performed immediately after the cooling process in the process of baking the bread. The final baked products from the untreated and scCO2-treated wheat flour were slightly different visually, as shown in the representative samples in 2-treated wheat flour was 9.7 cm. However, a slightly lower bread was expected with the scCO2-treated white wheat flour, as the enzyme activity in the scCO2-treated white wheat flour was reduced due to the impact of the scCO2. Measurements of the specific volume of the bread were also carried out, and it was found that the bread from the untreated white wheat flour has a specific volume 2.77 ± 0.15 cm3/g, which is slightly higher than bread baked from the scCO2-treated white wheat flour with a specific volume 2.61 ± 0.18 cm3/g. Besides the effect of scCO2 on the baking properties of white wheat flour, the reduced activity of α-amylase affected the baking properties significantly. Certainly, it has been proven that it is possible to prepare the final product with scCO2-treated flour successfully. Bread baked under the same conditions is only slightly lower, and retains all the same properties as bread baked with untreated white wheat flour. Furthermore, the air spaces in the bread are comparable, which is a confirmation of a successful fermentation process.Baked loaves of bread from untreated and scCO2 process presents a sustainable and green method for enzymes’ inactivation in flour, which was presented for the first time in this study. The protein concentration was determined for six different types of flour, and the activities were determined of the enzymes α-amylase, protease, peroxidase, and lipase in white wheat flour. In the present study, for the first time, attempts were made to investigate the factors leading to enzyme inactivation by the interaction between proteins and scCO2 in a high-pressure batch reactor, to assess the impact of scCO2 conditions on flour quality, and, at the same time, to prolonged flour storage. As expected, it was found that scCO2 treatment causes the inactivation of both native enzymes and enzymes in wheat flour under certain conditions. It was found that the scCO2 treatment does not change or affect the quality of the treated flour negatively, which was confirmed using different analytical methods. In particular, it was observed on the ESEM images that, due to the influence of pressure and the addition of CO2 after exposure of wheat flour to scCO2, the granules bound together and increased in volume. However, after a certain time, the shape and size returned to their original states, which are equal to those of the non-treated sample. Moreover, an important finding that has not been reported before in the literature is that the solubility of CO2 in various flour types goes hand-in-hand with the protein content of the flour sample. In this study, the potential was also evaluated of scCO2 treated white wheat flour as a raw material for bakery products, and the development of a low-fat white wheat flour. One of the most useful findings is the wheat flour’s lower lipid content after scCO2 treatment. The energy value of scCO2 treated wheat flour is less than that of untreated white wheat flour, and, thus, pre-treated flour has a longer shelf life.The scCO


C. elegans. However, PAD-1 is large, and the domains required to regulate EV release were unknown. Here, we reveal that the conserved N-terminal EWAD motif and C-terminal leucine zippers are required to inhibit EV release from the plasma membrane. Revealing a role for these domains is an important first step to identifying how EV release is regulated.Cells release extracellular vesicles (EVs) carrying cargos that can influence development and disease, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. We previously showed that the Dopey protein PAD-1 inhibits EV release from the plasma membrane in Caenorhabditis elegans . PAD-1 is thought to inhibit EV release by activating the phospholipid flippase TAT-5 to maintain phosphatidylethanolamine (PE) asymmetry in the plasma membrane. When PAD-1 is disrupted, cytofacial PE is externalized, membrane-sculpting ESCRT complexes are recruited to the plasma membrane, and EVs bud from the plasma membrane by ectocytosis . However, which domains of PAD-1 are important to regulate EV release was unknown.The Dopey family protein PAD-1 is a key regulator of extracellular vesicle (EV) release inAspergillus nidulans . Leucine zippers are often involved in protein-protein interactions , but it is unknown whether specific proteins interact with this domain. Furthermore, whether the N-terminal EWAD motif or C-terminal leucine zippers are required to inhibit EV release was unknown.PAD-1 has a conserved N-terminal Dopey domain and C-terminal leucine zipper-like domain, similar to other large Dopey proteins . In mammals, the N-terminal Dopey domain binds to the kinesin light chain KLC2 via a tryptophan surrounded by acidic residues (EWAD motif) , which is proposed to link Kinesin-1 to membranes for organelle trafficking . Indeed, an LKRL motif in the C-terminal leucine zipper region of Dopey1 has been shown to bind lipids . Furthermore, the leucine zippers play essential roles in Dopey protein function, as a mutation in the leucine zipper domain of the Dopey protein DopA (I1695R) disrupts cellular morphogenesis inpad-1(W39A)mutants were mostly sterile and that their few embryos were embryonic lethal, similar topad-1deletion mutants . To test for an increase in EV release, we crossed the pad-1(W39A)mutants with a degron-tagged plasma membrane reporter, mCh::PH::CTPD. The CTPD degron targets proteins for degradation in the cytosol starting at the first mitotic division, allowing us to specifically label EVs released from the plasma membrane starting after the 2-cell stage . We observed a 50-fold increase in EV puncta on the surface ofpad-1(W39A)mutant embryos compared to control embryos . These results demonstrate that the N-terminal EWAD motif is required to inhibit EV release, suggesting that PAD-1 binding to Kinesin-1 or other proteins through its Dopey domain regulates EV release.To determine the role of the N-terminal Dopey domain in EV release, we used CRISPR/Cas9-mediated genome editing to mutate tryptophan 39 to alanine (W39A) in the EWAD motif of PAD-1 . We found thatAspergillus, and an LKRL motif mutant. We discovered thatpad-1(M2244R)mutants were mostly sterile and that their few embryos were embryonic lethal, similar topad-1(W39A) and deletion mutants . In contrast,pad-1(LKRL-AAAA)mutants were entirely sterile, precluding the investigation of embryonic EVs. After crossing themCh::PH::CTPD EV reporter with thepad-1(M2244R)mutants, we found that the M2244R point mutation resulted in a >60-fold increase in EV puncta . These results suggest that the C-terminal leucine zippers of PAD-1 are crucial for inhibiting EV release by ectocytosis.To test a role for the C-terminal leucine zippers in EV release, we next created a PAD-1(M2244R) allele , which corresponds to the DopA I1695R mutant inin vitro, it remains unclear whether this is the only binding partner of the highly conserved EWAD motif. Furthermore, it will be important to test whether KLC2 plays a role in EV regulation as part of Kinesin-1. It is also unclear what molecules interact with the conserved C-terminal leucine zipper domain of PAD-1, although this region is important for membrane binding . Further investigation into the molecular interactions of the N- and C-terminal PAD-1 domains will help us understand the mechanisms that govern EV release as well as Dopey protein function.By characterizing the effects of point mutations in PAD-1, we demonstrate that both the conserved N-terminal EWAD motif in the Dopey domain and C-terminal leucine zippers of PAD-1 are required to inhibit EV release from the plasma membrane. Although a mammalian Dopey domain binds KLC2Worm Strains and MaintenanceC. elegansstrains were maintained and crossed on Nematode Growth