K and the viscosity exponent n of soybean–protein–isolate (SPI)/wheat–gluten (WG)/corn–starch (CS) blends were investigated through capillary rheometry. All blends exhibited shear-thinning behavior at 80 °C and 50% moisture. The CS content in SPI/CS blends or WG content in SPI/WG blends showed a positive relation to the viscosity exponent n and a negative relation to the consistency factor K. However, there was no correlation between the CS content in WG/CS blends and n or K. The coefficient of determination of the linear relationship between K and mass fraction in SPI/CS, SPI/WG/CS, SPI/WG and WG/CS decreased from 0.872 to 0.073. SPI was more likely to form a non-interactive structure, while wheat-gluten was more likely to form a highly interactive structure. It turned out that the materials with globular morphology, such as soybean-protein-isolate and corn-starch, are likely to form a non-interactive structure.Blends with different proportions of protein or starch show different rheological behaviors, which may be related to the fibrous structure formation of extruded textured plant proteins. The consistency factor  Stretching natural polymer proteins need to overcome entropy elasticity; that is, the product (S × S) and the relaxation time , and is quadratically correlated with the n value (R2 = 0.9934) [The viscosity exponent perature ,6 but al 0.9934) .The mixed materials in the phase separation or compatible state have different flow behaviors. The flow behavior of whey protein isolate and cross-linked waxy corn–starch blends exhibit that, with the increase in the mass fraction of cross-linked waxy corn–starch from 0 to 1, the G’ of blends indicate the range of phase compatible states. When the mass fraction of cross-linked waxy corn–starch is between 0.4 and 0.8, the G’ is relatively low, indicating the system may be composed of two continuous phases in a compatible state; when the mass fraction of cross-linked waxy corn-starch is less than 0.4 or higher than 0.8, the G’ is relatively low, indicating the system is filled by dispersed cross-linked waxy corn-starch particles in the continuous whey protein network, or the aggregated protein particles fill the space between the gelatinized starch particle matrix, and the system is in a state of phase separation [K and n values, which may be related to the fibrous structure formation of extruded, textured plant proteins. In this study, soybean–protein–isolate, wheat–gluten and corn–starch, which are commonly used in plant protein extrusion, were used as experimental materials to study the consistency factor K and the viscosity exponent n of the protein–starch blends with different mass fraction components. The degree to which the relationship between flow behavior and mass fractions deviated from the non-interactive model was evaluated. This helps in understanding and controlling the rheological behavior of protein–starch blends and may further control the formation of the fibrous structure.In the plant protein extrusion process, protein and starch are the main components, and each of those may form the continuous phase . With thp) for SPI and CS (both with 50% moisture) was 106 °C and 68 °C, respectively, while there was no distinct thermal transition peak for WG. This indicates that 80 °C is not enough to totally denature the SPI; the CS probably was in a partially gelatinized state, and the WG has been completely denatured.The DSC results of soybean–protein–isolate (SPI), corn–starch (CS) and wheat-gluten (WG) are shown in −1 and with CS content increasing from 0 to 100% in the blends.Viscosity as a function of shear rate for different soybean–protein–isolate (SPI)/ corn–starch (CS) blends with 50% moisture content at 80 °C barrel temperature is shown in n values increased from 0.297 to 0.464, indicating a marked shear-thinning behavior in the blends, and appeared positively correlated with CS content (R2 = 0.843). The consistency factor K values decreased from 119,492 to 3004 Pa·s. The K value of blends decreased with the increase in CS content from 0 to 100% and was negatively correlated with the CS content (R2 = 0.872).Power law parameters as a function of CS content in the SPI/CS blends are shown in −1 and as WG content increased from 0 to 100%.Viscosity as functions of shear rate for different soybean–protein–isolate (SPI)/wheat-gluten (WG) blends with 50% moisture at 80 °C is shown in n value increased from 0.297 to 0.419, not only showing a shear-thinning behavior but also suggesting the formation of some network [2 = 0.837). The consistency factor K decreased from 119,492 to 3,939 Pa·s with WG content increasing from 0 to 100%, and in a negative relationship with WG content (R2 = 0.575).The relation between power law parameters of different SPI/WG blends and WG content is shown in Viscosity as a function of shear rate for different WG/CS blends is shown in The power law parameters of different WG/CS blends are shown in The viscosity of different SPI/WG/CS blends obtained at 50% moisture and 80 °C is shown in n decreased from 0.404 to 0.246, and the consistency factor K increased from 6,087 to 119,492 Pa·s with increasing SPI content. The same trend was observed at 3/1 and 1/3 ratios for the consistency factor K, which increased from 10,837 to 119,492 Pa·s and from 4,101 to 119,492 Pa·s, respectively.The power law parameters of the different SPI/WG/CS blends are shown in no-interη, no-intern or no-interK of each blend were calculated according to the non-interactive model. The fitted equations between the mass fraction of components and no-intern or no-interK are shown in no-intern, in which the first two equations made sense. The decisive factors R2 of the linear fitted equation between the mass fractions of components and the no-intern were 0.625, 0.631, 0.997, 0.881, 0.862 and 0.866, respectively, and the R2 values of the exponential fitted equations were 0.652, 0.652, 0.999, 0.885, 0.867 and 0.870, respectively, indicating that the relationship between the mass fraction of components and no-intern was unstable under linear or exponential equations. This suggested that the viscosity exponent n of the blends was complicated. There was no simple model which could demonstrate the relationship between the mass fraction and the viscosity exponent n, even with the no-intern of none interaction model.The no-interK, the decisive factors R2 of the linear fitted equation were 0.9999, 1.0000, 0.9993, 1.0000, 1.0000, and 1.0000, respectively, while the R2 of the exponential fitted equation was 0.813, 0.828, 1.000, 0.931, 0.923, and 0.926 for each blend, indicating that linear equation was suitable for describing the relationship between mass fraction and no-interK, for the R2 was stable and was almost 1.0000, which suggested the R2 of linear equations between mass fraction and measured K values may serve as the index presenting the interaction degree of components in the blend. In the linear equation between mass fraction and K, a further R2 from 1 indicates a higher degree of interaction.As for the consistency factor K and n values, which might be related to the fibrous structure formation of extruded textured plant proteins.Protein and starch are the main components in plant protein extrusion, and each of them may form a continuous phase . Blends K and the viscosity exponent n of soybean-protein-isolate/wheat-gluten/corn-starch blends were investigated.To study a protein/starch blend’s rheological behavior, the consistency factor n increases and K decreases as corn–starch (CS) content increases in soybean-protein-isolate (SPI)/CS blends, presenting similar trends to our results. Reference [n and decreasing K in SPI/wheat–gluten (WG) blends when WG increases, also presenting similar trends to our results. The authors of [n increased and consistency factor K decreased with the increase in wheat–starch content, which is different from our results, and we believe this is because the wheat–starch had totally gelatinized at 140 °C and replaced gluten to become the dominant continuous phase in the blends [Reference  illustraeference  found inthors of  studied e blends ,13.no-interK or viscosity exponent no-intern of a non-interactive model was evaluated with linear, exponential, power function and logarithmic equations. Only the linear relationship between mass fraction and no-interK was stable, and all the R2s of the equations were almost 1.000. Thus, the R2 of the linear equations between mass fraction and measured K values may act as the index presenting the interaction degree of components in the blend, where a further R2 from 1 indicates a higher degree of interaction. After the linear relationship between measured consistency factor K and the mass fraction of components was calculated and the deviation from R2 = 1 was evaluated, we found that the R2 of the