The production of factory-made mortars is a multicomponent system. Viscosity-modifying admixtures (VMAs) are an inherent ingredient of these materials. The correct choice of the amount and type of these admixtures is important from the practical and scientific points of view. In this article, the use of cellulose ether (CE) and starch ether (SE) in cement pastes and mortars is studied. This research focuses on the hydration process and fresh-state properties of mortars because this subject determines the correct choice of the amount and type of admixture used, and the results determine the application and properties of hardened mortars. Polymers were added in the range from 0.056% to 0.22% in relation to the dry ingredients of the mortar. The research showed that cellulose ether had the greatest impact on the consistency, air content, bulk density, and water retention of ordinary dry-mix mortars. On the other hand, starch ether affected the hydration process, delaying the setting and hardening processes much more than cellulose ether. The action of these admixtures rose with the increase in the amount of polymer used in different ways (depending on the type of ether). Chemical admixtures are widely used to modify mortars and concretes in order to obtain standard or special properties. Mortar formulations quite often comprise from a few to over a dozen different ingredients, so the development of such recipes for mortar requires great knowledge and practice. Even small amounts of some admixtures can have a significant effect on the properties of fresh and hardened mortars. Among the wide range of these substances used in the production of factory-made masonry, plastering, and adhesive mortars, there are polymer admixtures, which include, among others, cellulose ethers and starch ethers ,3,4,5,6.Cellulose is a natural polysaccharide. It commonly occurs as the basic construction material in plant tissues. Pure cellulose is a white, water-insoluble substance, which must be further processed to be used as a chemical admixture. Cellulose ethers arise from a chemical industrial process. The plants etherified to obtain polymer admixtures include pure cotton (about 90% cellulose), wood (about 40–55% cellulose), and straw or reed (30–40% cellulose) ,15,16,17Starch is a natural polymer derived from the polysaccharide group. It is most often obtained from potatoes or corn. It is found in tubers, seeds, and plant roots. The main component of starch is amylopectin ,11,34,353A or cement hydration have been examined. These works have shown that cellulose ether has an influence on these processes. This polymer leads to a gradual slowing down of hydration dependent on cellulose ether chemistry. Water retention and rheological measurements have been tested in various reports [In the literature, one can usually find research on the properties of mortars modified with only one admixture—either starch ether or cellulose ether. Analyses often concern the influence of one type of admixture on the properties of pastes or mortars  ,6,10. So reports ,22,26. T reports ,18,30.Some research on pastes and mortars can be found in which the authors compare the actions of different VMAs. In , mortarsThe goal of the present work is the study and comparison of the uses of two types of viscosity-modifying polymer admixtures, which have important roles in dry-mixed mortars. The variables in the study are the type of admixture used and its amount. The paper reports different properties between mortars with cellulose ether and starch ether, as well as consistency, air content, and bulk density. The WRV value is defined as water retention measurements tested after, 10, 20, and 30 min. On this basis, the application properties of these materials are analyzed. Additionally, the influences of admixtures on the setting and hardening processes of the pastes are examined on the basis of calorimetric measurements and setting-time research. Setting-time research gives information about the initial, final, and total setting times, but the calorimetric method allows the full introduction of the hydration process in the first several hours of research. These parameters can allow a full understanding of the actions of cellulose ether and starch ether, which is necessary for the correct choice of the type and amount of admixture used. Additionally, if starch ether is added to dry-mix mortars with cellulose ether, understanding of its action and choosing the amount of starch ether should be preceded by research on the separate effect of each ether on the properties of pastes and mortars. In the present work, we assess how the same amount of cellulose ether and starch ether admixture affects the tested properties, as well as whether the scope of their activity is the same. In addition, the statistical influence of the amount of admixture on the tested parameters of pastes and mortars is verified, and on this basis, trends in changes of the tested parameters are determined. In summary, the article supplements the knowledge on the scope of operation of cellulose ether and starch ether based on the standard and nonstandard properties of pastes and mortars in the plastic state, as well as during the setting and hardening processes. On the basis of the research performed, it can be stated how the properties of pastes and mortars change, taking into account variable doses of admixtures, and it can be assessed how the same amount of both cellulose ether and starch ether affects the tested parameters, which complements the knowledge on the scope of operation of these polymers.CEM I Portland cement of the 42.5 R class , natural chalcedonite sand with a fraction of 0.1–1.0 mm as a fine aggregate , and tap water were the basic ingredients in the mortars. Selected physicochemical properties of the cement are given in The compositions of mortars were modified with the following commercial polymers: cellulose ether or starch ether as VMAs. Admixtures recommended by the producer for the production of cement-based mortars were selected for this research. The physical and chemical properties of CE and SE are shown in The amounts of binder, water, and sand were constant for all the mortars. The proportions by weight were one part cement to three parts fine aggregate. The amount of cellulose ether and starch ether used varied, and it constituted from 1 g to 4 g (from 0.056% to 0.22% in relation to the dry ingredients of the mortar—cement and sand). The water-to-cement ratio was 1.33 for all the mortars. The amount of water in the reference sample (REF) was chosen to obtain a constant flow table of 165 ± 5 mm. The mix proportions of the cement mortars are shown in The binder, fine aggregate, and water were weighed with an accuracy of 0.1 g, and the polymer admixtures were weighed with an accuracy of 0.0001 g. Water was added to the weighed, pre-mixed components, and everything was mixed mechanically for 3 min with an interval of 30 s in the middle of mixing. The samples prepared in this way were used for testing consistency, bulk density, air content, and WRV values.Paste samples were produced by adding the appropriate amount of polymer admixture (from 0 to 4 g) to 500 g cement and mixing (with water) mechanically in the same way as mixing mortars. Then, the setting-time test was performed for each of the nine samples. For CE and SE, eight pastes were prepared with the following proportions: 5 g cement, 2.5 mL distilled water, and 0, 0.01, 0.02, 0.03, or 0.04 g of admixture. Then, the samples were prepared for calorimetric measurements. The ninth reference sample (REF) did not contain any admixture. The hydration process was finished after 72 h. The setting time and the heat of cement hydration were measured for the pastes with a water-to-cement ratio of 0.5. A constant ratio of 0.5 for water-to-binder for these two types of research was chosen due to the possibility of comparison of the obtained results with the literature data.Preparation of samples and testing were performed in a laboratory at a temperature of 20 ± 2 °C and a relative air humidity of 65 ± 5%.After mixing the components, the consistency of the fresh mortars was tested by two methods. The flow table was determined by the PN-EN 1015-3 method  as the sThe air content was standardized by a pressure method according to PN-EN 1015-7 . The fin3 for each sample.The bulk density of fresh mortars was determined according to PN-EN 1015-6 . The finWater retention research was conducted using a nonstandard method following . The watThe setting times, as well as the beginnings and ends of the setting times, of cement pastes were measured according to the PN-EN 196-3 standard .SH is the degree of hydration, x1 is the value measured after a specified hydration time, and x2 is the value measured after full hydration or after a specified hydration time taken as a point of reference.The rate of cement hydration and the total heat evolution were assessed by heat evolution with a nonadiabatic–nonisothermal microcalorimeter. The measurements were obtained using a BT 2.15 low-temperature differential scanning microcalorimeter . The samples were prepared by blending the dry components with distilled water. The pastes were stored in small, sealed PE zip-bags and placed in the calorimeter. The changes in heat evolution were recorded every 30 s for a period of 72 h of measurement and saved automatically in a computer’s memory. On the basis of these data, the heat evolution curves and the total heat evolution curves as a function of time for all pastes were obtained. Additionally, the degree of hydration was calculated and compared using  and the x2 value read for REF. However, value x1 was applied as the cumulative amount of heat hydration after 12, 24, 36, 48, 60, and 72 h, so Formula (1) took the following form:SH is the degree of hydration; tQ is the cumulative amount of heat hydration after 12, 24, 36, 48, 60, 72 h for each sample; and Q72h is the cumulative amount of heat hydration after 72 h for the reference sample (REF).In this article, the cumulative amount of heat hydration after 72 h was assumed as the result of the The results of flow for all the samples are presented in The flow of the reference mortar (REF) was 166 mm. The results of the studies on the flow of mortars modified with cellulose ether ranged from 154 mm down to 144 mm (decreasing from 7% to 13%). The flow value of the CE4 sample was 144 mm, which was about 13% smaller than that of the REF sample. The results of the studies on the flow of samples modified with starch ether ranged from 148 mm down to 137 mm (decreasing from 11% to 17%). The flow value of the SE4 sample was 137 mm, which was about 17% smaller than that of the REF sample. As can be seen, the addition of even a small amount of starch ether significantly decreased flow. The consistency decreased as the amount of admixture increased, regardless of the type of admixture, but in the case of the action of starch ether, this effect was more noticeable. Both cellulose ether and starch ether have the effect of reducing flow, which must be taken into account when selecting the appropriate amount of admixture for a mortar. By analyzing all the results, it can be said that the used admixtures in amounts of 0.056–0.22% behaved as thickeners. These test results are consistent with those obtained in various publications ). The SEThe results of cone penetration for all the mortars are presented in The cone penetration of the reference mortar (REF) was 5.5 cm. The results of the studies on the consistency of mortars modified with cellulose ether ranged from 4.7 cm to 5.6 cm, and the consistency of mortars modified with starch ether ranged from 2.4 cm to 3.2 cm. It is clearly visible that starch ether reduced the cone penetration (by even twice as much compared to the reference sample). On the other