Media (NGM) seeded with OP50 bacteria and grown at room temperature using standard protocols (Brenner 1974). See Table 1 for a list of strains used in this study.Table 1: Worm strainsGenome Editingpad-1(syb2032[W39A])was generated using the guide RNA ATTCGAAACACCCAATGAATGGG and the repair template ACGCAAAAGCCATCGATCAGGCGTTGAAAACATTCGAAACACCCAATGAAGCCGCGGATCTCATTTCGGCACTCGGAAAATTGGCTAAAGTGGGTTTTGTTAG.The W39A, M2244R, and LKRL mutants were created by SunyBiotech using CRISPR-Cas9-mediated genome editing .pad-1(syb1647[M2244R])was generated using the guide RNA GATTGGTGTGTGGCCTATTATGG and the repair template GTACTTCTTCTCCGACTCCGCCCACACAGTTTGATTGGTGTGTGGCCTATACGCGTTACAGAGCTCGTTCACGCACTATCACAGCTTGAACAACAATTACAAAG.pad-1(syb2521[LKRLmut]) was generated using the repair templateGCCGCAGCAGCTACTTTTGTCGTTTTTGGTAGTCAATTAGATCAATATCACGGGCAGATGAA. Mutations are underlined and in bold.TCAATTATGACATCAAAAGAGCAAGAATACGAAGCACGTGCTCAAGCALight MicroscopyFluorescence images were taken with a Zeiss Axio Observer 7 inverted microscope with a Plan-Apo 40X 1.4 NA oil objective with Excelitas Technologies X-Cite 120LED Boost illumination, and a Hamamatsu ORCA-Fusion sCMOS camera controlled by 3i SlideBook6 software over multiple days .Image ManipulationImages were rotated, cropped, and inverted for clarity, and the intensity was adjusted using Adobe Photoshop 2022. AlphaFold2 models  were colorized and segmented in PyMol 2.3.2 .EV CountsmCh::PH::CTPD puncta were marked and counted on the top surface of 3- to 15-cell embryos using the ImageJ Cell Counter function (FIJI 2.3.0). The number of fluorescent puncta in clusters were estimated according to the average size of discrete mCh::PH::CTPD puncta.StatisticsStatistical significance was tested using Student’s one-tailed t-test with Bonferroni correction to adjust for multiple comparisons.


The 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the central enzyme of the shikimate pathway to synthesize the three aromatic amino acids in fungi, plants, and prokaryotes. This enzyme is the target of the herbicide glyphosate. In most plants and prokaryotes, the EPSPS protein is constituted by a single domain family, the EPSP synthase (PF00275) domain, whereas in fungi, the protein is formed by a multi-domain structure from combinations of 22 EPSPS-associated domains. The most common multi-domain EPSPS structure in fungi involves five EPSPS-associated domains of the shikimate pathway. In this article, we analyze 390 EPSPS proteins of fungi to determine the extent of the EPSPS-associated domains. Based on the current classification of the EPSPS protein, most fungal species are intrinsically sensitive to glyphosate. However, complex domain architectures may have multiple responses to the herbicide. Further empirical studies are needed to determine the effect of glyphosate on fungi, taking into account the diversity of multi-domain architectures of the EPSPS. This research opens the door to novel biotechnological applications for microbial degradation of glyphosate. However, the epsps gene is also present in most fungal and bacterial species. Thus, glyphosate may have an effect on microbial communities of free-living and host-associated microorganisms [Glyphosate-based products are the herbicide most used against weeds worldwide. The herbicide targets an almost universal enzyme in plants, the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), also known as aroA ,2. The Erganisms ,5,6,7. Rrganisms . A comprrganisms ,5 . The cuepsps gene) that highly contribute to modulating the response of organisms to the herbicide [n = 789; 726 sensitive, 6 resistant, and 57 unclassified) [Here, we analyze the evolution of the EPSPS domain in fungi and the distribution of additional EPSPS-associated domains. The EPSPS enzyme, at least in its single-domain structure characteristic of plants and prokaryotes, closes after its interaction with the two substrates, shikimate 3-phosphate (S3P) and phosphoenol pyruvate (PEP) . Most oferbicide ,12,13. Terbicide ,5,14, anerbicide ,17,18,19http://pfam.xfam.org, accessed on 8 July 2022), a comprehensive database of protein domains [https://ppuigbo.me/programs/EPSPSClass, accessed on 20 July 2022 [EPSPS-associated domains present in EPSPS proteins were obtained from the PFAM  [The potential sensitivity to glyphosate was estimated using the EPSPSClass web server (ly 2022) . EPSPS pEPSPS proteins were defined by the EPSPS domain, which is approximately 1350 nucleotides long (450 amino acids) [The EPSPS-associated domains can be classified into four main functional categories: shikimate (involved in shikimate pathway proteins), enzymes , expression (domains involved in gene expression), and structural function . The distribution of the EPSPS-associated domains in a dataset of 1175 multi-domain proteins showed certain dominance of the domains EPSP_synthase (as a marker of the EPSPS proteins), Shikimate kinase (SKI), 3-dehydroquinate synthase (DHQ_synthase), 3-dehydroquinate dehydratase (DHquinase_I), and Shikimate dehydrogenase substrate binding domain ; DHQ_synthase (n = 374); DHquinase_I (n = 367); Shikimate_dh_N (n = 366); and Shikimate/quinate 5-dehydrogenase . In fungi, 16 out of 22 EPSPS-associated domains were only present in less than three proteins  and in the synthesis of aromatic amino acids . There were also some promiscuous domains  associated with the EPSPS in some fungal species. Infrequent, but amply distributed, EPSPS-associated domains in fungi were involved in DNA modification and gene expression. A total of 22 domains were present in diverse domain architectures of the EPSPS protein, across 390 fungal species . Howeverproteins .n = 220) contained structures with five domains involved in the shikimate pathway, and approximately 1/3 of the protein sequences (n = 134) had all six domains of the shikimate pathway. Thus, the overall trend in fungal EPSPS proteins, as shown in the bipartite network, was an association of the EPSPS domain with other domains of the shikimate pathway.We have analyzed the distribution of the EPSPS-associated domains in different taxonomic groups of fungi  . AscomycThe EPSPS multi-domain structure in fungi was heterogeneous across the phylogenetic tree. However, most of the species had five or six domains in the EPSPS protein . Our anaPurpureocillium lilacinum is able to degrade glyphosate and use glyphosate as a nutritional source [P. lilacinum (PWI66746.1) is sensitive to glyphosate. Moreover, it has been suggested that the carbon-phosphorus bond in glyphosate is the major metabolic degradation mechanism utilized by fungi [Here, we analyzed the frequency and evolution of EPSPS-associated domains to determine variations in the intrinsic sensitivity of the EPSPS protein to glyphosate. In bacteria and plants, the EPSPS protein sequence has a single domain, whereas fungal EPSPS proteins contain several domains . Therefol source ). The EPby fungi .The EPSPS is a two-substrate enzyme with an open (without ligand) and closed (with ligand) conformation. Glyphosate’s mode of action is competitive against the PEP and noncompetitive against the S3P . HoweverIn fungi, the most common multi-domain structure of the EPSPS ranges from two to eight domains. The ancestral state of the EPSPS protein included six domains , as shown in the phylogenetic analysis. The wide diversity of EPSPS multi-domain structure in fungi is the product of several independent rearrangements of domains throughout evolution. Analyses of the EPSPS enzyme showed that most fungi are potentially sensitive to glyphosate. However, the total number of EPSPS-associated domains have an effect on the potential sensitivity status. Future analyses will be necessary to determine how different EPSPS multi-domain architectures affect the sensitivity of the EPSPS enzyme to glyphosate. These studies may have a substantial contribution to the development of novel biotechnological applications for microbial degradation of glyphosate.