relationship between measured K and mass fraction in SPI/CS, SPI/WG/CS (WG/CS = 3/1), SPI/WG/CS (WG/CS = 1/3), SPI/WG/CS (WG/CS = 1/1), SPI/WG, and WG/CS were 0.872, 0.785, 0.697, 0.597, 0.574, and 0.073, respectively, and the deviation from R2 = 1 was 0.128, 0.215, 0.303, 0.403, 0.426, and 0.927, respectively. Those may indicate that it is also the sequence of the increasing degree of the binary interaction.Two different polymers will interact with each other when mixed, either by combining them together or by separating them ,15. To tp of the SPI sample in the experiment with 50% moisture was 106 °C, which indicates that SPI retains a partially globular morphology under 50% moisture at 80 °C. The microstructure of gluten networks has revealed that the interacting and interpenetrating proteins could form a continuous network [Different from a low concentrated system, the degree of binary interaction in a highly concentrated system is related to the molecular morphology. SPI is mainly composed of globulins corresponding to storage proteins of soybeans, including 7S and 11S, whose thermal transforming temperature ranges between 89.97 °C(7S) and 108.79 ℃(11S) with 50% moisture ,17. The  network ,19. The  network . Imaging network . The WG/ network ,23. As fSoybean-protein-isolate (SPI) was supplied by DuPont , with protein and water content of 89.43% (dry basis) and 5.79%, respectively. Corn-starch (CS) was supplied by Ingredien , with corn and water content of 99.38% (dry basis) and 11.85%, respectively. Wheat–gluten (WG) was supplied by Lianhua Group Ltd. , with protein and water content of 77.30% (dry basis) and 7.49%, respectively.−1 between 20 and 130 °C under an inert atmosphere (50 mL min−1 of dry N2). The reference was a void aluminum TA pan. The onset temperature (To), peak temperature (Tp), and enthalpy . Samples were conditioned in hermetic aluminum TA pans with 50% moisture, weighed (8–10 mg) using a precision balance , and heated at a rate of 10 °C minThe blends of SPI, CS and WG were made at the ratios listed in −1) corresponding to increasing piston speeds. The pressure needed to extrude the sample through the capillary was recorded. The tolerance of the measuring pressure was set as 2%, and the pressure was recorded upon reaching a steady state within this tolerance.A capillary rheometry  of a dual barrel system was used to measure the shear viscosity of the samples. Round hole capillaries with an inside diameter (1 mm) and length (10 mm) were used. The length/diameter (L/D) value of the selected capillary was 10. The capillary was fixed at the bottom of the barrel. Generally, during high moisture extrusion of plant-based proteins, the barrel temperature is higher than 100 °C, which will cause rapid moisture dissipation and bake the samples during rheology measurements. Thus, the temperature of 80 °C was selected, which was close to 100 °C and caused slow dissipation. The temperature was set at 80 °C along the barrel during the experiment. The barrel was filled with a similar quantity of each sample, and it took about 6 min to complete a filling. The piston was allowed to contact the sample in the barrel; the piston moved down and stopped at the first appearance of dough at the bottom of the capillary. Then, the sample was allowed to equilibrate for 5 min before starting the test. The experiments were performed at increasing apparent shear rates , was used to fit the viscosity results of blends .(1)η=τγ−1. Using the more accurate models to fit the two flow behaviors will make the study complex; the universal power law equation was used to describe the rheology characterization. The power law equation and parameters  of samples were obtained via Origin 2022, and all the coefficients of determination (R2) of fitting models are no less than 0.9860. In detail, fit the shear rate and its corresponding viscosity data with the power law function and obtain K and n values using the nonlinear curve fit function of Origin 2022. With the power law model of each component, the viscosity data of each component at a certain shear rate can be calculated. To assess the repeatability of the capillary rheometry measurement, the measurements with 100% SPI, 100% WG, or 100% CS were repeated in separate runs. The power law equation and parameters  of three samples were repeatable, with variances less than 1%. Thus, for all other blends, one capillary rheometry measurement was carried out.There are Newton regions and non-Newton regions for all samples, with shear rates increasing from 0 to 1000 sno-interη is the calculated blend viscosity, nW is the mass fraction of each component, and nη is the viscosity of each component at a certain shear rate, which is calculated with the power law model of each component.The blend viscosity was supposed to be related to the proportion and the viscosity of each component when there was no interaction . A non-ino-interK and no-intern were fitted from the power law model with no-interη and the corresponding shear rate. These no-interK and no-intern of the non-interactive model were merely used as a standard to compare with the measured K and n values of all samples. The larger the difference between the measured values and the values from the non-interactive model, the higher the degree of interaction within the components of the blends.Then, K or n and mass fraction of SPI/WG/CS were drawn and fitted by EXCEL. The relationship between the mass fraction of components and K or n included linear equations, exponential equations, power function equations, and logarithmic equations.All the statistics in this study were calculated by PASW Statistics 18.0 software, and the equation fitting was achieved by origin 2022 . The content scatter diagrams and the relationship between n ranging from 0.245 to 0.466. CS content in SPI/CS blends and WG content in SPI/WG blends showed a positive correlation with the viscosity exponent n and a negative correlation with the consistency factor K. However, there was no correlation between the WG content and n or K in WG/CS blends. The coefficient of determination of the linear relationship between K and mass fraction in SPI/CS, SPI/WG/CS, SPI/WG and WG/CS decreased from 0.872 to 0.073. SPI is more likely to form a non-interactive structure, while wheat–gluten is more likely to form a highly interactive structure. It turned out that both binary and globular morphology, such as soybean–protein–isolate and corn–starch, are likely to form a non-interactive structure.The rheological properties of different blends with different soybean–protein–isolate (SPI)/wheat–gluten (WG)/corn–starch (CS) ratios were investigated through capillary rheometry. The blends exhibited shear thinning behavior at 50% moisture and 80 °C, with the viscosity exponent


SSIIIa protein was undetectable by immunoblot analysis in triple ssIIIa mutants carrying mutations in each homoeologous copy of ssIIIa . Loss of SSIIIa in triple mutants led to significant changes in starch phenotype including smaller A-type granules and altered granule morphology. Starch chain-length distributions of double and triple mutants indicated greater levels of amylose than sibling controls  and fewer long amylopectin chains. Wholemeal flour of triple mutants had more resistant starch (6.0% vs. 2.9% in sibling controls) and greater levels of non-starch polysaccharides; the grains appeared shrunken and weighed ~ 11% less than the sibling control which was partially explained by loss in starch content. Interestingly, our study revealed gene dosage effects which could be useful for fine-tuning starch properties in wheat breeding applications while minimizing impact on grain weight and quality.Starch synthase III plays a key role in starch biosynthesis and is highly expressed in developing wheat grains. To understand the contribution of SSIII to starch and grain properties, we developed wheat  Triticum aestivum) is a staple cereal crop that provides nearly 20% of the calories consumed worldwide1. Although wheat is a valuable source of nutrition, there is growing concern about the effects of wheat-based foods on health including the rising incidence of obesity and type 2 diabetes3. Most wheat food products are made from refined white flour which is mainly comprised of starch (~ 85% dry weight); they tend to be low in fibre and are rapidly digested in the upper gastrointestinal tract4. Thus, elevating the levels of resistant starch, an important component of dietary fibre, is a focus of many wheat improvement programs.Wheat . Starch polymers form granules in the amyloplasts of endosperm cells in developing wheat grains. The granules have a highly organized structure with amorphous lamellae that contain amylose polymers and branch points of amylopectin and crystalline lamellae that form from the ordered packing of