hand, the effect of cellulose ether did not change the consistency significantly.The obtained test results are also worth supplementing with visual observations of the samples during the consistency test using the flow table method. The mortar with 4 g of cellulose ether (CE4) stuck to the walls of the mold. It can be assumed that its dosage was so high that it could cause problems with mortar application. This phenomenon was not observed for the mortar with the same amount of starch ether (SE4). On the other hand, the SE4 sample was more “dry” and less consistent in whole mass than the CE4 sample. It can be assumed that the doses of 0.22% of each ether were too much. In this case, the application of these admixtures seemed ineffective.The results presented on 3. The parameter ranged from 1540 kg/m3 (CE4 sample) to 1790 kg/m3 (CE1 sample) for mortars containing cellulose ether. Adding 4 g of cellulose ether even caused a 17% reduction in bulk density. The parameter ranged from 1820 kg/m3 (SE4 sample) to 1840 kg/m3 (SE1 sample) for mortars containing starch ether. The addition of the admixture of starch ether led to a slight change in the bulk density. On the other hand, higher the content of cellulose ether in the mortar composition caused definitely smaller bulk density, which favorably affected the output. This is especially important from economic and practical points of view. The entrained air had an effect on the reduction of density of the mixture, which is important in terms of improved workability and, especially, during the application of mortar on walls or ceilings [As can be seen, the bulk density values of all the mortars with cellulose ether and starch ether were smaller than that of the reference sample. This parameter of the REF mortar was 1860 kg/mceilings . Figure w/w polymer: dry mortar).The results of water retention for REF were 80.3% after 10 min measurements, followed by 74.2% and 71.1% after 20 and 30 min, respectively. Thus, the mortar without admixtures obtained the lowest parameters. This sample had the smallest water retention. The water loss after 30 min was about 29%. As shown in In summary, the effect of water retention was more visible and noticeable for mortars modified with CE than for samples modified with SE (comparing the amounts of admixtures used in the tests).In The initial setting time for REF was noted after 325 min, but the final setting time was after 405 min, so the setting time for the reference mortar was 80 min. Both cellulose ether and starch ether influenced the binding and hardening processes of the tested pastes. However, this effect was more noticeable with starch ether. Especially large differences in the impacts of the ethers can be observed by comparing the results in On the basis of the calorimetric measurements shown in A supplementary analysis of the obtained research results is introduced in this chapter, including an assessment of the effects of the amount and type of polymer VMA on the selected properties of mortars and pastes. An increase in the amount of polymer admixture resulted in a decrease in flow. Starch ether had a bigger effect on consistency changes. On the other hand, cone penetration was almost the same for mortars modified with cellulose ether and mortars without admixture. Starch ether had a significant influence on the reduction in cone penetration.It was noted that adding cellulose ether admixture could affect the air content, even quadrupling it. In contrast, mortars modified with starch ether had almost two times bigger air content compared with the reference sample.Increase in the dosages of both admixtures decreased bulk density, but the use of cellulose ether was of greater importance, so the workability of these mortars was the biggest. Correlation between the bulk density and air content of the tested mortars showed decreases in bulk density with increases in air content. On the other hand, when air content increased, compressive strength decreased.Mortars modified with cellulose ether and starch ether were characterized by high water retention values. For example, the water retention after 10 min increased from 80.3% with a 0% dose of cellulose ether to 99.5% with 0.22% CE (CE4). Generally, the effectiveness of cellulose ether increased with increase in the dose of this admixture. The WRV value of mortars modified with starch ether ranged from 83.8% (SE1) to 88.1% (SE4). The water loss after 30 min was almost 29% for the reference mortar (REF), which was a negative phenomenon. Mortars modified with 3 and 4 g of cellulose ether had the smallest water loss values. The flow test results correlated with the water retention measurements, especially in the case of the cellulose ether admixture.It was clearly noticeable that both admixtures delayed the initial and final setting times, as well as lengthened the total setting time. Bigger differences in the results between the reference and the modified samples were obtained as a result of adding starch ether. A larger amount of admixture extended and delayed the tested parameters.Cellulose ether and starch ether both led to a gradual slowing of cement hydration and decreased heat evolution, which was also confirmed by the calculation of the degree of hydration.In addition to the results of the cone penetration tests, the statistical influence of the amount of admixture used was noted for the tested parameters.Polymer admixtures acted with different intensity levels on the tested properties of the pastes and mortars. Additionally, the tendencies of changes in the test results were different for each ether.In this paper, research was carried out on mortars modified with cellulose ether and starch ether. The purpose of this study was to evaluate the effect of the amount and type of polymer admixture used on selected properties of cement pastes and mortars. An evaluation was conducted on the fresh-state properties of mortars and the hydration processes of pastes. It was found that the amount, as well as the type of ether used, affected the properties of the tested materials differently. The following particular conclusions were drawn from this research:Due to the differential effects of cellulose ether and starch ether on the properties of mortars in the plastic state, as well as on the setting processes of pastes, the selection of the type of polymer and the amount of admixture used should be preceded by thorough analysis, which allows for obtaining the desired properties of a finished product .


Investigating starch properties of different adzuki beans provides an important theoretical basis for its application. A comparative study was conducted to evaluate the starch content, processing, digestion, and structural quality of 12 adzuki bean varieties. The variation ranges of the 12 adzuki bean varieties with specific analyzed parameters, including the amylose/amylopectin (AM/AP) ratio, bean paste rate, water separation rate, solubility, swelling power and resistant starch (RS) content level, were 5.52–39.05%, 44.7–68.2%, 45.56–54.29%, 6.79–12.07%, 11.83–15.39%, and 2.02–14.634%, respectively. The crystallinity varied from 20.92 to 37.38%, belonging to type BC. In correlation analysis, red and blue represent positive and negative correlation, respectively. Correlation analysis indicated that the termination temperature of adzuki bean starch was positively correlated with AM/AP ratio. Therefore, the higher the melting temperature, the better the freeze–thaw stability. The 12 varieties were divided into Class I, Class II, and Class III by cluster analysis, based on application field. Class I was unsuitable for the diabetics’ diet; Class II was suitable for a stabilizer; and Class III was suitable for bean paste, mixtures, and thickeners. The present study could provide a theoretical basis for their application in the nutritional and nutraceutical field. Vigna angularis. L) originated in China, whose planting area and total output have always ranked top worldwide. China can produce hundreds of flavored foods and is the world’s preeminent exporter of adzuki bean, with an annual export volume of 40,000–60,000 tons. As one of the most popular beans, it has the reputation of “red pearls” in international markets; thus, it is a good source of nutrients [Adzuki bean  is known to resist digestion in the small intestine and further ferments in the gut. RS has been reported to reduce postprandial blood sugar-insulin and plasma cholesterol, as well as increase insulin sensitivity . FermentDuring the change in thermal and crystalline properties of adzuki bean starch, the enthalpy of the starch melting endotherm is the same as the melting temperature of the crystal structure, which reflects the degree of starch crystallization; therefore, the enthalpy of fusion is usually related to the content of crystals in starch. The gelatinization temperature of starch is closely related to the X-ray diffraction (XRD) results of the starch crystal form, and it has a great relationship with the content of other processing quality indicators, such as amylopectin in starch ,16. ReseMost research on adzuki beans has concentrated on the functional and structural relationships of a single species. These functional characteristics include swelling power, solubility, light transmittance, freeze–thaw stability, and in vitro digestibility. Notably, their structural properties and thermal features were investigated and determined via XRD and differential scanning calorimetry (DSC) , respectThe present study aimed to compare the processing quality, which entails the amylose (AM) and amylopectin (AP) content, whiteness, gel texture properties, bean paste rate, freeze–thaw stability, solubility, and swelling power, as well as the digestible quality, which involves an in vitro digestibility analysis of starch, in order to determine the thermal property and crystallization characteristics of 12 Chinese adzuki bean varieties via DSC and XRD. Furthermore, it aimed to conduct the correlation and cluster analyses of their processing, digestible, and structural activities. We originally pointed out the end-products application classifications of some specific varieties of adzuki beans. Finally, we aimed to provide a theoretical basis for the better applications of adzuki bean starch in food and non-food industries.In this experiment, 12 adzuki bean varieties from different ecological regions were selected, and the varieties were selected from local varieties cultivated in different ecological regions in China. The sources, maturity and grain photo of different varieties are shown in 2O5-K2O = 24:4:8) before planting.The test field texture was loam, and the 0–20 cm soil composition of the test field in 2019 and 2020 were as follows: the content of alkali-hydrolyzable nitrogen; 88.37 and 84.76 mg/kg, the content of available phosphorus; 66.21 and 65.525 mg/kg, and the content of available potassium; 178.6 and 181.3 mg/kg, respectively. Fertilizer application in each plot (5 m × 1 m) was 400 g of compound fertilizer (N-P2 concentration (ppm) of 451.49 ± 19.63 ppm, the relative humidity of 75.51 ± 4.87%, the temperature of 26.59 ± 0.81 °C.The microclimate characteristics of the shaded area are shown as follows: the illumination intensity of 58,142.45 ± 1778.58 lx, the COThe experiment was carried out at the teaching and experimental base of Hebei Agricultural University in Baoding from 2019 to 2020 . Twelve adzuki bean varieties from different ecological regions in China were selected, and the natural light in the experiment season was approximately every day, with an illumination time of 14.5 h.2) and watering technique  during the flowering period. Additionally, insecticide  was sprayed during the flowering and podding period.The experiment adopted a randomized block design, with a total of 33 plots, and each