Due to the inherent toxicity of protein aggregates, the propensity of natural, functional amyloidogenic proteins to aggregate must be tightly controlled to avoid negative consequences on cellular viability. The importance of controlled aggregation in biological processes is illustrated by spidroins, which are functional amyloidogenic proteins that form the basis for spider silk. Premature aggregation of spidroins is prevented by the N-terminal NT domain. Here we explored the potential of the engineered, spidroin-based NT* domain in preventing protein aggregation in the intracellular environment of human cells. We show that the NT* domain increases the soluble pool of a reporter protein carrying a ligand-regulatable aggregation domain. Interestingly, the NT* domain prevents the formation of aggregates independent of its position in the aggregation-prone protein. The ability of the NT* domain to inhibit ligand-regulated aggregation was evident both in the cytosolic and nuclear compartments, which are both highly relevant for human disorders linked to non-physiological protein aggregation. We conclude that the spidroin-derived NT* domain has a generic anti-aggregation activity, independent of position or subcellular location, that is also active in human cells and propose that the NT* domain can potentially be exploited in controlling protein aggregation of disease-associated proteins. Spider-silk protein increases the solubility of an aggregation-prone reporter protein, showing potential applications in controlling aggregation of disease-associated proteins by natural solubility domains. The devastating effects of aggregation-prone proteins are clearly illustrated by neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease, which are characterized by an age-dependent accumulation of insoluble protein aggregates in affected neurons or brain parenchyma2. Although neurological disorders may be overrepresented, probably due to the limited possibilities of long-lived, post-mitotic neurons to eliminate protein aggregates, protein misfolding disorders are by no means limited to neurodegeneration, but also include diseases in which other tissues are affected by aggregation-prone proteins3. Even though the exact nature of the toxic species is still subject to debate, there is a large body of evidence suggesting that the propensity of proteins to aggregate is the primary determinant for the toxicity they elicit in these diseases4. Accordingly, keeping the disease-associated proteins in a soluble state is expected to minimize their cytotoxic effects and is, as such, a desirable but challenging objective from a therapeutical perspective.The plethora of human diseases that are linked to proteins that precipitate into insoluble aggregates highlights the importance for cells to keep their intracellular environment free of these inherently toxic protein species5. This exquisite characteristic explains the presence of amyloidogenic domains in a variety of natural proteins for which the unique features of these domains outweigh the costs of keeping the potential toxicity of these proteins at bay. A prime example is the family of amyloidogenic proteins called spidroins, which form the amyloid-like fibers that are the main constituent of spider silk, giving this biological product its unique properties and exceptional strength7.The strength, robustness and hard-to-disentangle nature of protein aggregates are at the same time unique properties that endow aggregation-prone proteins with the ability to form exceptionally rigid structures that can deal with forces that would be difficult, if not impossible, to resist by natively folded, globular proteins2-dependent fashion8. The NT domain has a dual function as it not only prevents premature aggregation but also, when appropriate, promotes aggregation by forming NT-dimers that promote oligomerization of the amyloidogenic domain9. An engineered NT domain, referred to as NT*, has been optimized to prevent protein aggregation. The NT* contains two amino acid substitutions, , which result in loss of the dipolar nature of wild-type NT that is required for domain-domain charge attraction11. Consequently, the NT* domain does not display the pH-dependent dimerization in vitro, while still preventing protein aggregation12. The NT* domain has been explored in various experimental models for its ability to prevent aggregation of proteins-of-interest18. The spidrion-derived NT* anti-aggregation domain has also been found to efficiently prevent aggregation of bacterially produced recombinant proteins, underscoring its usefulness as a solubility tag14. Interestingly, the ability of the NT domain to prevent protein aggregation appears to be its primary, native function, unlike other commonly used solubility tags, such as glutathione S transferase (GST), maltose binding protein (MBP), and ubiquitin19, which motivated us to examine its anti-aggregation potential in the natural environment where disease-associated amyloidogenic proteins have devastating effects.Spidroins are present at very high concentrations in the spider’s gland but only form safely amyloid-like fibers when secreted, which demands a stringent and tightly controlled mechanism to prevent premature aggregation in the gland. This is accomplished by the N-terminal (NT) domain of spidroins that precedes the highly repetitive amyloidogenic domain and regulates the formation of amyloid fibrils in a pH- and CO20. The AgDD domain, which is derived from the human FK506 binding protein 12 (FKBP12) protein21, has the unique feature that its aggregation state can be regulated through administration of the stabilizing ligand Shield-120. Using this experimental model, we found that the spider silk-derived NT* domain can efficiently prevent protein aggregation in cellular compartments relevant for protein misfolding diseases. This proof-of-principle study suggests that the generic anti-aggregation domain of a natural spider protein may be exploitable to counteract the toxic properties of disease-associated, aggregation-prone proteins.In this study, we explored the anti-aggregation properties of the NT* domain in the cytosolic and nuclear compartment of human cells using the aggregation destabilization domain (AgDD)22. By adding an additional 10 amino-acid sequence to the N-terminus of the DD signal, this motif has been converted to a Shield-1-regulatable aggregation domain that allows rapid induction of protein aggregation by omission of the ligand20. We chose this engineered aggregation domain because of its regulatable nature and its ability to promote the formation of filamentous protein aggregates that resemble the aggregates observed with disease-associated proteins20. The finding that AgDD induces protein aggregation irrespective of its position in the host protein also allowed a modular approach. For easy detection and to avoid interference of protein aggregation on the folding and maturation of the fluorescent protein, we combined these domains with superfolder green fluorescent protein (sfGFP)23. Thus, the reporter proteins were composed of three modules: the AgDD aggregation domain, the NT* anti-aggregation domain, and sfGFP  that targets proteins for degradation in the absence of the ligand Shield-1GFP Fig. .Fig. 1Sp20. The fusion proteins were transiently expressed in HeLa cells and analyzed by western blotting for protein aggregation. As expected, AgDD-sfGFP displayed Shield-1-regulatable aggregation as omission of Shield-1 resulted in a significant decrease in the pool of soluble AgDD-sfGFP  and transiently expressed these proteins in HeLa cells. The NLS-AgDD-sfGFP fusion precipitated in large intranuclear aggregates in the absence of Shield-1  and 5’-CAT GGT GGC CGA ATT TGA ATT CCC AGC ACT GAC-3’ (rev). The PCR products were purified from a 1% agarose gel using the Qiagen gel extraction kit  and inserted in EcoR1 digested AgDD-sfGFP using NEbuilder