short amylopectin chains6.The major component of the wheat grain is starch, a mixture of two glucose polymers, amylose (~ 20 to 30%) and amylopectin (~ 70 to 80%), which differ in size, glucose chain-length, and degree of branching7. Starch Synthase IIIa (SSIIIa) is strongly expressed in developing wheat endosperm cells and thus likely to make a major contribution to starch synthesis8.Three types of enzymes are required for amylose and amylopectin synthesis from adenosine diphosphate glucose (ADP-glucose) which is made in the endosperm from sucrose produced during photosynthesis and transported to the developing grain: soluble starch synthases and granule-bound starch synthases (SSs and GBSSs) which elongate glucose chains, starch branching enzymes (SBEs) which introduce branch points, and starch debranching enzymes (DBEs) which trim branched chains to create a structure that can crystallize to form the granule matrix9. Growing evidence suggests resistant starch is associated with health benefits such as reduced glycaemic response, thus elevating the resistant starch content of wheat grains has potential for improving their nutritional value9. For example, recent studies of wheat bread and pasta with resistant starch levels ranging from ~ 7 to 9% led to reductions in postprandial glycaemic response in vivo11. Starches with high levels of amylose tend to have a greater content of resistant starch than those with low amylose content, so a key strategy to increase resistant starch in wheat is to develop varieties that produce high-amylose starch12. Amylose and resistant starch levels can be elevated in wheat by combining induced mutations in SSIIa or SBEII genes17. However, initial analyses of some mutants have revealed adverse effects on yield components including grain weight and on grain quality characteristics, so it is not yet clear if these mutants can be used to develop commercially viable wheat lines19. Therefore, targeting other key starch biosynthesis genes might provide a better route to resistant starch.Resistant starch is a type of starch that resists digestion in the small intestine and reaches the colon where it is further metabolized by gut microbesSSIII. Studies of ssIIIa mutants in potato, rice and maize and of SSIII in vitro have suggested it has a key role in synthesis of long amylopectin chains (reviewed in Nakamura7). Manipulation of SSIII genes affects amylose and resistant starch content in other cereal species. For example, dull1 maize mutants deficient in starch synthase III have higher amylose contents than wild-type controls (25.4–30.2% vs. 21.5% in wildtype)20. Similarly, the barley amo1 mutant has higher levels of amylose relative to a wild-type control 21. Furthermore, a mutation in a SSIIIa gene confers high levels of resistant starch in cooked rice (~ 6.0% vs. ~ 1.5% in wild-type controls)22. There is also evidence that manipulation of SSIIIa in rice can influence starch structure indirectly, through pleiotropic effects on activities of other starch-synthesizing enzymes including the amylose-synthesizing enzyme GBSS23. Likewise, the lack of SSIII in maize leads to a decrease in starch branching enzyme IIa activity and increased starch synthase I activity25. The reported pleiotropic effects of loss of SSIII are unsurprising given the evidence that starch biosynthetic enzymes work together in complexes and that their activities are likely coordinated rather than independent26. For wheat, there is no peer-reviewed literature on the impact of loss of ssIIIa on starch accumulation and properties. Information in a patent application indicates that combined mutations in wheat ssIIIa homoeologues can lead to elevated levels of amylose and components of dietary fibre27.A possible candidate gene is 28. However, the recent advances in wheat genomics resources, specifically the exome capture and re-sequencing of wheat TILLING  mutants, allow induced mutations in different homoeologues and genes to be combined by crossing wheat mutants29. The functions and interactions of individual genes and homoeologues can then be investigated.Elucidating the roles of starch-related genes in wheat presents challenges in identifying loss-of-function mutations, which are likely to be masked by functional gene copies found in the other homoeologous genomesSSIIIa homoeologues. Here we describe the development and characterization of the starch and grain properties of the ssIIIa TILLING mutants.To discover whether manipulation of SSIIIa expression can alter starch properties and elevate levels of resistant starch in wheat we have generated a novel set of mutants in the elite bread wheat cultivar Cadenza carrying combinations of null mutations in SSIIIa and SSIIIb homoeologues in the elite bread wheat cultivar Cadenza were identified using the Grassroot Genomics BLAST Search (https://grassroots.tools/service/blast-blastn) to find homologous sequence to the SSIII genomic DNA sequence previously isolated by Li et al.8 (GenBank accession no. AF258609). Using the Wheat Expression Browser (http://www.wheat-expression.com/) in silico expression profiling of SSIIIa and SSIIIb paralogs and homoeologues was performed to investigate expression patterns for distinct plant tissues and for different stages during grain development. A heat-map comparing expression of SSIIIa and SSIIIb indicated that expression of SSIIIa is mainly limited to the grain, whereas SSIIIb is expressed in the roots, leaves/shoots, and spike as well as the grain  but lower expression throughout the rest of development. Given these results we supposed that SSIIIa is the predominant form expressed in the grain and corresponds to the cDNA and genomic DNA sequences previously isolated by Li et al.8. In light of these data we focused on developing mutants for SSIIIa homoeologues which we presumed would have the greatest impact on starch properties in the grain.Genomic DNA sequences for ain Fig. . SSIIIa SSIIIa  (Table http://www.wheat-tilling.com/). To predict the effect of the mutations on protein function we used the reference SSIII amino acid sequence (1628 residues) from a hexaploid wheat SSIII cDNA clone (GenBank accession AF258608), and previously defined protein domain regions described by Li et al.8. Each of our selected mutations was predicted to result in a protein truncated before the catalytic domain or within it, rendering the mutant protein inactive.Mutations causing premature stop codons in the A, B and D genome copies of D) Table  were idessIIIa  were crossed to create a line lacking active SSIIIa protein. Plants heterozygous for mutations in all three ssIIIa homoeologues were self-fertilized to produce segregating F2 populations. Using KASP markers . The number of plants selected for each genotype class is included in Table ssIIIa-AD for which only five plants were identified. Despite fewer ssIIIa-AD plants, a Chi-square test showed no evidence of segregation distortion (P = 0.8034). In addition to mutant genotypes, siblings without mutations in SSIIIa were selected to provide an ideal control; these lines are expected to have the same level of segregating background mutations left over from the TILLING process as the ssIIIa mutants. Genome-specific sequencing primers were designed to validate the selected mutations .The TILLING mutants for each homoeologue of rs Table , 724 segns Table  and all ssIIIa mutations on SSIIIa protein, immunoblot analysis was performed using maize polyclonal antiserum for SSIIIa (anti-DU1N)25 ; the average width of triple mutant grains was also 5% lower than that of the sibling control (P < 0.001) and the area was 5% smaller (P = 0.04) (Tables The average thousand grain weight (TGW) of triple mutants was 11% lower than that of sibling controls  vs. 59.6 (SD = 9.7) for controls, (P = 0.400) (Table There was no evidence for an effect of genotype on mature grain protein content (estimated by measuring nitrogen released by combustion) , compared to 9.91% (SD = 0.75%) in the sibling control; ANOVA across all genotypes 0) Table .P < 0.001) (Table P < 0.001), as were the ssIIIa-BD double mutants   Table a. The fl1) Table a. Althou1) Table a. The flP < 0.001), but the amylose contents of starch from single and double mutants were not different from that of the sibling controls  (Table P < 0.001)  was more than twice that of the sibling control (2.86 g/100 g flour); 1) Table c. ResistssIIIa mutants and sibling controls. In the triple mutants many of the A-type granules were smaller and more elongated than those of sibling controls, and many granules had protrusions  in the triple mutant compared to the sibling control  was used to measure particle volumes in purified starch. All of the starch samples showed a bimodal size distribution as expected for wheatP < 0.001) and BD double mutant  compared to sibling controls   