variety was repeated thrice. The plot area was 5 × 1 m, sown in holes; 2 grains per hole. The plant spacing was 25 cm, with the row spacing as 50 cm, and two rows were planted in each plot, employing 50 seedlings. The soil was loosened prior to cultivation, and after the seedlings emerged, the two true leaves were unfolded; the seedlings were set when the first compound leaves appeared. Other field management was referred to as conventional field management, which includes field weeded during the growth period, topdressing nitrogen fertilizer application . The glucose content (X1) in the sample was calculated according to the following formula, which was then converted to starch content [The enzymatic hydrolysis method according to the Chinese National Standard (GB 5009.9-2016) was adopted for the determination of total starch content. The basic principle is that after removing fat and soluble sugars from the sample, starch is hydrolyzed into small molecular sugars with amylase , followed by hydrolyzing into glucose with hydrochloric acid (6 mol/L), and finally determined with the content of glucose. Then, 5.00 mL of alkaline copper tartrate solution A (15 g of copper sulfate and 0.050 g of methylene blue were dissolved in water and fixed to 1000 mL) and 5.00 mL of alkaline copper tartrate solution B  were placed in a 150 mL conical flask, and then 10 mL of water was added. Two glass beads were put into the mixture, and about 9 mL of glucose standard solution from the burette was dropped, controlling the mixture to boil within 2 min. The solution was kept boiling, continuing to drop glucose at the rate of one drop every two seconds until the blue color of the solution faded as the endpoint, recording the total volume of glucose standard solution consumed, and making triplicate, taking the average value to calculate every 10 mL (5 mL for solution A and 5 mL for solution B) of alkaline copper tartrate solution equivalent to the mass of glucose, m content .(1)X1=m1 is the amount of glucose in the sample (mg); X0 is the reagent blank (mg); m is the sample weight (g); m1 is a 10 mL alkaline copper tartrate solution that is equivalent to the mass of glucose (mg); V1 is an average volume of sample solution consumed during determination (mL).In the formula: X is the content of starch in the sample (g/100g); XThe Chinese national standard method (GB 7648-87) was adopted for the determination of amylose. The basic principle entails that starch and iodine form an iodine–starch complex, which has a special color reaction. Amylopectin and iodine form a brown-red complex, while amylose and iodine form a dark blue complex. Under the condition that the total amount of starch remains unchanged, the two starch dispersions are mixed in different proportions, and react with iodine under certain wavelength and acidity conditions to generate a series of colors, from purple red to dark blue, representing their different amylose and branched starch. Amylose content ratio, according to absorbance and amylose concentration, has a linear relationship, which can be measured by spectrophotometer .1 is the mass of crude starch contained in the weighed sample, 100 mg.In the formula: G is amylose content calculated by regression equation, mg. mThe size of starch granules was measured with a micrometer under an optical microscope . Four effective fields of view were selected (at least five starch granules in each field) from the starch granule samples of each variety, followed by measuring the length and width of the largest starch granule in each effective field of view, using the starch granule length  and the geometric mean of the width .The determination method employed for whiteness evaluation was consistent with the Chinese national standard (GB/T 22427.6–2008). The whiteness of adzuki bean whole powder was measured using an intelligent whiteness tester . The intelligent whiteness tester was calibrated using a white tile . The whiteness values were measured thrice, and an average value was calculated. Several studies regarding the evaluation of whiteness have been reported for various food substances, such as cassava raw materials, avocado seed starch, rice, wheat, etc. ,20.Adzuki beans were grounded with a 300 W grinder and then passed through a 40-mesh sieve to obtain adzuki bean flour. Subsequently, 50 g of adzuki bean flour was placed in a glass dish , followed by mixing with 20 mL of distilled water. The adzuki bean flour mixture was steamed for 45 min in an atmospheric pressure hot water boiler , and cooled at room temperature. The texture characteristics analysis of adzuki bean gel was conducted, adopting the TMS-PRO Food Property Analyzer . The texture analysis parameters were set as follows: the test speed was 1 mm/s, deformation was 50%, and the starting force was 0.5 N. Notably, each sample was measured 6 times. The maximum and minimum values were removed, and the average values were taken as the final test result. From the texture profile curve, the following parameters were calculated: hardness, cohesiveness, adhesiveness, springiness, and chewiness.0, g). The bean paste rate  was calculated according to the following formula:The adzuki bean paste was isolated by the method adopted by our predecessors in this research field , with a 0). Subsequently, 300 mg of adzuki bean starch was dissolved in 5 mL of distilled water to prepare the starch suspension. The suspension was kept at 100 °C for 30 min. Thereafter, it was cooled at room temperature and put in a freezer at −20 °C for 24 h. The suspension was taken out, thawed naturally at room temperature, and weighed (m1). The suspension was centrifuged at 3000× g for 15 min. The supernatant was discarded, and the sediment with centrifuge tube was weighed (m2). The water separation rate was calculated as follows:The empty 10 mL centrifuge tube with a lid was weighed . The supernatant was collected in the pre-numbered constant weight aluminum box and placed in a drying oven at 105 °C to dry to a constant weight. Finally, the box was taken out and cooled at room temperature for being weighed.Thereafter, 100 mg of adzuki bean starch was dissolved in 5 mL of distilled water to prepare the starch suspension. The suspension was kept at 90 °C for 30 min, with subsequent stirring at every 2 min. Following stirring, it was cooled at 25 °C and centrifuged (3500× v/v) to inactivate the enzyme. The digestibility of starch was determined by 3,5-dinitrosalicylic acid (DNS). Based on the rate of hydrolysis, starch was defined as rapidly digestible starch , slowly digestible starch , and resistant starch . The RDS, SDS, and RS fractions were determined by the following formulas:This analysis was performed according to the method adopted by Hu et al., Xu et al. and Ge et al. ,6,13, amIn the formula: Gt denotes the reducing sugar content at each interval. Accordingly, RDS, SDS, and RS are the rapidly digestible starch, slowly digestible starch, and resistant starch, respectively. Lastly, T expresses total starch weight.Thermal properties were conducted according to Ge et al.  and HongThe X-ray diffraction pattern (XRD) was measured using the Bruker D8 Advance instrument produced in Germany. A fully water-balanced starch sample (equilibrated for 72 h in an environment with a relative humidity of 100%) was tightly packed into a plastic abrasive tool , and measured under X-ray. The determination was operated at 40 kV and 40 mA with Cu Kα radiation (λ) at 0.1542 nm. XRD patterns were acquired for a 2θ range of 4~45° with a step size of 0.02 and a step rate of 3/min. The degree of crystallinity was calculated according to the method of Zeng et al. .p < 0.05. All data were processed by Origin . All values were expressed as mean ± SD. Notably, SPSS  software was used for correlation and cluster analysis. Among them, cluster analysis adopts the method of hierarchical cluster, adopts between-groups linkage, and the measurement interval is squared Euclidean distance. Correlation analysis was performed using bivariate correlation, and the correlation coefficient was Pearson. Furthermore, Dunnett’s multiple range tests were used to determine the significant differences between a group means at The starch granules were observed, measured, and photographed by Olympus BX51 optical microscope. Lastly, MDI Jade  software was adopted for the calculation of crystallinity.(The contents of amylose (AM) and amylopectin (AP) are respectively shown in C, those The content of amylose in adzuki beans varies depending on the variety. Previous studies have reported that the native red adzuki bean starch had apparent amylose contents of 24.33% to 26.64% . NotablyAs shown in The biosynthesis of starch is a complex biochemical process, and a series of enzymes are involved in the formation of starch granules. The biosynthesis of AP was mainly composed of soluble starch synthases (SS), which were catalyzed by starch-branching enzymes (SBEs) and starch-debranching enzymes (DBE), while AM was mainly catalyzed by granule-bound starch synthase (GBSS) encoded by waxy genes catalytic synthesis .The photosynthetic products of leaves mainly exist in the form of sucrose and were translocated into the grains, followed by degradation to generate uridine diphosphate glucose (UDPG) and fructose before they could be used to synthesize starch. Illustratively, the synthesis of sucrose was controlled by sucrose phosphate synthase (SPS), while an enzyme–sucrose synthase (SS) catalyzed the sucrose degradation. Finally, under the action of these starch synthases, the synthetic starch was stored in the grain .The differences in starch granule content and starch granule size distribution among the adzuki bean varieties were mainly due to different genotypes, as well as differences in various cultivation parameters, including field temperature, light, water, fertilizer, among others. Adzuki bean is a typical short-day crop, such as Baihong 2, which is planted in Baoding. Invariably, these species are poised with shortened sunshine hours during the day, resulting in insufficient vegetative growth and accelerated reproductive growth. Shading can improve the activities of SS and SPS enzymes and can strengthen the synthesis of starch. Per the total starch content data, the northern variety Baihong 2 (31.258%) had significantly higher values than that of the southern variety Yuhong 2 (29.776%). Notably, more research results reported in this experiment further clarified the differences in AM and AP contents in total starch of various adzuki bean varieties. Although the research on starch metabolism pathways has been widely conducted, with the involved enzymes and genes explicitly elucidated , the mecFrom Commercially, adzuki beans are mainly employed for their desirable gel quality; therefore, the gel produced by adzuki bean flour was determined. Among the various indicators of texture properties, the hardness of each adzuki bean variety varies from 56.23 to 176.33 N; with the tenderest evaluated as Baihong 2, while the hardest is determined as Jihong 9218. The variation range of cohesion was between 0.06 and 0.19; with the least determined as Baihong 2, while the greatest was Tangshanhong. The variation range of elasticity sets between 0.56 and 2.79 mm; with the lowest being Baihong 2, while the greatest was Tangshanhong. The variation range of adhesiveness was between 3.38 and 34.07 N, with the least determined as Baihong 2 and the greatest being Jihong 9218. The variation range of chewiness sets between 2.35 and 88.98 mJ; with the least evaluated as Baihong 2, and the highest being Tangshanhong. The differences between the indicators of varieties are shown in Lupinus albus L.) flour, as measured by texture indices [The study found that flour processed from adzuki beans was less cohesive, firmer, and chewier than lupin  to 54.06 °C (Baohong 