HiFi DNA Assembly Master Mix . In order to clone AgDD-NT*-sfGFP, a similar procedure was performed with the primers 5’-AAA CCG GAA GAA TTC ATG TCA CAC ACT ACA CCA TG-3’ (fwd) and 5’-AGT TCT TCT CCT TTG CTG AAG CTA GCT GAA TTC CCA GC-3’ (rev). For the DD domain, the primers 5’-GTC TCA TCA TTT TGG CAA AGA ATT CGC CAC CAT GCT GG-3’ (fwd) and 5’-TAG TGT GTG ACA TGA ATT CTT CCG GTT TTA GAA GCT CCA C-3’ (rev) were used. The AgDD-sfGFP-NT* was originally constructed in the EGFP-N1 vector  by digesting EGFP-N1 with the restriction enzymes BamHI and NotI. The AgDD-sfGFP was PCR amplified using the primers 5’-CGA CGG TAC CGC GGG CCC GGG ATC CAT GCT GGC CCT GAA GCT G-3’ (fwd) and 5’-TAG TGT GTG ACA TGA ATT CGG ATC CTT TGT AGA GCT CAT CCA TG-3’ (rev). NT* was amplified using the primers 5’-TGA GCT CTA CAA AGG ATC CGA ATT CAT GTC ACA CAC TAC ACC-3’ (fwd) and 5’-TGA TTA TGA TCT AGA GTC GCG GCC GCT TAT GAA TTC CCA GCA CTG AC-3’ (rev). The AgDD-sfGFP and NT* PCR products were inserted in the EGFP-N1 backbone according to the procedure outlined above giving rise to AgDDsfGFP-NT* (EGFP-N1). The AgDD-sfGFP-NT* open reading frame was PCR amplified from AgDD-sfGFP-NT* (EGFP-N1) the using the primers 5’-AAA CAT TCT TGG ACA CAA ACT CGA GTA CAA CTT TAA CTC ACA C-3’ (fwd) and 5’-TAA GCT GCA ATA AAC AAG TTA ACT TAT GAA TTC CCA GCA CTG-3’ (rev). The PCR product was used to swap the insert in AgDD-sfGFP plasmid, which was excising with the restriction enzymes HpaI and XhoI, with AgDD-sfGFP-NT* using the procedure outlined above. NLS-AgDD-sfGFP was received from Add-gene (#80625). The NLS-AgDD-NT*-sfGFP was generated by PCR amplifying the open reading frame from NLS-AgDD-sfGFP (Addgene #80625) using the primers 5’-GTC TCA TCA TTT TGG CAA AGA ATT CGC CAC CAT GCC AC-3’ (fwd) and 5’-TAG TGT GTG ACA TGAA TTC TTC CGG TTT TAG AAG CTC CAC-3’ (rev). The PCR product was inserted with the PCR amplified NT*-tag using NEbuilder HiFi DNA Assembly Master Mix. The NT*-sfGFP was generated by assembling single strand DNA oligo 5’-GTA TGA ATG ATG TCA GTG CTG GGA ATT CAG CTA GCT TCA GCA AAG GAG AAG AAC TTT TCA C-3’ (NT*-sfGFP) with EcoRI digested NT*-AgDD-sfGFP plasmid, using NEbuilder HiFi DNA Assembly Master Mix.The AgDD-sfGFP construct was purchased from Addgene (#78289). All PCR amplifications were done according to the manufacturer’s instructions for Phusion HF DNA polymerase . The DD domain was PCR amplified using the primers 5’-CAG TGC TGG GAA TTC AAA TTC GGC CAC CAT GCT GG-3’ (fwd) and 5’-AAG TTC TTC TCC TTT GCT GAA TTC TTC CGG TTT TAG AAG CTC CAC-3’ (rev). The NT*-tag was PCR amplified using the previously described NT*-Aβ42 constructParental HeLa cells and DAOY cells were transfected with either AgDD-sfGFP, NT*-AGDD-sfGFP, AgDD-NT*-sfGFP or AgDD-sfGFP-NT* together with 0,2 µg PiggyBac Transposase Expression Vector using Lipofectamine 3000  according to manufacturer’s instructions. Transfected cells were successively sorted by flow cytometry for sfGFP expression at 3 and 10 days (HeLa cells) or only at 3 days (DAOY cells) after transfection using the ARIA III FACS sorter followed by expansion of the sorted cells.Stable cell lines were seeded on coverslips in the presence or absence of 1 µM Shield-1 . Cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 15 min at 24 or 48 h postseeding. Coverslips were washed, stained with 2 µg/ml Hoechst 33342 (Thermo Fischer Scientific) in PBS for 15 min and mounted with homemade Mowiol/DABCO. Coverslips were imaged using a Zeiss LSM 880 confocal microscope with a Plan-Apo 40x/1.3 Oil objective  or a Zeiss LSM 710 confocal microscope equipped with a C-Apochromat 40X/1.2 water objective.oC for 30 min. The supernatant was mixed with 0.5 of its volume of 4x reducing NuPAGE LDS sample and boiled (“Soluble fraction”). The pellet was washed twice with lysis buffer and resuspended in equal volumes of lysis buffer and 4x reducing NuPAGE LDS sample buffer. These samples were boiled and sonicated (“Insoluble fraction”). Samples were run on 4–12% NuPAGE Bis-Tris gels  with MOPS running buffer and transferred to PVDF membranes. The membranes were blocked in 5% milk dissolved in Tris-buffered saline (TBS) + 0,1% Tween and incubated first with primary antibodies against GFP , against the NT*-tag38  and GAPDH , followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG secondary antibodies (both 1:5000)  and imaged with the Odyssey infrared imaging system . The intensity of the GFP bands in the “Soluble fraction” and “Insoluble fraction” of lysates collected in three independent experiments - performed on separate days - of the soluble and insoluble fractions were quantified using ImageJ.HeLa cells stably expressing AgDD-sfGFP, NT*-AgDD-sfGFP, AgDD-NT*-sfGFP or AgDD-sfGFP-NT* were incubated in the absence or presence of 1 µM Shield-1 for 16-24 h. Cells were washed twice with PBS, harvested in lysis buffer A , 10 µM MG132) and incubated on ice for 50 min. A small part of the sample was taken and mixed with 0.5 of its volume of 4x reducing NuPAGE LDS sample buffer  and boiled (“Input”). The remaining sample was spun down at 21.130 g and 4 Quantifications of western blots were obtained from three independent experiments. All experiments have been checked for reproducibility.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Domain adaptation aims at reducing the domain shift between a labeled source domain and an unlabeled target domain, so that the source model can be generalized to target domains without fine tuning. In this paper, we propose to evaluate the cross-domain transferability between source and target samples by domain prediction uncertainty, which is quantified via Wasserstein gradient flows. Further, we exploit it for reweighting the training samples to alleviate the issue of domain shift. The proposed mechanism provides a meaningful curriculum for cross-domain transfer and adaptively rules out samples that contain too much domain specific information during domain adaptation. Experiments on several benchmark datasets demonstrate that our reweighting mechanism can achieve improved results in both balanced and partial domain adaptation. Unsupervised domain adaptation transfers knowledge from a labeled source domain to an unlabeled target domain. The goal is to learn a shared latent representation of source and target samples, complemented with a classifier for accurate classification using the latent representation as input. During learning, the differences between source and target representations are minimized at a population (distribution) level, while the discriminative ability of the classifier is maximized using only the labeled source data. Subsequently, the learned classifier and representation can be used to predict on target samples without the need of manual labeling effort. With the popularity of Generative Adversarial Nets (GANs)  The datasets may include samples that contain too much domain specific information. Matching with such samples may cause unreliable gradient and deterioration during training  Most of these approaches assume that the source and target are generated from the same set of classes, i.e., the balanced domain adaptation. In many real applications, however, in order to for the source knowledge to cover the target but with no information on target labels, we may have to collected a much larger source dataset with classes that do not present in the target domain. Such problem is described as the partial domain adaptation , then move to domain specific samples (difficult to transfer). Such a strategy can improve the stability of the adversarial learning process . We seek to leverage information in the source and a set of (unlabeled) target inputs, Xt, to develop a target label classification model without knowing the target label Yt. Similarly, Yt ∈ t can take one of Kt distinct labels with probability P(Yt). Here we not only consider the standard scenario, denoted as balanced domain