Table . There w8) Table .ssIIIa mutants had different chain-length distributions (CLD) compared to the sibling control (measured on debranched starch). Analysis of CLD data indicated fewer long amylopectin chains and more amylose chains in the double and triple mutants compared to the sibling controls. The CLDs showed three distinct peaks, the first around a degree of polymerization (dp) of 10–12 corresponding to short amylopectin chains within a single crystalline lamella of starch, the second between 37 and 100 dp corresponding to longer chains that span multiple lamellae as defined by Hanashiro, et al.32, and the third around 2500 dp corresponding to long amylose chains32 . The relative heights of the peaks were significantly different between genotypes . The proportion of area > 100 dp (corresponding to proportion amylose by mass) was higher in the double (mean = 29.4%) and triple mutants (mean = 33.8%) than the sibling control . Within chains < 100 dp; the proportion of the area corresponding to longer chains (37 to 100 dp) was lower in the double (30.0%) and triple mutants (28.3%) than the sibling control , indicating a reduction in longer amylopectin chains compared to shorter amylopectin chains in the double and triple mutants ; for conclusion: 75.4 °C vs. 70.5 °C in sibling controls (P = 0.001)) . The peak and conclusion temperatures of gelatinization for the triple mutants were significantly greater than those of the sibling controls (for peak: 61.8 °C vs. 58.4 °C in sibling controls ) Table . There w)) Table .P < 0.001) (Table P = 0.042) and sucrose  (Table P = 0.0001 and P = 0.0005 respectively)  were measured in grains of triple mutant and wild-type sibling controls harvested at 25 days after anthesis (DAA) and maturity. At 25 DAA, sucrose levels in the triple mutant were significantly greater, 3.04 mg/grain vs 1.00 mg/grain in the sibling control, (1) Table . At matu7) Table . Overally) Table .ssIIIa mutations on non-starch polysaccharides in the grain, enzyme fingerprinting was used to measure relative levels of arabinoxylan oligosaccharides (AXOS) and mixed-linked β-glucan oligosaccharides in wholemeal flour samples following enzymatic digestion. Total AXOS  was significantly higher in the triple mutants  and in BD double mutants (17% more P = 0.001) relative to the wild-type sibling controls (Table P = 0.008) (Table To determine pleiotropic effects of 8) Table .Table 3AP < 0.001), AB and BD double mutants , and D-genome single mutant , with some evidence for an increase in the B and AD mutants  in the triple mutant  mutants presented differential effects of distinct Waxy proteins  on amylose content35. These findings suggest that starch gene homoeologues do not always show functional redundancy but can also present dosage and homoeologue-specific effects. This presents new opportunities for fine-tuning starch phenotypes in breeding applications to reach desired effects on starch structure which is not possible in diploid crops like rice or maize.The aim of our study was to estimate the nature and extent of alterations in starch and grain properties brought about by loss of SSIIIa in wheat. Research in other cereal crops has shown major effects of loss of SSIII that include increased resistant starch contentssIIIa mutants differed strongly from controls with respect to starch granule morphology and size distribution. Wheat has two distinct types of starch granules, disc-shaped A-type granules of 5–40 μm in diameter and more spherical B-type granules which are smaller (< 5 μm) and more abundant36. The smaller A-type granules observed in the triple mutant suggest that SSIIIa plays a major role in the growth of A-type granules. Many of the A-type granules also had an unusual shape; this could be due to an effect on granule growth and/or the changes in starch composition (more amylose) and amylopectin structure (fewer long chains). The change in birefringence pattern observed in the A-type granules of triple mutants is also consistent with irregular patterns of granule growth. In contrast, the B-type granules in the triple mutant appeared normal which could suggest a more specific role of SSIIIa in the synthesis of A-type granules. Peng, et al.37 previously showed preferential binding of starch branching enzyme isoforms to A-type granules and proposed the proteins could play a role in the growth of small A-type granules into full-sized A-type granules; it is possible that SSIIIa could play a similar role. We also observed A-type granules with a more normal shape; the mixture of normal and abnormal granules may reflect an increasing influence of SSIIIa on granule growth as cellularisation and granule initiation proceeds during the very early stages of endosperm development. Initiation of A-type granules begins very early in development when SSIIIa expression is relatively low and continues for a few days as SSIIIa transcript levels rise and SSIIIb transcript levels fall. It is thus possible that the granules formed early in development in the triple mutant have fewer growth defects than those formed towards the end of the A-type granule initiation period38.The triple 39 previously reported that du1 mutants in maize had smaller starch granule size distributions (4–11 μm) than a control (6–17 μm). In rice, the starch granules of a ssIIIa mutant were variable in size and more rounded in shape than normal polyhedral starch granules40. Ahmed, et al.41 reported A-granules with ‘bumps’ in barley amo1 mutants like the protrusions we observed in the wheat triple ssIIIa mutants. It is worth noting that starch granule size distribution can affect physicochemical properties of flour and breadmaking properties, which may be an important consideration for possible applications of the ssIIIa mutant42.Disruption of granule size and morphology due to loss of SSIIIa has been observed in other cereals. Wang, et al.ssIIIa mutant had a relative increase of ~ 35% compared to the sibling control (measured by iodine binding method)  Table . ElevatessIIIa mutants were different from that of the sibling control . It is likely that this increase is mainly due to the elevated proportion of amylose, which is less readily digested than amylopectin. The amylose content of starch typically has a positive association with resistant starch9. This elevation of resistant starch in the triple mutant is within the range of values reported for other starch biosynthesis mutants in wheat. For example, ssIIa and sbeIIa triple mutants produced using the cultivar Cadenza have 1.4% and 7.2% resistant starch respectively. However the amylose levels of those mutants (45.7% for ssIIa and 78.7% for sbeIIa) are greater than the ssIIIa triple mutant (35.1%), which suggests other factors besides amylose are likely contributing to the change in resistant starch such as changes in amylopectin structure and possibly interactions with other components of the flour17. The lower resistant starch levels observed in purified starch compared to wholemeal flour (1.70 g/100 g of starch vs 6.01 g/100 g of flour in the triple ssIIIa mutant) also highlights the importance of flour matrix. Furthermore, in this study we found that mutations in ssIIIa genes also led to greater levels of arabinoxylans and β-glucans  were higher than those in raw flour (0.4 g/100 g) used to bake bread19.The greater peak and conclusion temperatures observed in DSC analyses suggest that the triple mutant starch behaves differently from the sibling control when subjected to hydrothermal treatment which is consistent with previous literature for starches from a range of botanical origins with elevated amylose contentsImportantly, our results show that lack of SSIIIa has effects on the grain beyond starch composition and structure that will need to be taken into consideration for breeding applications. The grains of the triple mutants weighed about 11% less than the grains of the sibling control Fig.  and S4. ssIIIa triple mutants were also very different from controls with respect to grain texture: mutant grains were harder and had vitreous endosperms. This is an important consideration in assessing the value of the triple mutants for wheat breeding as their hardness index was at the high end of the range considered acceptable for bread wheats; hard grains impact important milling and baking quality factors like milling energy, starch damage and water absorption48. Variation in grain texture of wheat can be caused by several genetic and environmental factors, including the nature and levels of puroindolines (proteins that bind to starch in flour), total protein levels and levels of water-soluble pentosans and lipids49. The two starch synthesis mutants of hexaploid wheat described previously—ssIIa and sbeIIa—both have hard endosperms associated with low starch contents and elevated levels of