876), with an average of 61.91 °C and had standard deviation of 5.557 °C. Furthermore, Baohong 876 presented the highest Tp (78.76 °C) among all the evaluated varieties, while Henong 3 had the lowest Tp (69.52 °C). The average value of Tp was 74.21 °C, and the standard deviation was calculated as 3.353 °C. The variation range of Tc sets was between 81.83 (Jihong 1) and 92.71 °C (Tangshanhong). The average value of Tc was 85.15 °C, and the standard deviation was evaluated as 3.809 °C. The highest enthalpy (5.80 J/g) was found in the Tongza 6 variety, while the lowest was presented by Tangshanhong (3.43 J/g), with an average of 3.43 J/g, and standard deviation determined as 0.987 J/g. The data of To, Tp, and Tc in this experiment are basically consistent with the research results of Honda et al. [The various indicators of the starch thermodynamic determination of each adzuki bean variety are shown in a et al.  and Xu eIn this study, 12 adzuki bean varieties from different ecotype regions were selected, and their starch solubility, swelling power, freeze-thaw stability, amylose and amylopectin content, AM/AP ratio, and texture properties were individually evaluated. The correlation analysis  revealedThe solubility was negatively correlated with the amylose content and AM/AP ratio. This may be attributed to the dissolution of starch granule crystals and the invariable dissociation of amylose during starch swelling. Swelling power was positively correlated with the amylose content and AM/AP ratio, had an insignificant negative correlation with amylopectin content and crystallinity, and was significantly negatively correlated with freeze-thaw stability and RDS. This may be attributed to the fact that as the temperature approaches the gelatinization temperature of starch, the microcrystalline bundle structure of starch begins to loosen, for the exposed polar groups of starch to combine with water, resulting in the rapid absorption of surrounding water by the starch granules, and invariably increasing its expansion potential .c was significantly positively correlated with AM/AP ratio, indicating that Tc was closely related to the structure and composition of starch. Both Tc and Tp indicated a very significant positive correlation, and their trend among the varieties showed similar changing alteration patterns, while To had a significant positive correlation with the freeze-thaw stability, indicating that the better the freeze-thaw stability, the higher the melting temperature of the starch crystals.Correlation analysis  showed tCluster analysis was performed on a total of 22 indicators to obtain the final pedigree map. As shown in o and Tp were all relatively high. Meanwhile, the degree of crystallinity sets between Class I and Class III. Therefore, Class II adzuki bean varieties were suitable as stabilizers, belonging to gel-type food additives, which can improve the texture properties of foods. This is conventionally applied in dairy products and sausages. Compared with the other two types, Class III adzuki bean varieties had relatively high amylopectin content, paste yield, Tc, and crystallinity. Hence, Class III varieties were suitable for making bean paste food. Moreover, the high content of amylopectin was suitable for application in food processing, serving as a water retention agent or as a sustained release agent, binder, and thickener in other aspects, and it was relatively easy to be gelatinized.As shown in The DSC conclusion temperature of adzuki bean starch was significantly positively correlated with AM/AP ratio, indicating that the higher the melting temperature of the crystals, the better the freeze–thaw stability of adzuki bean starch.The crystallinity varies from 20.92 to 37.38%. According to the analysis of X-ray diffraction pattern, strong diffraction peaks appear at 15°, 17°, 23°, which confirmed that the crystal types of adzuki bean starch belonged to type BC .Cluster analysis was performed on a total of 24 indicators, and 12 different adzuki bean varieties were divided into Class I, II, and III as follows: Class I included Henong 1, Henong 2, Henong 3, Liaohong 08712, Tongza 6, Pinhong 2011, Jihong 1, with lower SDS content and higher RDS content, which are unsuitable for the dietary requirements of diabetics; Class II contained Tangshanhong and Jihong 9218, which were suitable to be added to gel food as a stabilizer, and thus improve the texture characteristics of food, and they could be employed in the processing of dairy products and sausages; lastly, Class III consisted of Baohong 876, Yuhong 2, and Baihong 2, which were suitable for bean paste food with high throughput and good commerciality, as well as for mixtures and thickeners owing to its relative potential for easy gelatinization.Overall, this study plays a positive guiding role toward the improvement of adzuki beans breeding quality, which can provide important reference significance for the development of adzuki bean variety resources and further present a theoretical basis for the application of adzuki bean starches in food and non-food fields.


This review describes the role of CXCL1, a chemokine crucial in inflammation as a chemoattractant for neutrophils, in physiology and in selected major non-cancer diseases. Due to the vast amount of available information, we focus on the role CXCL1 plays in the physiology of bones, bone marrow, muscle and the nervous system. For this reason, we describe its effects on hematopoietic stem cells, myoblasts, oligodendrocyte progenitors and osteoclast precursors. We also present the involvement of CXCL1 in diseases of selected tissues and organs including Alzheimer’s disease, epilepsy, herpes simplex virus type 1 (HSV-1) encephalitis, ischemic stroke, major depression, multiple sclerosis, neuromyelitis optica, neuropathic pain, osteoporosis, prion diseases, rheumatoid arthritis, tick-borne encephalitis (TBE), traumatic spinal cord injury and West Nile fever. CX3C chemokines (1 representative in humans),CXC chemokines (17 in humans),CC chemokines (26 in humans),XC chemokines (2 in humans).Chemokines are chemotactic cytokines , whose mCXC motif chemokine ligand 1 (CXCL1), a CXC chemokine  and melahttps://pubmed.ncbi.nlm.nih.gov; accessed on 30 December 2021). The search phrases included: This review is based on articles available in PubMed ((CXCL1 or CINC-1 or MIP-2 or MIP2 or (KC chemokine) or MGSA or gro-a or gro-alpha) and (bone or muscle or brain or astrocyte or microglia or neuron) not review.The results included all articles with the title, abstract or keywords mentioning selected organs and CXCL1 or paralogs of this chemokine in rodents. Review papers were not searched. Over 1700 articles were searched and preselected based on the title of the paper. Based on the literature searched, subsections were written about the physiological role of CXCL1 in given tissues and organs. After selecting the most exhaustive 100 articles, 15 diseases were selected in which CXCL1, or its mouse or rat paralog, plays an important role. Then to write each of the subsections on the role of CXCL1 in diseases, articles were searched by the phrase: “the name of the disease” and (CXCL1 or MIP-2 or MIP2 or (KC chemokine) or MGSA or gro-a or gro-alpha) and not review.Next, a selection of search articles was made by title. The papers were then read and searched for new and interesing literature references.https://pubmed.ncbi.nlm.nih.gov; accessed on 30 December 2021) was used and the phrase: In addition, each subsection contains a brief introduction about the disease being discussed. To write these introductions, PubMed  in rats ,10, and Due to these discrepancies, we adopted the following principles when writing this review. First, to show whether CXCL1 has a role in a given process in humans, we showed elevated CXCL1 expression in patients with a given disease or at the site of given physiological processes. Then, we described the mechanism of CXCL1 action, including in vivo studies on experimental animals with elevated expression of chemokines that are ligands for CXCR2, namely, KC or macrophage inflammatory protein-2 (MIP-2) in the mouse and CINC-1 in the rat. It can be assumed that if KC, MIP-2, CINC-1 and CXCL1 appear in different species in the same disease, then they play the same role in disease mechanisms. Importantly, in the present study we focus only on human CXCL1, although it is possible that other human ligands for CXCR2 also have the same function in the diseases we discuss.In some studies on mice, KC has a significant function in a given disease, even though in humans CXCL1 does not. In such a case, we describe the mechanism of action of CXCR2 ligands in a given disease and then provide possible CXC chemokines that play some role in humans, most frequently CXCL8/IL-8. The large majority of literature does not examine all ligands for CXCR2 and for this reason it is possible that other CXC chemokines, not just CXCL8/IL-8, may also be involved in the mechanisms of a given disease.+ monocytes . γ. γ134]. Expression of ligands for CXCR2 during ischemic stroke may also depend on miRNAs. In rats, an ischemic stroke is associated with promoter hypermethylation of miR-532-5p , which rMajor depression is a mental illness that affects approximately 6% of the population and its pathogenesis may be related to inflammation . In miceThe mechanism of CXCL1 involvement in major depression in humans appears to differ significantly from the animal model. Depressed suicidal persons experience a reduction in CXCL1 expression in the prefrontal cortex, similar to the reduction in the expression of CXCL2 and CXCL3 . Plasma There are no studies showing correlations between depression on CXCL1 expression in the hippocampus in humans. It is possible that, as in experimental animals, depression is associated with an increase in CXCL1 expression in this brain structure in humans. If, in contrast, the human hippocampus showed a decrease in CXCL1 expression, it would indicate that CXCL1 plays an important role in brain function in humans and a decrease in the expression of this chemokine could cause depression. This would likely be related to hippocampal neurogenesis  or impaiMultiple sclerosis is a neurodegenerative and autoimmune disease of the brain and spinal cord, with an estimated incidence of 0.2 to 240 people per 100,000, depending on the population studied . Myelin-CXCL1 is a significant element in the course of multiple sclerosis. This chemokine has been shown to have both neuroprotective and inhibitory properties in the progression of multiple sclerosis , as wellCXCL1 expression is increased in the brains of patients with multiple sclerosis . In part+ T helper type 17 (Th17) cells which produce IL-17 [In multiple sclerosis, CXCL1 is produced by activated microglia  and astrce IL-17 ,164,165.ce IL-17 . IL-17 ace IL-17 . As the ce IL-17 . In micece IL-17 ,168, whiInflammation has a destructive effect on oligodendrocyte progenitor cells as shown by experiments on human embryonic stem cell-derived oligodendrocyte progenitor cells  and muriCXCL1 also participates in the progression of multiple sclerosis . CXCR2 aNeuromyelitis optica is a neurodegenerative and autoimmune disease. Auto-antibodies to aquaporin 4 (AQP4) are responsible in over 80% of cases of this disease . The incChemokines, such as CXCR2 ligands, are important in the development of neuropathic pain. However, due to bioethical constraints, all knowledge about the importance of CXC chemokines in the development of neuropathic pain is based on experiments on laboratory animals. Due to the fact that CXCR2 ligand systems differ significantly between humans and mice, the presented mechanism of neuropathic pain gives only a hint of the role that CXCL1 may play in this disease in humans.Following nerve injury in mice, KC expression increases in spinal astrocytes ,181,182.