adaptation, where s = t and P(Ys) = P(Yt), but also the partial domain adaptation, where t ⊂ s, i.e., the target labels are a true subset of the source labels, so Kt < Ks. This is a common scenario in practice, where we need to transfer from a large source dataset to a smaller target with fewer number of classes.Assume we have a labeled C(·), and a domain discriminator D(·).Previous methods, such as Tzeng et al. , performxs; ψs) and label predictor Cϕ(·) are trained in a supervised-learning manner on The source encoder Enc(Cϕ(·) is assumed to perform a softmax activation operation and s) and Cϕ(·) will be fixed during adaptation.where D, until D can no longer set an effective boundary between the source and target. During training, the discriminator is trained by minimizing the adversarial objective, To minimize the impact of the discrepancy of features between source and target domains, the discriminator is learnt to classify the source and target feature domains, while the target encoder is trained to fool And the target encoder is separately minimized by,where we have inverted the labels relative to Equation (2) as in Goodfellow et al. , which hWasserstein uncertainty estimation is achieved via Wasserstein gradient flows (WGFs) Villani, . It is aθ, θ′) such that the two marginals equal μ and ν, respectively. The Wasserstein distance defines an optimal-transport problem, where one wants to transform μ to ν with minimum cost  in a metric space. If μ is absolutely continuous w.r.t. the Lebesgue measure, there is a unique optimal transport plan from μ to ν, i.e., a mapping T:ℝd → ℝd pushing μ onto ν satisfying T#μ = ν. Here T#μ denotes the pushforward measure  is the set of joint distributions over . Motivated by the Euclidean-space case, if we define velocity of the particle, a gradient flow can be defined on Consider Lemma 1. τ}τ ∈ μ be an absolutely-continuous curve in vτ defines a gradient flow on τμτ+∇θ·(vτμτ) = 0, where aLet {.F above is lifted to be a functional in the space of probability measures, mapping a probability measure μ to a real value, i.e., F is the energy functional of a gradient flow on vτ in Lemma 1 has the form first variation of F at μτ  defines a solution path on this manifold. Usually, by choosing appropriate F, the landscape is convex, e.g., the Itó-diffusion case  distribution for θ, denoted as μ(θ), is learned by solving an appropriate gradient-flow problem. To make the stationary distribution of the WGF consistent with the target posterior distribution, we define an energy functional characterizing the similarity between the current variational distribution and the true distribution pθ as:where potential energy is based on an i.i.d. assumption of the model, and E2 is the energy functional of a pure Brownian motion  = 0 in Equation 7). According to Equation (5), the first variation of functional E1 and E2 can be calculated as:Note F(μ) defines a landscape determined by pθ.Substituting Equations (8) into (5) yields the specific PDE form of the WGF. The energy functional M particles To solve the above WGF problem (Equation 5) we proposed to use particles, approximating μ with θk is a delta function with a spike at θk. Consequently, solving for the optimal μ is equivalent to updating the particles. We investigate the numerical solution to solve (Equation 5) via the discrete-gradient-flow method.where δt into a piece-wise linear curve, leading to an iterative optimization problem to solve the intermediate points denoted as k denotes the discrete points, and h is referred to as the stepsize parameter. The iterative optimization problem is also known as the minimizing movement scheme (MMS)  approximate (Equation 5) by discretizing the continuous curve μF1 ≜ KL(μ||pθ) can be easily approximated as:With particles approximating the μ in Equation (9), the evolution of distributions described by Equation (5) can be approximated with gradient descent on particles. According to Liu and Wang , the grawhere κ is the kernel function, which typically is the radial basis function (RBF) kernel defined as T*. IPOT provides a solution to the original Wasserstein distance specified in Equation (4). Specifically, IPOT iteratively solves the following optimization problem using the proximal point method  is the entropy function and cov(·) is the covariance operator. The norm of the covariance matrix corresponds to its largest eigenvalue, which is identical to the largest variance among all 1D projections of the particle outputs Uent(x) and Uvar(x) are called Wasserstein uncertainty estimation. They describes the domain prediction uncertainty of the input samples with a domain discriminator.where For adversarial domain matching, we propose to reweight the source and target samples according to our domain prediction uncertainty. Given a training sample, its uncertainty weight can be evaluated as,where k for the discriminator, k = 1, …, M, the adversarial objective can be modified as,We integrate our uncertainty weights into the adversarial domain matching in Section 2.1. For each particle The loss for the target encoder is modified as,The complete procedure for the proposed adversarial domain matching is illustrated in Remark 2. The proposed method can be regarded as a scheme of adaptive importance sampling, which excludes the difficult instances at the earlier stage of adaptation and stabilize the adversarial training. Further, in partial domain adaptation, source classes not included in target will be predicted with very low domain uncertainty throughout training. These classes will be down weighted and ruled out during domain matching.Unsupervised domain adaptation is based on the appropriate matching between the source and target distributions . We evaluate WUDM on three domain adaptation benchmark datasets: the digits datasets, Office31 and Visda2017. In order to evaluate the effectiveness of our proposed Wasserstein uncertain estimation, we conduct an ablation test by using a domain discriminator with point estimation, denoted as UDM, where the uncertainty is estimated only with its prediction entropy as in Equation (13). UDM is an ablation study of our proposed Wasserstein Uncertainty Estimation, which is equivalent to estimating the domain uncertaintyusing the entropy uncertainty , Webcam (W) and DSLR (D). This is a relatively difficult dataset since the Webcam and DSLR contains very small amount of images, i.e., less than 10 for some classes, which may easily lead to overfitting during the adaptation process. In order to explore different combinations of large and small datasets for the source and target, we consider three transfer directions: A → D , W → A  and W → D .The data pre-processing and experiment setting are the same as above for Visda2017 except that we use ResNet-50 with 31-dimensional output instead of 12. Due to the small size of Office31, we approach the task as fully transductive, where all labeled instances from the source and all unlabeled instances from the target are used during training and adaptation. This is the same for the experiments in Long et al. , Ganin eWe conduct experiments with all the 10 digits in the balanced setting. The results are shown in Note that the proposed method outperforms simple model (point estimation) with a large margin. This indicated that our uncertainty weights with Equation (15) is more accurate in representing sample uncertainty, demonstrating the effectiveness of our Wasserstein uncertainty estimation.Following Cao et al. , we onlyt-SNE. It can be shown that other methods tend to negatively transfer the target samples toward the redundant source classes. These samples will be misclassified by the source classifier and caused degraded performance for the adaptation. Our method promotes transfer within the same class, which preserves intra-class structure of target representation during domain adaptation. The resulting target representation is more discriminative and