protein and water-soluble pentosans19. It seems likely that the hardness and vitreosity of SSIIIa grains are attributable to compositional changes other than protein since the levels of protein were not found to differ.The 19. Further studies using backcrossed and independent triple mutant lines and evaluation of field performance and end-use functionality will shed light on whether the ssIIIa mutant offers any advantages for commercial breeding.In this study we show that lack of SSIIIa in hexaploid wheat has significant effects on starch and grain properties. Our analyses revealed interesting gene dosage effects which could be used for fine-tuning starch properties in wheat breeding applications. Although amylose and RS were elevated in mutants, the altered grain characteristics, may limit their usefulness for wheat breeding. Reductions in starch content and grain weight are a common effect of starch biosynthesis mutations and are likely to lead to yield penaltiesAll methods were carried out in accordance with relevant guidelines and regulation.SSIIIa homoeologue , TILLING mutants carrying premature stop mutations were identified in the hexaploid wheat variety Cadenza using the in silico Wheat TILLING database (http://www.wheat-tilling.com/)29. Grains for each mutant were ordered from the Germplasm Resources Unit  using the publicly accessible SeedStor system https://www.seedstor.ac.uk/; permission to use the materials for research purposes was obtained. The mutants were crossed, and the single, double, and triple mutants were selected from segregating F2 populations along with sibling controls not carrying the mutations in SSIIIa. Selections were made using KASP genotyping  with homoeologue specific primers designed using the program PolyMarker50. Mutations were validated in triple mutants by Sanger sequencing; the sequencing primers are available in Table For each 2 plants were grown in the glasshouse in a randomized block design and grains harvested at maturity. To make wholemeal flour, 5 grain samples, each from a different plant, were selected at random and ground in a UDY Cyclone Mill  using a 0.5 mm screen. The same grain samples were used for starch purification. For each outcome measured, biological replicates are defined as samples originating from separate F2 plants.  protein, then a goat anti-Rabbit IgG-Alkaline Phosphatase antibody. Blots were developed with BCIP/NBT Colour Development Substrate (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium) following manufacturer instructions (Promega S3771).Developing grains at 25 days after anthesis were harvested from Fhttps://www.marvitech.de). The protein content of mature grains was measured using the AACC 46–30 Crude protein—combustion method51 on a CE440 Elemental Analyser . Grain hardness index was determined using a Perten Single Grain Characterization System ; the average of 100 grains was measured for each biological replicate.Thousand grain weight, grain length, grain width and grain area were estimated from the weight of 387–1171 mature grains per plant using a MARVIN seed analyzer  50 grains were surface sterilized and incubated at 17 °C on filter paper in the dark. The germination index was calculated using the method of Walker‐Simmonsg for 5 min. The pellet was washed twice with 2% (w/v) SDS then twice with ice cold acetone by resuspension followed by centrifugation. The final pellet was air-dried.For each sample approximately 50 wheat grains were soaked overnight then homogenized in a small volume of water using a mortar and pestle. The homogenate was filtered through Miracloth and the filtrate centrifuged at 3000https://www.megazyme.com/) following the manufacturer’s instructions for the recommended KOH assay format. The resistant starch content (amount of resistant starch measured on a g/100 g ‘as is’ basis) was measured for 100 mg samples of wholemeal flour and 100 mg samples of purified starch using the Resistant Starch Assay Kit (Rapid) .Total starch content was measured for 100 mg samples of wholemeal flour using the Total Starch Assay kit , which corrects for absorbance from amylopectin binding iodine53.The apparent amylose content was determined using an iodine dye binding method using a potato amylose standard curve55. Starch chain-length distributions were analyzed according to the HPLC-SEC procedures detailed in Supplementary Material. Three parameters were estimated for each genotype; these were (1) the relative heights of the peaks corresponding to long amylopectin chains and amylose, (2) the ratio of the area under the curve > 100 dp (amylose) to total area, and (3) the ratio of the area between 37 and 100 dp (long amylopectin chains) to the area between 0 and 100 dp  as defined by Hanashiro, et al.32. Since each outcome was relative within the sample, no cross-sample normalisation was necessary. These ratios were then compared as other outcomes as described in the Data analysis section below.Purified starch isolated from whole grains was solubilized in DMSO containing 0.5% (w/v) LiBr followed by enzymatic debranching with isoamylaseDifferential scanning calorimetry was used to characterize the thermal properties of starch using a MCDSC instrument, TA Instruments . Starch (50 mg) was accurately weighed in a high-pressure pan  then 1 mL of degassed water added and the pan lid hermetically sealed. The furnace was purged with dry nitrogen gas at a flow rate of 50 mL/min. The measurement procedure started with temperature equilibration at 20 °C for one hour, followed by a temperature ramp up to 140 °C at the rate of 1 °C/min. DSC parameters were analysed using TA NanoAnalyze and TA Advantage/Universal Analysis.Starch granule size distribution was measured using a Coulter counter (Beckman Coulter Multisizer 4e). The average of two technical replicates was used for each biological replicate. The relative total volume of A-type and B-type starch granules was estimated for each technical replicate by fitting the distribution of diameters (weighted by particle volume) to a mixture of Gaussian distributions, which was observed to fit the data well in all cases. From each mixture model we extracted the relative proportion of total starch volume accounted for by B-type granules, and the mean diameters of the A-type and B-type granules. These were then compared across genotypes as other outcomes using the linear mixed models described in the Data analysis section.57, except that recombinant enzymes were used for digestion. Briefly, wholemeal flour samples were digested with endo 1,4 β-xylanase (E.C.3.2.1.8)  and endo 1,3(4) glucanase (‘lichenase’) (E.C.3.2.1.73)  to digest AX and MLG, respectively. After digestion, the samples were filtered, diluted in 10 μM melibiose , and analysed using a CarboPac PA1 analytical column  on a Dionex ICS-3000 (Thermo Scientific). The peak areas of the oligosaccharides released by enzyme digestion were expressed as percentages of the total peak areas of all AXOS and ‘total AXOS’ was calculated as the sum of all known AXOS peaks. Two major β-glucan oligosaccharides, G3 and G4, were released by enzymatic digestion and total MLG was calculated as the sum of G3 + G4 peak areas.Enzyme fingerprinting was used to measure relative values of arabinoxylan oligosaccharides (AXOS), and mixed-linked β-glucan (MLG) oligosaccharides following the methods by Ordaz-Ortiz et al. (2004 and 2005)58.Developing grains at 25 days after anthesis and mature grains were harvested from F2 plants into liquid nitrogen. Extracts of soluble sugars were prepared and assayed enzymatically following methods reported in Fahy, et al.Two-µM sections of mature grains prepared using a microtome were placed in a drop of 50% (v/v) ethanol on a microscope slide and dried at room temperature. Grain sections and isolated starch were stained with diluted Lugol’s iodine solution (4% v/v for sections and 6.25% v/v for isolated starch) (Sigma L6146) and viewed on a light microscope. A polarizing filter was used to examine the birefringence patterns of starch granules.Dried isolated starch was gently brushed onto a carbon tab and fixed to a SEM pin stub. Samples were viewed using a Zeiss Supra 55 VP FEG SEM.All outcomes were compared across genotypes using linear mixed effects regression models, with the sibling control chosen as the reference group. Replicates in analysis correspond to individual plants. Between 3 and 13 replicates were included for each genotype in each analysis depending on available material  R package was used to estimate mixture models62. All analysis code is available at https://github.com/quadram-institute-bioscience/SSIIIa-Rscripts.All analyses were conducted using R (versions 3.6.1 and 4.1.1) , using the lme4 (version 1.1-27.1) and lmerTest (version 3.1-3) packages for estimating mixed modelsSupplementary Information.