+ currents [+ currents in sensory neurons [CXCR2 activation on neurons and spinal microglia has been found to lead to neuropathic pain ,186,187.currents  and upre neurons . Another neurons . CGRP is neurons .It has been found that KC can also cause neuropathic pain by recruiting neutrophils ,192,193.Also, studies with laboratory animals show that CXCR2 ligands reduce spontaneous activity . This inSc) [C) into PrPSc. This is followed by aggregation of PrPSc in neural tissue which leads to spongiform degeneration. Examples of prion diseases include sporadic Creutzfeldt-Jakob disease (sCJD), fatal familial insomnia and kuru. An important element in the course of prion diseases is neuroinflammation [Prion diseases form a group of neurodegenerative diseases caused by scrapie-associated prion protein (PrPSc) . This prammation  involvinammation ,200,201.ammation . In contAs KC is a murine paralog for human CXCL1 ,11, it mSc reduces the activity of these cells [CXCL1 does not play an important role in prion diseases. In multiple sclerosis, an increase in CXCL1 expression in the brain has a destructive effect on neural tissue through infiltration of neural tissue by neutrophils ,173. Evese cells  and therFlavivirus. It infects animals in central and eastern Europe and northern Asia. In humans, TBEV infection usually gives mild symptoms; however, some 2% of cases of TBEV infection are fatal. TBEV is a neurotropic pathogen that attacks neural tissue, including the brain. The vector for this virus is the Ixodes sp. tick, which means the virus enters the body of the host during feeding by these parasites. Tick-borne encephalitis (TBE) is a disease caused by the tick-borne encephalitis virus (TBEV) . Its genAt the same time, the feeding of the tick involves a strong immune response . The ticOther evasins inactivate other groups of chemokines. For example, Evasin-1 inactivates the proinflammatory chemokines CCL3 and CCL4 . EvasinsTBEV is a neurotropic pathogen causing a severe neuropathological disorder . One comTraumatic spinal cord injury involves damage to the spinal cord as a result of a violent accident or fall. It is estimated that there are approximately 17,000 cases of traumatic spinal cord injury per year in the United States alone . ApproxiFlavivirus [West Nile fever is caused by West Nile Virus (WNV) from the genus avivirus . Its genavivirus .Culex sp. For this reason it is found in warm countries in Africa, Asia, Australia and Europe. When bitten by the mosquito, the virus is introduced into the skin [WNV acts as a neurotropic pathogen that affects the nervous system and also other tissues and organs such as the skin, kidney and gastrointestinal tract. WNV is a zoonotic pathogen that is transmitted to humans by mosquitoes, mainly by the skin . It infethe skin , causingthe skin . This reAnti-inflammatory substances in the mosquito saliva reduce inflammatory responses , which aWNV is a neurotropic pathogen that can get into the brain and spinal cord, although this form of infection is rare in humans . There aThe role of CXCL1 in physiology and in non-cancer diseases has been fairly well studied. Nevertheless, the biggest shortcoming in knowledge of this chemokine is the huge difference in CXCR2 ligand systems between humans and rodents. For this reason, human CXCL1 does not have functions identical to those of murine KC and rat CINC-1. The results obtained in studies on animals cannot be completely and unqualifiedly compared to a human patient’s condition during a given disease. For this reason, until new research models are invented and disseminated, certain areas of knowledge about CXCL1 are still in the realm of conjecture.One direction of CXCL1 research should be the application of existing knowledge in practice. CXCL1 plays an important role in the pathogenesis of many non-cancer diseases. Therefore, it is possible to use either CXCR2 inhibitors or anti-CXCL1 antibodies in the therapy of these diseases. As there are 7 chemokines that activate CXCR2, it seems that the use of CXCR2 inhibitors is a better option. However, the chemokines that are CXCR2 activators differ in their sites of expression, among other things. For this reason, antibodies to individual CXCR2 activators may also be a convenient therapeutic option, provided we learn the exact differences between individual CXCR2 activators in a given disease.


Therefore, our vaccine may have potential protection against SARS-CoV-2 and its variants.The development of an effective multivalent vaccine against SARS-CoV-2 variants is an important means to improve the global public health situation caused by COVID-19. In this study, we identified the antigen epitopes of the main global epidemic SARS-CoV-2 and mutated virus strains using immunoinformatics approach, and screened out 8 cytotoxic T lymphocyte epitopes (CTLEs), 17 helper T lymphocyte epitopes (HTLEs), 9 linear B-cell epitopes (LBEs) and 4 conformational B-cell epitopes (CBEs). The global population coverage of CTLEs and HTLEs was 93.16% and 99.9% respectively. These epitopes were spliced together by corresponding linkers and recombined into multivalent vaccine. In silico tests, the vaccine protein was a non-allergen and the docking with TLR-3 molecule showed a strong interaction. The results of immune simulation showed that the vaccine may be helpful to initiate both cellular and humoral immunity against all VOC. The optimistic immunogenicity of the vaccine was confirmed  In December 2019, the world witnessed the first coronavirus pandemic in human history. The coronavirus is a novel virus which was named as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV), and caused corona virus disease 2019 (COVID-19) which was named by the World Health Organization (WHO) , 4. The The SARS-CoV-2 is a single and positive stranded RNA virus with an enveloped structure . The lenThe variant named B.1.1.7 first appeared in the United Kingdom and had 10 characteristic mutations in S protein, including D614G and N501Y mutations in ACE2 receptor binding domain (RBD) and two characteristic mutations in Protein N , 12. It in vitro is the most widely used , mRNA vaccine (Moderna Corp), AZD1222 adenovirus-based vaccine (Astrazeneca Corp), JNJ-78436735 adenovirus-based vaccine (Johnson & Johnson Corp), NVX-CoV2373 nanoparticle-based vaccine (Novavax Corp), etc. However, with the emergence of SARS-CoV-2 variants, the epitopes of original virus have also changed, threatening the effectiveness of existing vaccines . In viewely used –28. In thttps://bigd.big.ac.cn/ncov/variation/annotation) had classified the SARS-CoV-2 variants. This study was graded according to the assessment of CNCB website, and the Class I variants (the population incidence was greater than 0.05) were selected to screen antigens for vaccine design. Finally, we selected 5 SARS-CoV-2 variants of different lineages with the best quality evaluation.China National Center for Bio-information (CNCB) (https://www.ncbi.nlm.nih.gov/). In addition, antigenicity of all S proteins was predicted using online software VaxiJen 2.0 (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html). The VaxiJen was the first server that did not rely on protein sequence alignment and only classified proteins according to their physicochemical properties, so as to predict protective antigens    .http://tools.iedb.org/mhci/) database (http://www.cbs.dtu.dk/services/NetCTLpan/) used artificial neural networks (ANNs), and it can simulate the binding between antigen peptide and major histocompatibility complex (MHC) for the prediction of MHC class I antigenic epitopes (The antigenic epitopes were restrictive in binding to human leukocyte antigen (HLA) molecules. In order to make the vaccine effect cover the global population more widely, we expanded the selection of HLA allele. According to the HLA allele reference set from the IEDB website    , there wpan-4.0)  was usedhttps://services.healthtech.dtu.dk/service.php?BepiPred-2.0) (The online Server BepiPred-2.0 (red-2.0)  was usedhttp://tools.iedb.org/ellipro/) was used to predict the conformational B-cell epitopes (CBEs). The predicted conformational epitopes of six virus strains were compared and synthesized. The predicted conformational epitopes of six virus strains were compared and synthesized, and finally used in the construction of vaccine. And the signal peptide sequences were removed when predicting epitopes in S proteins.The online software IEDB . The SOMPA adopted the self-optimized prediction method (SOPM) to improve the accuracy of prediction by multiple alignment with protein sequences of the same family . The GalaxyRefine adopted ab initio modeling, and refined the loop or terminus regions in the primary protein 3D model (https://prosa.services.came.sbg.ac.at/prosa.php) and the Structure Assessment service of SWISS-MODEL (https://swissmodel.expasy.org/assess) were used to verify the tertiary structure quality of vaccine construct. The ProSA-web could evaluate the overall model quality by Z-Score as well as the local model quality. In addition, the SWISS-MODEL had drawn Ramachandran plot that displayed favorable areas of backbone dihedral angles against amino acid residues in tertiary structure  , charged polar, atomic composition, half-life, stability, hydrophobicity and so on. According to the physicochemical properties of vaccine protein, it can guide the preparation of vaccine solution and the vaccination strategy in vivo experiment.The online tool ProtParam (tparam/)  was usedhttp://www.ddg-pharmfac.net/AllerTOP/) was used to predict the allergenicity of rSMEV. This prediction tool is based on auto cross covariance (ACC) transformation of protein sequences into uniform equal-length vectors, and the target protein was compared with allergens and non-allergens data sets for predicting allergen properties (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html)   was usedrSMEV had contained both HLA-I and HLA-II antigen epitopes. In addition, a wide range of HLA-I and HLA-II alleles were selected when predicting antigen epitopes. In order to verify the coverage of rSMEV to the world population, the IEDB (http://tools.iedb.org/population/)   was usedrSMEV with TLR-3 was confirmed by the molecular docking approach. The tertiary structure of TLR-3 molecular was obtained from the PDB database (http://www.wwpdb.org/). The molecular docking works were performed by ClusPro Server (https://cluspro.bu.edu/home.php) (The toll like receptor (TLR) was a class of important protein molecules involved in innate immunity, and it was also a bridge between nonspecific immunity and specific immunity . The TLRome.php) . And the- or Na+ ions were added into the protein surface to neutralize the total charges of the systems. The Amber ff14SB force field  was used to perform the immune simulation  (To detect the immune response of mulation . The C-Imulation . In this to 8 h) . In addi to 8 h) .in vivo experiment, we analyzed the adaptability and preference of the codon of rSMEV. The online tool JCat (JAVA Codon Adaptation Tool) (http://www.jcat.de/Start.jsp) was used to optimize the codon .The pET-28a (+) was selected as the vector that expressed the The eight-week-old SPF BALB/c mice provided by the Animal Experiment Center of Xinjiang Medical University were randomly divided into rSMEV and Healthy control (HC) two groups. The mice were dripped intranasally with vaccine protein in rSMEV group (n=8), and the mice was dripped intranasally with normal saline in HC group (n=8). The concentration of vaccine protein solution was 500 μg/mL, each mouse was given 60 μL each time (30 μL each nostril), once every 2 weeks, 3 times in total and the vaccine protein was detected as endotoxin-negative by limulus amebocyte lysate (LAL) before using. Two weeks after the third immunization, the mouse