less entangled.In We select the 10 classes shared by Office31 and Caltech-256 as our target labels. For each direction of adaptation, we use all the images of these 10 classes in the target split as the target domain , and images from all the 31 classes in the source split as the source domain . In Combined with the experiments of Visda2017, our method tend to produce larger performance improvement compared with the results from balanced domain adaptation. This is because partial domain adaptation suffers from larger degree of negative transfer, and our method can alleviate such effect by ruling out the irrelevant source classes and focusing on source samples that are more informative for target classification. The average accuracies of WUDM are higher then UDM in both Visda2017 and Office31. This validates the usefulness of our Wasserstein uncertainty estimation in the case of partial domain adaptation.In this paper, we propose to reweight the source and target samples in domain adaptation with domain prediction uncertainty. For estimation of domain uncertainty, we employ a probabilistic domain discriminator and develop the Wasserstein uncertainty estimation, which can be easily integrated into concurrent adversarial domain matching. The resulting uncertainty weights impose an adaptive curriculum on domain adaptation that stabilize adversarial training and alleviate the effect of negative transfer in the case of partial domain adaptation. Experiments on several benchmarks show that our method achieves improved results on both balanced and partial domain adaptation.http://yann.lecun.com/exdb/mnist/, https://paperswithcode.com/dataset/office-31, http://ai.bu.edu/visda-2017/.Publicly available datasets were analyzed in this study. This data can be found here: All authors contributed to the article and approved the submitted version.It is funded by ECE, Duke University.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. The handling editor SX is currently organizing a Research Topic with the author(s) RZ.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. 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Liquid sodium‐potassium (Na‐K) alloy has the characteristics of high abundance, low redox potential, high capacity, and no dendrites, which has become an ideal alternative material for potassium/sodium metal anodes. However, the high surface tension of liquid sodium potassium alloy at room temperature makes it inconvenient in practical use. Here, the Na‐K as reducing agent treats with hydrazone linkages of covalent organic frameworks (COFs) and obtain the carbon‐oxygen radical COFs (COR‐Tf‐DHzDM‐COFs). The preparation method solves the problems that the preparation process of the traditional Na‐K composite anode is complex and has high cost. The structures of the COR‐Tf‐DHzDM‐COFs are characterized by X‐ray diffraction （XRD), fourier transform infrared (FT‐IR), electron paramagnetic resonance (EPR), and solid‐state NMR measurements. It is the first time that carbon‐oxygen radical COFs from bulk COFs are constructed by one‐step method and the operation is flexible, convenient, and high rate of quality, which is suitable for big production and widely used. The cycle stability of the composite Na‐K anode is improved, which provides a new idea for the design of high‐performance liquid metal anode. Radical COFs realized by the incorporation of Na‐K alloy are applied as non‐Newtonian viscous liquid anode. Due to the high surface tension and good structural stability, the new non‐Newtonian fluid COR‐Tf‐DHzDM‐COFs@NaK exhibits excellent electrochemical performance. Among them, the liquid metal anode has deformation and self‐healing properties, which can slow down the volume change caused by metal dendrite growth during the cycle. For example, a new flexible lithium‐ion battery anode was developed by room temperature gallium‐based liquid metal and two‐dimensional material of MXene. As for the more economical alkali metal of sodium and potassium would be more versatile. However, obtaining common metals in liquid phase usually requires high temperature operation, which will bring safety problems and additional energy consumption. Unlike ordinary metals, sodium potassium (Na‐K) alloys can be stable in liquid phase at room temperature. These excellent properties make Na‐K alloy an excellent anode material in alkali metal‐based batteries, and alleviate the formation of dendrites and short circuit.With the wide application of portable electronic equipment and electric vehicles, higher requirements are put forward for high energy density batteries.1 Alth Therefore, regulating the stability of radical intermediates by grafting the radical monomer to covalent organic frameworks (COFs) is one of the ways to optimize the performance. The previous of study pointed out that nitrogen‐oxygen/carbon‐oxygen/carbon/nitrogen radical COFs for catalyst and electrode have a highly active and fast reaction kinetics, which is helpful in rate of reaction. The spatial effect of unpaired electrons can be de‐localized by ionization and π–π conjugate interaction, and the self‐exchange behavior of intermolecular electrons can be restricted. Rigid big rings are often used to regulate the stability of radical intermediates, which can inhibit dissolution in electrolyte, transport charge by π conjugated frameworks and transport ions by a large number of nanoporous. However, the current synthesis of radical‐based COFs requires linking radical units, which increases the threshold. In addition, the preparation process of the traditional direct synthetic radical COFs are complex and has high cost, which greatly limits their application. the radical monomer is unstable and complex synthesis process, which hinders the research work to go deeper. Hence, here reports a special method that used a liquid alkali metal  reducing agent, the successfully in situ construction of carbon‐oxygen radical COFs (COR‐Tf‐DHzDM‐COFs) in bulk quantities using a simple and cheap process. The whole preparation process (mixing and filtering) is simple and feasible, does not need special apparatuses, has low cost and is suitable for mass production and promotion. Significantly, the raw COFs of Tf‐DHzDM‐COFs ) don't need a higher temperatures and long reaction time , which can be removed from the crystalline structure through sufficient washed with water. This method is suitable for large‐scale production of carbon‐oxygen radical COFs materials. Depending on all above studies and experiments, we can conclude that using Na‐K alloy treatment rules in carbon‐oxygen radical synthesis is viable. More than anything, the proposed method can also be applied to similar tasks.The crystal structure of the COR‐Tf‐DHzDM‐COFs was further tested by X‐ray powder diffraction (XRD) instrument Figure . All dif12 ind In addiction, the electrochemical performance of half‐cell with COFs and the composites anode was tested. CV test showed that a Na+/K+ intercalation potential of 0.5–1 V  and scanning electron microscopy (SEM), as shown in Figure 14 In Figure  There are slight changes for the carbonyl group both in the FT‐IR and NMR spectra. Close analysis of the FT‐IR spectra, the peak at 1305–1200 cm−1 was flattened compared to the raw material; and in the NMR spectra the peak at 127.45 ppm was also overlapped by the neighboring peak. All this indicating the occurrence of the carbon‐oxygen radical, which weakens the original signal.A better understanding of the effects of Na‐K alloy treatment on the presence form of O element in COR‐Tf‐DHzDM‐COFs at molecular levels is performed by Fourier Transform Infrared Spectroscopy  in the COR‐Tf‐DHzDM‐COFs, indicating that all the alkali‐metal had been removed. Furthermore, Figure  indicating that the chemical environment around N element changes. Obliviously, it can be seen that the proportion of oxygen vacancy in the COR‐Tf‐DHzDM‐COFs is significantly higher than that of Tf‐DHzDM‐COFs, indicating that the carbon‐oxygen radical concentration increases. It can be understood as a short time after the treatment of Na‐K alloy reducing, some position of oxygen can under strong reduction and leaving the original position left oxygen vacancy, which also verified from the EPR. The results further prove that the COR‐Tf‐DHzDM‐COFs is a kind of porous crystallinity material with carbon‐oxygen radical.Furthermore, XPS experiment was used to characterize the O, C element of COR‐Tf‐DHzDM‐COFs and Tf‐DHzDM‐COFs, and the fitting spectrum obtained is shown in Figure 16 Them sulfuric acid and 1 m hydrochloric acid, for 24 h, and then the solid was filtered and dried. The X‐ray powder diffraction (XRD) was used for analysis and comparison the anti‐acidity of carbon‐oxygen radical in COR‐Tf‐DHzDM‐COFs  and room temperature. Therefore, it can be considered that the COR‐Tf‐DHzDM‐COFs studied in this paper brings new possibilities for defect engineering to improve material properties, and this work has universal significance in many research fields.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.