Policymakers, practitioners, and researchers need a balanced, thoughtful, and analytical resource to meet the challenge of global aging at a rate that’s historically unprecedented. The Journal of Aging & Social Policy (JASP), which was founded in 1989, serves this role by drawing contributions from an international panel of policy analysts and scholars who assume an interdisciplinary perspective in examining and analyzing critical phenomena that affect aging and the development and implementation of programs for older adults from a global perspective. Study settings extend beyond the United States to include Europe, the Middle East, Australia, Latin America, Asia, and the Asia-Pacific rim. This presentation will document the scope, content, and focus of JASP, including the rise of international submissions, which now account for approximately half of articles published. Opportunities for publishing in JASP will be discussed; so too will strategies for navigating the peer-review process successfully.


In this study, digital light processing (DLP) was used to achieve 3D-printed composite materials containing photosensitive resin blended with starch and hemp fibers. The synthesis of 3D-printed composites was performed without heating, according to various material combinations ranging from pure photosensitive resin to a mixture of three phases, including resin, starch, and hemp fibers, with the weight content for each reinforcing phase reaching up to a third of the formulation. The morphology, composition, and structure of the 3D-printed composites were assessed using infrared spectroscopy, laser granulometry, X-ray diffraction, and optical and scanning electron microscopy. In addition, thermal behavior and mechanical performance were studied using calorimetry, differential scanning calorimetry, and tensile testing combined with high-speed optical imaging. The results showed that the post-curing step is a leading factor for improving the mechanical performance of the 3D-printed composites. In addition, hemp fiber or starch did not alter the tensile strength. However, the largest reinforcing effect in terms of stiffness improvement was obtained with starch. Additionally, starchy composites demonstrated the strongest dependence of heat capacity on operating temperature. Additive manufacturing (AM) is one of the major breakthrough technologies that have received major attention in recent decades ,2. It isDigital light processing (DLP) is another AM route that shares the same rationale as stereolithography but solves the problem of long term scanning by using a digital screen that projects an image of the layer under construction . TherefoIn this work, we report for the first time a successful attempt to process starch and hemp fiber fillers in additive manufacturing using the photopolymerization route. Starch is considered as a solid filler in a sensitive resin, and the evaluation of the composite performance and the cost point are discussed. In fact, photosensitive resins are known to be expensive feedstock materials, and only a limited choice of materials can be processed. Blending can be a solution in designing new structures with specific performance requirements, such as water sorption/desorption capability, while maintaining a reasonable printing cost. In addition, the use of biomass has proven to be a strong trend in the plastics industry because of the environmental footprint, high specific mechanical properties, biocompatibility, and transfer properties. For instance, Azmin et al.  developeThe other motivation behind this study was to bypass the difficulty of processing starch by using other AM routes. Indeed, starch is not suitable for FDM, as the heating of starch beyond the glass transition results in a large drop in mechanical stability, making it impossible to support any layering during the printing process.3  purchased from the Longer company  under the tradename of Longer (LNG). The physical properties of the resin are provided in −1 and 400 cm−1 at 8 cm−1 resolution using a Smart iTX–ATR diamond accessory. The infrared spectra were obtained from 200 coadded scans using OMNIC 9.2.41 software . All spectra in the 4000 cm−1 to 700 cm−1 region were baseline-corrected and unit vector normalized, and the mean spectra were calculated for three repetitions using OPUS 7.5 software . A comparison of the spectra with those from the OPUS library enabled us to identify the main components present in the resin.Liquid resin and solid extract of the 3D-printed sample (LNG+STR) were analyzed by mid-infrared spectroscopy with a Thermo Nicolet IS50 spectrometer . The spectra were collected in reflexion mode between 4000 cmMechanical testing was performed using a Zwick Roell universal machine equipped with a 10 kN load cell . A fixedThe microstructure of the resin/starch composites and rupture patterns were observed using a scanning electron microscope (SEM) in environmental mode (FEI Quanta 200). With this mode, high-resolution observations were made without the need for prior treatment and preparation of the sample surface. This was possible due to the presence of a low pressure inert gas and/or water vapor in the sample chamber, avoiding vacuum stresses. To do so, an accelerated voltage of 20 kV with different levels of magnifications was selected. Images were obtained with a resolution varying from 1280 × 1024 pixels, corresponding to a physical pixel size from 0.11 µm to 3.13 µm. Samples were prepared by gold/palladium coating prior to observation.® device”. The measurement principle for this device involved determining the quantity of adsorbed and desorbed water vapor by means of the acquisition of water vapor pressure and application of the ideal gas law. The measurements were performed at the equilibrium state for the samples and at a constant temperature of 23 °C. Prior to the test, samples with a height and diameter of 40 mm and 5 mm, respectively, were dried in a vacuum oven at 40 °C until their mass became stable.The water vapor sorption isotherms for the studied materials were obtained by a volumetric method using the “Belsorp Aqua 3The crystallinity analysis of the studied composites was carried out using the X-ray diffraction technique with INEL EQUINOX 600 X-ray equipment (France). The tested samples had the form of solid platelets. A copper (Cu) radiation source was used with a wavelength of 1.54060, voltage of 40 kV, and current intensity of 40 mA. The diffraction patterns were obtained after a 2 h exposure time and normalized with respect to the total scattering value. The 2θ range was selected between 5° and 50°. The thermal behavior of both starch powder and photosensitive composites was determined using differential scanning calorimetry (DSC). The thermoanalytical analysis was conducted using DSC3+ equipment from METTLER Toledo. Temperature scanning was carried out using the following temperature profile: the isotherm stage at 25 °C was maintained for 5 min, followed by a heating stage from 25 °C to 400 °C, with a heating rate of 10 °C/min under a controlled argon ambiance with a flow rate of 25 mL/min.® for a temperature range between −10 °C and 45 °C and a speed of 0.1 °C/min. The major advantage of this device lies in the fact that it uses 3D fluxmetric sensors that completely surround the sample, which allows for a more accurate result. The morphology of the starch powder was determined using a numerical optical microscope (Keyence) with a typical resolution of 2880 × 2160 pixels. Observations were performed under different magnification levels (from 20× to 400×) with a pixel size varying from 0.26 µm to 5.2 µm. The size distribution of the starch granules was assessed using laser granulometry equipment (CILAS 1090), for which the measurement size range was 0.04 µm to 500 µm. The measurement of the size distribution was conducted under dry mode. Four replicates from different batches were used, and excellent reproducibility was achieved. The heat capacity of the samples was measured using a Calorimeter CalvetThe main components present in the LNG resin are acrylic compounds, such as butyl-, ethyl- and methyl-acrylate in greater proportion.