splenocytes were collected aseptically in rSMEV and HC groups for experimental detection. The study was approved by the animal ethics committee of Xinjiang Medical University, and the whole animal experiment process was carried out in strict accordance with the experimental requirements and operation guidelines of the animal ethics committee of Xinjiang Medical University.rSMEV vaccine protein solution (10 μg/mL) was coated on the ELISPOT plate and incubated overnight at 4 °C. Then the plate was washed for 5 times by Microplate washer  and blocked with 200 μl RPMI-1640 medium  containing 10% fetal calf serum (FCS) (Sigma) for 30 minutes at 37 °C. After the plate was washed by PBS containing 0.05% Tween 20, the vaccine protein and mice splenocytes suspension prepared (2×105) were added into the ELISPOT plate3. Three repeat wells were set for each sample and the plate was incubated for 20 hours in a 37 °C humidified incubator with 5% CO2. After the plate was washed for 5 times, the biotinylated detecting antibody (anti-mouse IgG) was added and the plate was incubated for 2 hours at room temperature. After the plate was washed, the Streptavidin-ALP was added and incubated at room temperature for 1 hour. The BCIP/NBT solution (MlBio) was added and incubated for chromogenic reaction. The plate was rinsed and dried, the antibody secreting cell (ASC) spot were counted using EliSpot Reader . In this experiment work, the Mouse IgG ELISpot BASIC kit (ALP)  was adopted for the ELISPOT experiment.The Enzyme-Linked Immunospot (ELISPOT) experiment was performed for evaluating the specific B-cell response in mice. First, the + T-cell response in mice. First, the splenocytes in HC and rSMEV groups were co-cultured with the rSMEV vaccine protein (5 μg/mL). The splenocytes were incubated with RPMI-1640 medium containing 10% FCS for 48 hours in a 37 °C under 5% CO2 humidified incubator. Then the culture supernatant was collected, and the samples and standards were added into the IFN-γ and IL-4 ELISA plates after plates were washed. The ELISA plates were incubated at 37 °C for 1.5 hours and washed for 5 times. The biotinylated detecting antibody was added to the plates and incubated at 37 °C for 1 hour. After the plates were washed for 5 times, the Streptavidin-ALP was added and incubated at 37 °C for 30 minutes. The BCIP/NBT solution were added for chromogenic reaction at 37 °C away from light for 15 minutes. Finally, the termination solution was added, and the OD450nm value was measured immediately by Spectrophotometer  for detecting the corresponding level of IFN-γ and IL-4. The Mouse IFN-γ ELISA Kit  and Mouse IL-4 ELISA Kit  were adopted for ELISA experiment. The enzyme-linked immunosorbent assay (ELISA) experiment was performed for evaluating the CD4The vaccine construction strategy of this study was shown in The GenBank search number of the six SARS-CoV-2 virus strains was NC_045512 (Wuhan-Hu-1), MW642026 (Lineage B.1.1.7), MW621453 (Lineage B.1.351), MW642250 (Lineage P.1), MW631888 (Lineage B.1.429) and MZ491545 (Lineage B.1.617.2) separately.The GenBank accession number of S protein derived from six SARS-CoV-2 virus strains was YP_009724390 (Wuhan-Hu-1), QRX39358 (Lineage B.1.1.7), QRV12312 (Lineage B.1.351), QRX39425 (Lineage P.1), QRW35522 (Lineage B.1.429) and QXF88313 (Lineage B.1.617.2). The result was shown in Compared with the original SARS-CoV-2 virus strain, the five virus variants had a total of 37 mutation sites. There were 25 mutation sites in the SARS-CoV-2 S1 region, among them, 20 mutation sites were in N-terminal domain (NTD) and five mutation sites in RBD. There were 17 mutation sites in the SARS-CoV-2 S2 region. Among them, two mutation sites were in heptad repeat region 1 (HR1) and one mutation site in HR2 Figure 2After the analysis, the S proteins of six different SARS-CoV-2 lineages all had signal peptide regions. The signal peptide region of Lineage Wuhan-Hu-1 was 1-15 (MFVFLVLLPLVSSQC), the signal peptide region of Lineage B.1.1.7 was 1-15 (mfvflvllplvssqc), the signal peptide region of Lineage B.1.351 (mfvflvllplvssqc), the signal peptide region of Lineage P.1 was (mfvflvllplvssqc), the signal peptide region of Lineage B.1.429 was 1-15 (mfvflvllplvsiqc), the signal peptide region of Lineage B.1.617.2 was (mfvflvllplvssqc) Figure 3In this part, the CTLEs of six SARS-CoV-2 virus strains were analyzed by online Server NetCTLpan 1.1 with each virus strain having 27 kinds of CTLEs of HLA-I alleles. The Rank of CTLEs was less than 1% in the prediction using IEDB. All CTLEs were ranked from small to large according to the sequence start position , 23.94% Extended strand (Ee), 6.51% Beta turn (Tt) and 49.51% Random coil (Cc)  of the improved sequence was 0.93. When the CAI-Value>0.8, the codon improved was considerate to have a high adaptability to the DNA sequence (Escherichia coli strain K12 used in the study was 50.73%.The rSMEV was inserted to the sites between XhoI (site 158) and BamHI (site 2006) in the pET-28a (+) vector. The DNA sequence of rSMEV was written in The DNA sequence of rSMEV induced B-cell response. The splenocytes of rSMEV group mice could secrete specific antibodies induced by vaccine protein, while the splenocytes of HC group mice did not secrete specific antibodies (P<0.001) . This suggested that rSMEV protein could activate CD4+ T-cell reaction. In the study, the ELISPOT experiment was performed to evaluate the in vivo and in vitro study. Theoretically, the vaccine designed by this study has a broader protective effect for SARS-CoV-2 infection.As you known, the decrease of vaccine protection ability elicited mainly by S protein mutation of SARS-CoV-2. There was no a multivalent epitope vaccine for S protein to resist the infection of SARS-CoV-2 and its variants at present. Therefore, we had contrived a multivalent epitope vaccine of S protein to cope with the problem of virus mutation more effectively. In this study, we took the VOC including Wuhan-Hu-1 and SARS-CoV-2 variants B.1.1.7, B.1.351, P.1, B.1.429, B.1.617.2, etc as the objects of study, and took S proteins of virus strains as the research target. Meanwhile, the immunogenicity of the vaccine was confirmed + and CD4+ T-cell epitopes for activating the cellular immunity. In order to expand population coverage of the vaccine, we predicted 27 kinds of HLA-I and 27 kinds of HLA-II T-cell epitopes respectively  should recognize both the antigenic peptide and the polymorphic parts of its MHC molecules. Therefore, MHC molecular restriction was fully considered when screening the CD8ectively . By comprSMEV was analyzed. The secondary structure analysis showed that the vaccine protein had 6.51% Beta turn and 49.51% Random coil. Because the spatial structure of Beta turn and random coil is loose, it is easy to form epitopes. Therefore, the rSMEV that containing a large amount of this structure has a good vaccine structural basis.To ensure the SARS-CoV-2 vaccine more reliable and effective, we spliced together the chosen epitopes that included 8 CTLEs, 17 HTLEs, 9 LBEs and 4 CBEs by linker sequences. The appropriate linkers could avoid the formation of new epitopes (Linker-epitope) and enhance the presentation of antigen epitopes by ensuring the full exposure of the screened target epitopes , 62. TherSMEV. Meanwhile, the unreliable loops or termini of the predicted 3D structure were reconstructed and refined by the GalaxyRefine Server. The good tertiary structure of rSMEV would be used for later molecular docking and MD simulation. We further analyzed the physicochemical properties and allergenicity of rSMEV. The physicochemical properties results showed that molecular weight of rSMEV was 65 KD. Previous studies have reported that it is reasonable when the molecular weight of vaccine protein is less than 110 KD (rSMEV was suitable for vaccine. In addition, the analysis of theoretical pI, amino acid acidity, alkalinity, stability, hydrophilicity and allergenicity also displayed that the rSMEV was reasonable and safe.In view of Rosettafold’s excellent performance in protein tertiary structure prediction , we usedn 110 KD , which hrSMEV had strong affinity with TLR-3 molecule, we conducted the molecular docking using ClusPro Server. The results showed that lowest energy value in the all outputted rSMEV-TLR-3 complexes was −1175.3 kcal/mol. The capacity of the system is lower, the structure of object is more stable (rSMEV-TLR-3 complex was quite stable. Moreover, the interaction interfaces proved that the binding affinity between rSMEV and TLR-3 was ionic and hydrogen bonds mainly. Due to the interaction of ionic bond and hydrogen bond is strong, this also indirectly indicates that the binding between rSMEV and TLR-3 is very close (When microorganisms break through the mucosal barrier, TLR can recognize them and initiating the adaptive immune response . Becausee stable . Therefory close .rSMEV-TLR-3 complex. The RMSD value represented the structural fluctuation of the overall structure of the vaccine and TLR-3 complex, which can evaluate the stability of the system during interaction. In this part, the floating range of 100 ns RMSD value was about 1 nm, suggesting that the structure of the complex is relatively stable. The RMSF value represented the flexibility of amino acid residues in the docking complex, which can evaluate the agility of each residues in the complex. The results suggested that the agility of receptor and ligand sites after 650 was higher than that of residues 0-650. The average binding free energy of the system calculated by the MMGBSA tool was −88.54 ± 6.91 kcal/mol. Therefore, MD simulation proved that rSMEV and TLR-3 docking complex in this study had reasonable structural stability and agility. The above results showed that rSMEV can closely bind to TLR-3, and the complex has high stability and agility.We also used MD simulation to analyze the structural stability and agility of rSMEV in vivo and in vitro. Firstly, the rSMEV can stimulate an excellent immune response confirmed by immune simulation. Then the mice were immunized with rSMEV through experiments in vivo and in vitro. The mice can produce specific ASCs and high levels of IFN-γ and IL-4, the result confirmed that the rSMEV can induce cellular and humoral immunity and has satisfied immunogenicity.That is more important, we had verified the immunogenicity of the in vivo and in vitro. Compared with other studies, our rSMEV is more significant in the current perspective. Obviously, the rSMEV designed by the study estimated that it may have a high potency against SARS-CoV-2 and its variants theoretically. However, this study did not include other antigens with important immunogenicity of SARS-CoV-2. At the same time, this study still needs to further carry out human clinical trials to ensure the effectiveness of the vaccine. All in all, we provided an effective immunological strategy and mean for the active prevention and control of COVID-19 worldwide.In many current studies that based on S proteins to design SARS-CoV-2 vaccine –51, 65, The original contributions presented in the study are included in the article/The animal study was reviewed and approved by The Animal Experiment Medical Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University.MY, YZ, YL, ZC, ZWL, JW, and ZNL performed the experiments. MY and YZ wrote and edited the paper. YL, ZWL, JW, and ZNL collated and verified the experimental data. FZ and JD provided experimental design and ideas, and reviewed and modified the paper. All the authors agreed to publish the final version of the paper.This study had been supported by National Natural Science Foundation of China  and The key R&D project of Xinjiang Autonomous Region: Research on key technologies of prevention and control for COVID-19 [Project number: 2021B03003-2].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.