Carbon fibers and resin used in manufacturing carbon fiber-reinforced plastic composite structures flow before the resin solidifies, resulting in disrupted fiber orientation and non-uniform thickness. This process, known as consolidation, is critical for the quality of the composite structure, but no technology exists to measure the deformation in situ. This study proposes a strip-type embeddable shape sensor based on fiber optics for in situ monitoring of consolidation deformation. The sensor consists of a thin, flexible sheet with optical fibers embedded in the upper and lower surfaces of the sheet, and it can monitor out-of-plane bending deformation in composite materials during consolidation. Finite element analysis and experiments are used to evaluate the basic performance of the shape sensor before it is applied to composite gap/lap monitoring. For the first time, the relaxation of consolidation deformation due to the flow of fiber-resin suspension is measured. The proposed sensor will be a powerful tool for elucidating consolidation mechanisms and for validating composite manufacturing simulations. Carbon fiber-reinforced plastic (CFRP) composites have several applications because of their high mechanical properties and excellent environmental durability. There are various methods for manufacturing CFRP structures, including the method that uses intermediate material sheets called prepreg, in which carbon fibers are impregnated with uncured resin, which can manufacture high-quality CFRP required for aerospace applications ,2. VoidsFor evaluating the cure-induced deformation of composite materials, embedded optical fiber sensors are effective ,19,20. FThis study proposes an in situ deformation measurement method for composite consolidation using a unique strip-type embeddable shape sensor based on fiber optics. First, an overview of the proposed method is described. Then, basic shape identification tests are conducted to evaluate the performance of the sensor. Finally, the effectiveness of the proposed sensor is demonstrated by monitoring composite gap/lap sections a.It is important to note that the 0° layer deformation that occurs during the consolidation process is dominated by out-of-plane bending deformation . The conTherefore, this study proposes a strip-type embeddable shape sensor based on fiber optics . This seThe Bernoulli–Euler beam theory is used to calculate the sensor out-of-plane displacement w . First, This study uses ODiSI A-50 , one of the most advanced commercially available instruments, to measure the strain distribution along the optical fiber embedded in the shape sensor . ODiSI uTo evaluate the validity of the proposed shape sensor, a finite element analysis was performed using Abaqus 2021 . The distance E11 = 180 GPa, thickness 180 μm) and the two shape sensors. Convex shapes of two different heights were generated on the shape sensors by vacuum bagging the entire specimen. Note that the composite sheet was used to prevent the sensor from breaking due to local deformation where the sensor and wires came in contact. The curvature was calculated from the strain measured by a distributed strain measurement system (ODiSI A-50) using Equation (1), and the deformation was calculated using Equation (3). The sampling interval for strain measurement was set to 1 mm for accurately and stably measuring changes in the strain distribution caused by deformation. Additionally, to evaluate the stability of the sensor response in the heating environment expected during composite curing, a test was conducted, in which the vacuum bagged specimen was heated to 100 °C in an oven.To measure deformation using the fabricated shape sensors , a demon−5 mm−1 for the 0-sensor and ±3 × 10−5 mm−1 for the 90-sensor, respectively. The micrometer-scale deformation caused sufficiently large curvature changes compared to the measurement error, confirming that the proposed sensor can sensitively detect micrometer-scale deformations. It is interesting to note that the 0-sensor had a slightly wider and smaller curvature than the 90-sensor, especially at the 100 μm wire position. This was because the bending rigidity of the 0-sensor was very high, which prevented the sensor from completely following the steep deformation of the composite sheet. Recently, large-scaled composite structures have been manufactured using an automated lay-up machine that aligns and laminates several narrow prepreg sheets known as slit tapes. However, gaps and overlaps occur between the slit tapes due to the limit of lay-up accuracy. These gaps and overlaps cause changes in fiber orientation and resin-rich areas during consolidation a, result2/902]S. The prepreg sheets were laid up by hand, with a 5 mm wide gap and lap on one ply of the 90° layer just below the upper 0° layer. Two 6 mm wide sections of one ply of 0° prepreg directly above the gap/lap were removed, and the two kinds of 6 mm wide shape sensors were inserted there. The entire specimen was vacuum bagged and then cured in an autoclave via heating. The curing process was as follows. Following vacuuming, the autoclave was pressurized to 0.3 MPa at 20 °C, then heated to 100 °C at 2 °C/min, maintained at 100 °C for 90 min, and then cooled to room temperature at 2 °C/min. The curvature of the shape sensor during this curing process was measured by the measurement system (ODiSI A-50). The measurements were taken manually when predetermined temperatures were reached, and each measurement took less than 5 s to complete. After curing, the thickness distribution of the specimen was measured using a 3D scanner , and the cross section of the specimen was polished for observation.Additionally, as a preliminary preparation, the viscosity of the epoxy resin during the curing process was evaluated using a rheometer . The resin was squeezed out of the T700S/2592 prepreg. This study proposed a strip-type embeddable shape sensor based on fiber optics that enables in situ monitoring of composite consolidation deformation. The sensor consists of a thin, flexible sheet with optical fibers embedded in the upper and lower surfaces of the sheet. The basic response performance of the shape sensor was first evaluated using finite element analysis and experiments. The results confirmed that the sensor with low bending rigidity is preferred. The sensor was then applied to composite gap/lap monitoring. For the first time, the relaxation of consolidation deformation due to the flow of fiber/resin suspension was measured. The shape obtained by the sensor and the deformation at the gap/lap section agreed well, confirming the validity of the proposed sensor system. The proposed sensor will be a powerful tool for elucidating consolidation deformation mechanisms and for validating composite manufacturing simulations.