−1, the ethylenic stretching C=C vibration at 1640 cm−1, the CH2 deformation at 1460 cm−1, and the stretching C-O band at 1990 cm−1. The associated peaks at 982 and 810 cm−1 are assigned according to the in- and out-of-plane C-H bending.The infrared-assigned features characteristic of acrylic compounds are the ester C=O band at 1720 cmOn the other hand, comparing longer resin spectra with an epoxy resin spectrum b for the−1 and 1515 cm−1, the C-O and C-C stretching bands of the epoxy group at 1035 cm−1, and the epoxy deformation bands at 570 cm−1.Revealed several common peaks: the aromatic C=C stretching vibrations bands at approximately 1600 cm−1, C-O and C-C stretching bands at 1035 and 993 cm−1 and C-CH, and C-OH deformation bands at 935 cm−1. The presence of starch did not alter the assignment identified for the photosensitive resin in Observation of the infrared response of LSC0 containing starch c revealeIn fact, around a wavelength of 410 nm, the absorbance of starch was limited, as shown by Wuttisela et al. . The autPreliminary tests conducted to increase the amount of starch from 10% to 40% in the formulation showed that the maximum amount of starch is approximately 30%. Beyond this limit, two main problems occur: the first issue is related to the drastic increase in the blend viscosity, which does not allow the printing platform to perform the prescribed height increments of 50 µm. This issue is further amplified by the starch sedimentation that creates a gradient for the starch content. The second problem is related to the UV absorbance of starch, which is estimated at the operating wavelength (355 nm to 410 nm) to be approximately 19% of the absorbance peak, according to Wuttisela et al. . A highe4 W/m², the polymerization depth of the pure resin can be as large as 70 µm, according to the model used by Sun et al. [4 W/m². In the present study, with a spot size of 50 µm and an optical laser power of 10 W to 11 W, the estimated curing depth was 55 µm after 0.5 s of exposure. By combining an exposure time ranging from 7 s up to 22 s and a layer height of 50 µm, only minor alteration of the curing occurred, due to the presence of second phase material in the resin formulation as long as the starch load was below 40%. In fact, the main two phenomena altering the curing were incident beam absorbance by starch and photon scattering by either starch or hemp. The absorbance represented nearly one-fifth of the incident beam, and the lateral laser beam scattering resulted in the loss of 16% of the maximum depth for a load content of 30%, according to the model of Sun et al. [4 W/m². This alteration is materialized by the loss at curing depth. Indeed, the polymerization depth depends on the laser intensity and exposure time. For a laser intensity of 5 × 10n et al. . This den et al.  for an eThe thermal properties of all modified and genuine resins were compared according to the evolution of the specific heat capacity as a function of temperature b. The reThe addition of starch or hemp fiber led to the same overall brittle behavior for the cured resin, as evidenced by the optical micrographs recorded during the tensile testing c,d. It mExamination of the tensile response  showed tTo further explore the interpretation of the tensile results, the arrangement produced by the use of starch granules and natural fibers was studied. This study showed the potential of using a mixture of photosensitive resin with natural fillers, such as hemp fibers and starch granules, to decrease the cost of feedstock materials and improve the environmental footprint of technical parts processed by additive manufacturing. DLP technology does not require the melting of starch, which is a significant problem in other technologies, such as fused filament deposition. DLP has led to successful attempts to print samples with a starch filler composition of up to 30%. The 3D printing process operated at room temperature with the help of photopolymerization did not alter the thermal stability of the fillers. The resulting tensile performance was found to depend primarily on the post-curing process, where the transition from an elastic—plastic material into a quasi-brittle material was observed for all composites. This demonstrated that the filler amounts considered in this study did not alter the photopolymerization process, irrespective of their shape and size. Only minor differences were observed between hemp-based and starch-based composites, which are explained by the relative homogeneity of starch-based composites. The most promising formulation, LSC0, resulted in a slightly improved stiffness compared with the reference photosensitive resin and the modulation of heat capacity with respect to the operating temperature.


Mycobacterium ulcerans (M. ulcerans). There are no known vectors or transmission methods, preventing the development of control methods. There are effective diagnostic techniques and treatment routines; however, several socioeconomic factors may limit patients’ abilities to receive these treatments. The Bacillus Calmette–Guérin vaccine developed against tuberculosis has shown limited efficacy, and no conventionally designed vaccines have passed clinical trials. This study aimed to generate a multi-epitope vaccine against M. ulcerans from the major facilitator superfamily transporter protein using an immunoinformatics approach. Twelve M. ulcerans genome assemblies were analyzed, resulting in the identification of 11 CD8+ and 7 CD4+ T-cell epitopes and 2 B-cell epitopes. These conserved epitopes were computationally predicted to be antigenic, immunogenic, non-allergenic, and non-toxic. The CD4+ T-cell epitopes were capable of inducing interferon-gamma and interleukin-4. They successfully bound to their respective human leukocyte antigens alleles in in silico docking studies. The expected global population coverage of the T-cell epitopes and their restricted human leukocyte antigens alleles was 99.90%. The population coverage of endemic regions ranged from 99.99% (Papua New Guinea) to 21.81% (Liberia). Two vaccine constructs were generated using the Toll-like receptors 2 and 4 agonists, LprG and RpfE, respectively. Both constructs were antigenic, non-allergenic, non-toxic, thermostable, basic, and hydrophilic. The DNA sequences of the vaccine constructs underwent optimization and were successfully in-silico cloned with the pET-28a(+) plasmid. The vaccine constructs were successfully docked to their respective toll-like receptors. Molecular dynamics simulations were carried out to analyze the binding interactions within the complex. The generated binding energies indicate the stability of both complexes. The constructs generated in this study display severable favorable properties, with construct one displaying a greater range of favorable properties. However, further analysis and laboratory validation are required.Buruli ulcer is a neglected tropical disease that is characterized by non-fatal lesion development. The causative agent is  Mycobacterium ulcerans, which is categorized as a mycolactone-producing-mycobacterium (MPM) (Buruli ulcer (BU) is a chronic, necrotizing disease that primarily affects the skin and occasionally bones . It is cum (MPM) . It is cum (MPM) , 4. It hum (MPM) . There aum (MPM) . The annum (MPM) . There wum (MPM) . The Worum (MPM) , 7. Thisum (MPM) . It is ium (MPM) . Experieum (MPM) .M. ulcerans, which hinders the development