Similarly, quinoa starch by enzymatic extraction had a higher onset (82.1 °C), peak (83.8 °C), and conclusion (86.3 °C) temperatures, as well as an enthalpy change (6.8 J/g). It further showed maximum hardness (238.8 N), gumminess (105.6 N), chewiness (80.2 N), SDS content (7.5% of raw and 4.8% of cooked), and RS content (15.4% of raw and 13.9% of cooked), whereas it contained minimum RDS content (77.1% of raw and 81.9% of cooked). The results suggest that extraction of starch by the enzymatic method could be a viable approach to retain the native structure of starch and may eventually improve the glycemic response.The purpose of this study is to investigate the effects of starch extraction methods  on the multi-scale structures and functional properties of quinoa starch. When the enzymatic method was compared with alkali and wet-milling, it showed higher protein content (2.4%), larger mean size of aggregated granules (44.1 μm), higher relative crystallinity (29.0%), scattering intensity (17.8 α.u.), absorbance ratio of 1047/1022 (0.9), single and double helical content (8.2% and 23.1%), FWHM ratio (1.5), and average molecular weight and radius of gyration (1.58 × 10 Chenopodium Quinoa Willd.) seeds are pseudo-cereals of the Chenopodiaceae family with dicotyledon plants and are widely grown as an edible and staple grain crop in South America  , and D , respectively, showed significant differences. The size distribution of starch granules extracted by alkali, wet-milling, and enzymatic methods was observed higher  are shown in d higher  than pregranules , and cougranules ,30, wher methods .θ, consistent with previous reports [In  reports ,2. All t reports , and amy reports ,30 in we−1, which is assumed to represent the crystalline and amorphous regions and corresponds to lamellae repeat distance or Bragg spacing [The double logarithmic SAXS patterns, including scattering vector (q), lamellar distance (D), fractal dimension (α), and scattering intensity (I) of quinoa starch extracted by different methods  are presented in  spacing . No diff spacing . Moreove spacing . The sca−1 were observed to be sensitive to starch conformation, which is related to amorphous regions of starch [−1 and the molecular order of double helices are positively correlated with relative crystallinity than the absorbance ratio of 1047/998 cm−1 [−1 and lower ratios of 1022/998 cm−1 than alkali (0.8 and 1.3). These results indicate that the alkali isolating method reduced the short-range molecular order of starch, which is consistent with the XRD data  changes of quinoa starch extracted by alkali, enzymatic, and wet-milling methods were analyzed by solid-state NMR spectra, and the results are shown in −1, 800–1500 cm−1 (fingerprint region), and 2800–3000 cm−1 (C–H stretch region). On the Raman spectra scale (0–3500 cm−1), the sharpening bands at 480, 865, 943, 1264, and 2900 cm−1 represent δ(CH2), νs(C1–O–C4′), νs(C1–O–C5), skeletal (C–C–O), and ν(C–H) modes, respectively, while the splitting bands at 1381 cm−1 and 1155 cm−1 are assigned to δ(C–OH) and δ(C–H) modes, and α-(C1–O–C4′) asymmetric stretch, respectively [−1 and 2900 cm−1 represent the molecular order and its changes obtained from native quinoa starch granules with narrower bond energy distribution. The intensity of 480 cm−1 and 2900 cm−1 band was reduced in the starch samples extracted by alkali and wet-milling compared to the enzymatic method. Furthermore, the short-range ordered structural variations were studied with full-width at half-maximum (FWHM) at 480 cm−1 and its results are presented in −1 was recorded as 2.1, 1.7, and 1.5 for granules isolated by alkali, wet-milling, and enzymatic methods, the higher FWHM value of the alkali method confirming the disruption of the starch short-range ordered structure, consistent with XRD and FTIR results. Generally, the low FWHM values of 480 cm−1 at short-range scale have been suggested to have a more ordered molecular structure with high relative crystallinity [The LCM-Raman spectra of quinoa starch extracted by alkali, wet-milling, and enzymatic methods are shown in ectively . The narallinity ,41.Mw), average radius of gyration (Rz), and molar mass ratio (Mw/Mn) of quinoa starch extracted by different methods  are presented in Mw (1.58 × 107 g/moL) and Rz (106.8 nm) than wet-milling (1.18 × 107 g/moL and 101.1 nm) and alkali (1.13 × 105 g/moL and 41.6 nm), whereas the Mw/Mn of the enzymatic method (1.3) was observed to be lower compared to alkali (2.7), but slightly higher than wet-milling (1.2). The higher Mw and Rz of the enzymatic method indicate the greater molecules with a larger molecular size of the amylopectin, whereas the low Mw and Rz values of the alkali and wet-milling methods suggest that amylose and amylopectin molecules of starch granules were possibly disintegrated into less super molecular components due to the high pH value in the alkali method [The average molecular weight , peak temperature (Tp), and conclusion temperature (Tc) of gelatinization represent the hydrothermal stability of the starch crystalline structure, and enthalpy change (ΔH) corresponds to the loss of the starch granule molecular  structure [To, Tp, Tc, and ΔH of the enzymatic  extraction method were significantly higher than wet-milling , and alkali . All the above characteristic temperatures and ΔH exhibited the order of enzymatic > wet-milling > alkali. The results contrast the previous study of Correia et al., who studied the thermal properties of Portuguese nuts starches isolated by alkaline and enzymatic methods and observed similar gelatinization temperatures (61.5–63.0 °C) [To, Tp, Tc, and ΔH exhibited by the enzymatic method containing large starch granules inhibited the gelatinization, water absorption, and eventually lowered granule swelling, which were most likely exerted by the minor components  [To, Tp, Tc, and ΔH of the enzymatic method are positively correlated to their particle size D50  . However63.0 °C) . The hig lipids) ,47. The size D50 . A similgranules . Another, and ΔH . The gelar order . Thus, eTextural properties are described in terms such as hard, soft, liquid, solid, rough, smooth, creamy, crispy, lumpy, gritty, etc., to assess the quality of a product, which is related to density, viscosity, surface tension, and other physical properties of the specific product . Table 3in vitro digestion properties  of raw (uncooked) and cooked quinoa starch was investigated, and the obtained results are presented in in vitro digestion in both uncooked and cooked conditions. Previous studies have reported that digestion of starch is affected by size, crystallinity, polymerization, non-starch components, and amylose/amylopectin ratio [The impact of starch extraction methods  on in ratio ,55. The in ratio . The smain ratio . Furtherin ratio .The multi-scale structure and functional characteristics of quinoa starch obtained by different methods of extraction  were compared by PCA. The similarities, differences, and distribution of quality attributes defined by the first and second PCA dimensions of the starch samples isolated by alkali, wet-milling, and enzymatic methods are shown in in vitro digestion analyses were subsequently performed. Starch granules extracted by the enzymatic method exhibited the larger size of aggregated granules, higher protein content, relative crystallinity, semi-crystalline lamellae, absorbance ratios of 1047/1022 and 1022/998 cm−1, helical structure content, Mw, and lower FWHM ratio at 480 cm−1 compared with wet-milling and alkali methods. Furthermore, the enzymatic method increased the texture properties , SDS, and RS content. The corresponding methods, on the other hand, reduced the gelatinization and RDS. From these results, it is concluded that extraction methods induce significant structural differences in starch granules. Enzymatic extraction is the more appropriate method to retain the intact structure of starch, whereas the alkali method is highly effective in the extraction and purification of starch, which may lead to different functional properties and industrial applications.In this study, quinoa starch was extracted by alkali, wet-milling, and enzymatic methods to investigate the impact of extraction methods on starch multi-scale structures and functional properties. The morphological, compositional, multi-scale structural , thermal, textural, and


Introduction and importance: This report details the clinical features and management in a case of Descemet stripping automated endothelial keratoplasty (DSAEK) which had primary graft failure (PGF) due to an inverted yet attached lenticule.Presentation of case: A 66-year-old gentleman had poor visual recovery in the right eye after undergoing cataract surgery 12 years prior to presentation. The visual acuity was counting fingers and examination revealed endothelial decompensation. The patient underwent a DSAEK and postoperatively had a well attached lenticule. However, the cornea was edematous three weeks after the surgery and optical coherence tomography (OCT) revealed a reversed lenticule. The patient underwent a repeat DSAEK and had an uneventful postoperative course. The visual acuity was 20/40 after 7 months with a clear cornea and a well attached graft.Discussion: PGF is a rare complication following DSAEK which occurs due to poor endothelial function of the donor graft. Insertion of a reversed lenticule may get overlooked as a cause of PGF unless the graft edge profile is examined on an OCT scan. The graft in the current case was well attached despite its inverted position suggesting that graft adherence is perhaps not a function of the corneal endothelial pumps in isolation and may be driven by factors such as the intraocular pressure.Conclusion: A reversed DSAEK lenticule may have normal adherence to the host stroma and must be considered in cases with PGF. OCT of the graft edge is required for diagnosis before performing a repeat keratoplasty. This intraoperative manipulation of the graft is done primarily to ensure the correct orientation of the lenticule. Although several approaches exist to facilitate the same, it may still get attached in an inverted fashion, especially with very thin lenticules or in eyes with significant corneal scarring., Insertion of an inverted graft has not been reported as a cause of primary failure and so this report intends to describe the clinical features and management of primary graft failure in an eye with an attached yet reversed DSAEK lenticule.The advent of endothelial keratoplasties (EK) has revolutionised the treatment of corneal endothelial disorders and has enabled the restoration of a near normal corneal anatomy and physiology.A 66-year-old gentleman had complaints of intermittent pain and watering in the right eye after having undergone a small incision cataract surgery with a posterior chamber intraocular lens twelve years before presenting to us. This complaint had exaggerated in the three months prior to presentation and was now associated with a decrease in vision. There was no past history of any prior interventions, and the patient did not give any relevant family history. At presentation, his visual acuity in the right eye was counting