Influence of strain accumulation schedule during hot rolling, under the thermomechanical control process (TMCP) on the structure evolution and carbide transformations of Cr-Ni-Ti austenitic stainless steel, is studied. The cellular, fragmented dislocation substructure and dynamically recrystallized (DRX) structure are observed in the steel with different strain accumulation schedules. It was found that the strain accumulation schedule, especially fractionality, affects the work-hardening and softening behavior quite significantly. The role of the strain accumulation schedule on the fragmented substructure and DRX structure evolution as well as carbide transformations and the relationship between the microstructure changes due to TMCP and the mechanical properties of studied steel, involving the recent ideas of the physics of large plastic strains, are considered. Austenitic stainless steels have been widely used in a variety of industrial fields due to their superior corrosion resistance and excellent mechanical properties. However, the relatively low yield strength of austenitic stainless steels is an obstacle to wide-spread applications. As well known, TMCP is one of the most advanced resource-saving technologies for the manufacture of metal blanks and parts ,2,3. A rThe role of DRX and some examples of the investigations dealing with the study of the evolution of grain boundaries and subboundaries in stainless steel during DRX have been described in ,15,16,17Under normal conditions, hot rolling is usually performed by several passes. At different ranges of strain accumulation schedules, the corrosion-resistant austenitic steel , of type Cr-Ni-Ti  has been studied. Specimens with a cross-section of 22 mm × 22 mm were heated for 40 min at 1180 °C. Then, they were air-cooled to the studied deformation temperature and rolled on a laboratory rolling mill  at 0.35 m/s. In the case of one-pass, the strain degree during rolling was r, %) along the section of the workpiece for all of the studied modes was estimated using the methods of stereometric analysis and computer metallography [12,31,51%, and during fractional rolling—10% per pass. One-time deformation may be applied with strain degrees of 31 and 51%. In the third (1060 °C) and fifth (1010 °C) passes, rolling was carried out after cooling from a heating temperature of 1180 °C to temperatures corresponding to the deformation temperature during the implementation of the fractional mode, respectively. Metallographic analysis has been performed using the light microscope Zeiss Axio Observer Dlm  with an image analysis system. The fraction of the recrystallized structure , the microstructure of steel, which is characterized by large grains containing annealing twins and round carbide inclusions of a few microns, was uniformly distributed throughout the sample volume. Under conditions of fractional reduction, an increase in the total degree of reduction results in the monotonic diminution of grain size and growth of precipitate density ρiε. A rise in the strain degree of one-pass deformation has a completely different effect on the initial structure. Both the grain size and the precipitate density change non-monotonically. The grain sizes are smallest at ε = 30%  is a fairly stable activity, and its particles of different degrees of dispersity are present in the steel up to 1150 °C.εΣ is Ton = 1150 °C. In one-time deformation, rolling with strain degree ε = 10% also starts at 1150 °C, but rolling with ε = 30 and 50% occurs after the cooling samples are preliminarily heated to 1150 °C to temperatures corresponding to the third (1070 °C) and fifth (1020 °C) pass in fractional deformation .The rolling temperature conditions in the given experiment are selected as a function of the accumulation method of deformation. With fractional deformation, the temperature of the onset of rolling for all of the total degrees of reduction In steel deformation, there is a solution of finely dispersed carbide, which is based on their interaction with the flux of moving dislocations. Freely moving carbon atoms enter the solid solution or segregate at dislocations in the form of Cottrell atmospheres. An increase in the magnitude or rate of deformation is accompanied by an increase in the degree of solution of the initial carbide phase.With preliminary cooling of the steel or cooling in the pauses between deformation, the solid solution corresponding to the limit of solubility of C, Ti atoms fractionally become supersaturated, and break down, with the deposition of carbide phase particles. As more dislocations and inter-fragment boundaries are present in the metal volume, the number of sites for carbide particle deposition increases and these particles are found to be more dispersed and homogeneously distributed throughout the volume.To explain the influence of the method of accumulating deformation on carbide transformations in the steel at the conditions of the experiment, the following factors must be considered.The aforementioned peculiarities have determined the kinetics of structure transformations. Therefore, it may be proposed that the lowering of strengthening tendency should take place when the solubility temperature of carbide decreases.Mechanical properties.The laws of structural changes observed in the present work with one-time and fractional accumulation of deformation in Cr-Ni-Ti austenitic stainless steel are of practical importance, since they permit the prediction of its behavior in rolling and mechanical tests. For example, with fractional accumulation of deformation, a monotonous increase in strength and decrease in ductility can be expected, which is associated with an increase in the density of dislocations, dispersed releases of the carbide phase, and the proportion of fragmented structure in the volume see . In the The strain accumulation schedule, during hot rolling under TMCP conditions, affects the strengthening and softening behavior of Cr-Ni-Ti austenitic stainless steel quite significantly. The monotonic growth in strength at the fractional accumulation of deformation results in an increase in the density of dislocations and precipitates, as well as a rise in fragmented substructure quantities. The magnification of one-time deformation reductions results in DRX volumes. As more dislocations and inter-fragment boundaries are present in the metal volume, the number of sites for carbide particle deposition increases. Furthermore, these particles are found to be more dispersed and homogeneously distributed throughout the volume.