fingers, while that in the left eye was 20/25. Slit lamp examination revealed an edematous cornea with epithelial bullae in the periphery . The ant– Briefly, the central 9 mm of the host Descemet’s membrane was stripped. An 8mm trephination was carried out on a pre-cut donor tissue with an endothelial count of 2508 cells/mm2. The graft was inserted with a Sheets glide and apposed to the host stroma with a full chamber air tamponade of ten minutes. Postoperatively the visual acuity was counting fingers and the graft was edematous but well attached on the optical coherence tomography (OCT) scan  scan . The patT) scan . FurtherT) scan .2 was inserted. The rest of the surgical technique was similar to that of the first procedure. On the first postoperative day, the patient had a visual acuity of 20/60 with a well attached graft and a significant decrease in the corneal edema. The correct apposition and orientation of the graft was confirmed on OCT  has also been described for the same purpose. Vaddavalli et al and Bhogal et al independently studied the role of IOP in graft adherence. The diagnosis of this entity is made in the presence of persistent corneal edema following a DSAEK. The normal time taken for the recovery of graft clarity following DSAEK ranges from three weeks to three months which can cause a delay in establishing the diagnosis of primary failure. As a result, the subsequent interventions required to visually rehabilitate the patient will also get stalled. However, a reversed lenticule is an exception to this as it can be detected in the early postoperative period and promptly managed. Several measures have been advocated to help ensure the insertion and attachment of the graft in its correct position. These include marking of the stromal surface, use of the double ring sign, intraoperative OCT, etc., However, the lenticule may get inverted despite these safeguards especially in cases with very thin grafts or in eyes with suboptimal clarity of the anterior chamber structures due to corneal scarring. And thus, caution must be exercised in such cases during graft insertion and more than one safeguard should be employed to ensure the correct graft orientation especially by novice EK surgeons.Primary graft failure is a rare complication following DSAEK, with a reported rate of around 1%. Alternatively, in centers with limited access to AS-OCT, slit lamp examination of the graft edge may also help assess its orientation in relatively clear corneas. This additional information is vital as the mere presence of an attached lenticule does not signify its correct orientation. Future studies that look into different insertion techniques and the associated rates of graft reversal can help identify the safest surgical procedure in eyes where in a graft reversal is anticipated. Although a repeat EK was carried out for the reversed lenticule in our case, this option may not be available in areas where corneal grafts are a scare resource. In such scenarios, reversal of the graft can be attempted within a short interval from the primary surgery especially in grafts with excellent preoperative endothelial cell counts.As highlighted by the current case, judicious use of an OCT in the weeks following the surgery can help identify the improper orientation of the lenticule in eyes with slow recession of corneal edema. Although documentation of an attached lenticule with an AS-OCT scan is a routine practice following endothelial keratoplasty, it is essential to include the peripheral graft area when capturing such images. An acute angle configuration of the graft edge has been described which indicates a stroma-to-stroma apposition with correct positioning of the endothelial layer.The current report highlights a reversed DSAEK lenticule as a cause of primary graft failure. This clinical entity can be diagnosed early with the help of an AS-OCT in eyes with slow resolution of the corneal edema and a repeat intervention can be planned subsequently. Adhesion of the graft in DSAEK may depend on factors beyond functional endothelial pumps and an improved understanding of the same will help improve outcomes in eyes which are at a greater risk of having graft detachments.Written informed consent was obtained from the patient for publication of this case report and accompanying images.SYB contributed to the study conceptualization, study investigation, methodology, manuscript editing and revision. AK contributed to the original draft preparation, study methodology and revisions. The authors describe an interesting case of Primary graft failure due to reversed lenticule in Descemet Stripping Automated Endothelial Keratoplasty and a very valuable use of the anterior segment OCT. Slit-lamp biomicroscopy may not be as precise in edematous corneas as details may be easily missed, even by expert clinicians. Anterior Segment Tomography is a very useful tool in searching for clinical cues that may guide the clinician in the diagnosis. As clearly demonstrated by the authors in this case report, OCT of the graft edge may demonstrate the reversed position of the lenticule a detail that would be overlooked by biomicroscopy.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?YesIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:Cornea, Corneal Transplant.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We thank the reviewer for their comments Summary Interesting paper. This case presents a patient who had primary graft failure after a DSAEK graft. Graft failure was secondary to reverse graft tissue. The patient had a repeat DSAEK graft after surgery with the correct corneal graft position with a successful outcome. I like how they describe what to look for in these specific situations on OCT images to confirm the diagnosis. I think this is important as many cases are difficult to distinguish if the graft is in the correct position even with the reference maker.Add in the Figure 2 legend the different types of angle configurations between cases. Maybe consider adding a geometrical line on the edges with acute and obtuse angles on OCT images. It will be helpful for the surgeon to picture the different findings on OCT by doing the following:Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?YesIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:OphthalmologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We would like to thank the reviewer for reading and commenting on our submission. The required change has been made to the figure 2 The authors reported a patient who underwent DSAEK and developed persistent corneal oedema after transplantation despite graft adherence.  In addition to the use of ASOCT, an attentive slit lamp examination with a narrow light beam and focusing on the graft edge may alert the clinician about a potential graft reversal. This may be highlighted as ASOCT may not be readily available in every eye centre.  In the report, the authors mentioned the use of Sheets glide in implanting the lenticule. Unlike in DMEK where some surgeons would use an S-stamp or an asymmetrical triangle mark at the graft periphery, graft orientation in DSAEK relies on a careful preparation and insertion process. It would be interesting to compare the different insertion techniques  and look at the rate/risk of graft lenticule reversal - but in reality, there are multiple factors that could interfere with the final outcome. Similarly, the authors may wish to elaborate on what in their opinion contributed to the graft reversal during the first transplantation, and what special precaution was taken during the second transplantation in order to avoid the same complication. This would be beneficial to novice EK surgeons in ensuring a correct graft insertion.  In places where grafts are scarce, reversal of a flipped endothelial keratoplasty may be attempted if the reversal had been noted earlier. In our experience, we had treated two flipped DSAEK buttons on postoperative day 1 by reversing the graft in the operating theatre. One of the two grafts cleared up after the reversal. This option may be considered especially in areas where corneal grafts are not as abundant.Are enough details provided of any physical examination and diagnostic tests, treatment given and outcomes?YesIs the case presented with sufficient detail to be useful for other practitioners?YesIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment?YesIs the background of the case’s history and progression described in sufficient detail?YesReviewer Expertise:CorneaI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1. In addition to the use of ASOCT, an attentive slit lamp examination with a narrow light beam and focusing on the graft edge may alert the clinician about a potential graft reversal. This may be highlighted as ASOCT may not be readily available in every eye centre. We thank the reviewer for their comment and agree that slit lamp examination can provide an understanding of the graft orientation especially in eyes with limited scarring and a good view of the anterior chamber. This point has been added to the discussion. 2. In the report, the authors mentioned the use of Sheets glide in implanting the lenticule. Unlike in DMEK where some surgeons would use an S-stamp or an asymmetrical triangle mark at the graft periphery, graft orientation in DSAEK relies on a careful preparation and insertion process. It would be interesting to compare the different insertion techniques  and look at the rate/risk of graft lenticule reversal - but in reality, there are multiple factors that could interfere with the final outcome.We concur with the reviewer that understanding the rates of graft reversal in different insertion techniques will help identify the one associated with the least risk of graft reversal and this procedure can be adopted in eyes where in a higher risk of reversal is anticipated. This has been added to the discussion. .  3. Similarly, the authors may wish to elaborate on what in their opinion contributed to the graft reversal during the first transplantation, and what special precaution was taken during the second transplantation in order to avoid the same complication. This would be beneficial to novice EK surgeons in ensuring a correct graft insertion The first procedure was performed by a surgeon with relative inexperience and marking of the graft was not carried out. This could have resulted in the inversion of the graft during its insertion. This point along with measures to prevent the same has been added to the discussion to help surgeons early in the course of their training.  4. In places where grafts are scarce, reversal of a flipped endothelial keratoplasty may be attempted if the reversal had been noted earlier. In our experience, we had treated two flipped DSAEK buttons on postoperative day 1 by reversing the graft in the operating theatre. One of the two grafts cleared up after the reversal. This option may be considered especially in areas where corneal grafts are not as abundant.We agree that attempting a graft reversal is a viable option in low resource settings provided a reasonable time interval exists between the primary EK and the secondary procedure. This point has been added to the discussion.