of prevention methods  vaccine was created against tuberculosis using live attenuated M. bovis . BCG wasbacteria . Howeverbacteria , 16. Vacvaccines . None ofvaccines . The advOne of these approaches is reverse vaccinology (RV). RV has been steadily evolving since the development of sequencing technologies . This meH cells)  . CTLs, a4+ cells . CD4+ ceesponses . B-cellsesponses . The ideantigens .M. ulcerans proteins  (https://www.ncbi.nlm.nih.gov/) in the protein format (http://metagenomics.iiserb.ac.in/mp3/index.php), with the threshold set to 0.5  for multiple sequence alignment  for topology analysis , with the peptide length set to 9  were filtered based on the allele frequency, in order of highest to lowest, and the population standard was set to gold only  for antigenicity analysis (http://tools.iedb.org/immunogenicity/)  . This wenicity/) . Peptidehttps://services.healthtech.dtu.dk/service.php?NetMHCIIpan-4.0), with the peptide length set to 15   for antigenicity analysis (The probable outer membrane proteins were submitted to NetMHCIIpan v 4.0 (et to 15 . NetMHCIet to 15 . The firult.asp) . Only poanalysis . The tar+ epitopes were submitted to the IFNepitope website (http://crdd.osdd.net/raghava/ifnepitope/predict.php) (https://webs.iiitd.edu.in/raghava/il4pred/predict.php) to predict epitopes that may be capable of inducing interleukin-4 (IL-4) (+ and IL-4 inducing CD4+ epitopes were submitted to the AllerTOP v. 2.0 webserver (https://www.ddg-pharmfac.net/AllerTOP/) for allergenicity analysis (https://webs.iiitd.edu.in/raghava/toxinpred/index.html)  (+ epitopes with 100% linear conservancy were selected and used to identify overlapping CD4+ epitopes.The antigenic CD4ict.php) . Epitope4 (IL-4) . The SVManalysis . AllerToanalysis , 49. Epiex.html) , 51. Nonrvancy/) . The Epirvancy/) . It defirvancy/) . CD8+ ep+ and 7 CD4+ epitopes were submitted to the Population Coverage website (http://tools.iedb.org/population/)  . The Poplation/) . This islation/) . The resepitopes . The folhttps://webs.iiitd.edu.in/raghava/abcpred/index.html) with default parameters (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html)  (+ epitopes with 100% conservancy were selected and used to identify overlapping B-cell epitopes. The conserved B-cell epitopes were submitted to the AllerTOP webserver (https://www.ddg-pharmfac.net/AllerTOP/) for allergenicity analysis and ToxinPred (https://webs.iiitd.edu.in/raghava/toxinpred/index.html) for toxicity analysis (http://tools.iedb.org/immunomebrowser/) to determine if the identified epitopes have been examined in immune assay studies, and if so, the results of the assays  . Bacterirvancy/) . CD8+ epanalysis , 50, 51.e assays . The par+ epitopes with an existing crystalline structure was HLA-A*02:06. The most common HLA allele for the virulent CD4+ epitopes was HLA-DRB1*01:01. The crystalline structures 3OXR was retrieved for HLA-A*02:06 (https://www.rcsb.org/) (http://www.attract.ph.tum.de/services/ATTRACT/peptide.html) for docking analysis. The docking was completed on the locally installed ATTRACT on Centre for High-Performance Computing (CHPC) South Africa. Following docking, 50 frames were generated for each docking interaction. The best frame for each docking model was determined based on the lowest energy value. Visual Molecular Dynamics (VMD) software v 1.9.3 was used to visualize the best model for each epitope, and UCSF Chimera v.1.14 was used to produce images of the epitope structures  , 63. Thesb.org/) , and chasb.org/) . The allructures , 66.+ and 7 CD4+ T-cell epitopes and 2 B-cell epitopes that were antigenic, immunogenic, non-allergenic and non-toxic were assembled to form two candidate sequences. The TLR2 and -4 agonists Lipoprotein LprG and the resuscitation-promoting factor (RpfE), respectively, were selected as adjuvants to boost the potential resulting immune response (https://www.ncbi.nlm.nih.gov/). The LprG adjuvant was added to construct one, and the RpfE adjuvant was added to construct two. The EAAAK linker was used to connect the respective adjuvants at the N-terminal of the vaccine constructs, the AAY linker between the CD8+ epitopes, the GPGPG linker between the CD4+ epitopes and the KK linker between the B-cell epitopes. The CD8+ and CD4+ epitopes were arranged in decreasing order of antigenicity values. The two models were submitted to AllerTOP v. 2.0 (https://www.ddg-pharmfac.net/AllerTOP/) for allergenicity analysis (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html), with a threshold of 0.5  to predict the secondary structures (https://yanglab.nankai.edu.cn/trRosetta/), and 3D models were generated  (https://prosa.services.came.sbg.ac.at/prosa.php) was used to validate the refined models by calculating the Z-scores (https://saves.mbi.ucla.edu/) for analysis and further validation using ERRAT and PROCHECK  , 76. ModZ-scores , 78. ThePROCHECK –81.in vitro (https://web.expasy.org/protparam/) (Escherichia coli (http://babylone.ulb.ac.be/SCooP/index.php) to determine various thermodynamic properties of the structures, such as the melting temperature (Tm), change in enthalpy (Δ Hm), change in specific heat upon folding (Δ Cp), and the folding free energy at room temperature (Δ Gr)  . This inhia coli . The mode (Δ Gr) –86.http://www.jcat.de/) for codon adaptation and vaccine optimization (Escherichia coli (strain K12). The XhoI (5’CTCGAG’3) and HindIII (5’AAGCTT’3) restriction sites were added to the N- and C-terminals of the first DNA optimized sequence, respectively. The HindIII (5’AAGCTT’3) and BamHI (5’GGATCC’3) restriction sites were added to the N- and C-terminals of the second DNA optimized sequence, respectively. The sequences were each inserted into the pET-28a(+) plasmid using SnapGene v.6.0.2 software .The models were submitted to the JAVA Codon Adaption Tool (JCat) (mization . Codon amization . JCat dehttps://www.rcsb.org/), respectively  for the analysis of the flexibility of the structures (https://www.ebi.ac.uk/thornton-srv/databases/profunc/) to analyze the binding interactions between the TLRs and MEV complexes (http://212.87.3.12/A3D2/)  , 98.https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php?page=1) to observe the simulation of the potential immune response to the designed constructs  simulation. The complexes were equilibrated at 300 K for an additional 2 ns at a pressure of 1 bar. MDS production was run for 100 ns at NVT. The simulations were run using the GPU (CUDA) version of PMEMD provided in AMBER 18  using the AMBER 14 and 18 packages , 102. Ths of 8 Å . InitialAMBER 18 –108.The CPPTRAJ and PTRAJ modules in AMBER 18 were used to carry out post-MDS analyses , 109. ThThe CPPTRAJ and PTRAJ modules were used to generate 2000 frames of the complexes and the unbound MEVs . VMD v 1A total of 9906 potentially virulent proteins were identified. There were 9 MFS transporter proteins identified as virulent. After alignment, 97 peptides of various lengths were identified. There were 49 peptides identified to have an outer topology.+ and CD4+ T-cell epitopes capable of binding to MHC I and II yielded 178 CD8+ and 245 CD4+ T-cell epitopes. Following antigenicity analysis, 80 CD8+ and 89 CD4+ antigens were identified. A total of 62 immunogenic CD8+ T-cell epitopes were identified and extracted. CD4+ epitopes capable of inducing IFN-γ and IL-4 amounted to 30 and 10, respectively. A total of 47 CD8+ and 7 CD4+ non-allergenic and non-toxic epitopes were detected. Upon conservancy analysis, 11 conserved CD8+ epitopes and 7 overlapping CD4+ epitopes were identified. The CD8+ epitope GVDGRLPLL had the highest antigenicity score, i.e., 1.80, while the epitope with the lowest antigenicity was YAQRAAHRL, with a score of 0.64  to 246.48nM (YAQRAAHRL)  had an ABCpred score of 0.56 and an antigenicity score of 0.78, while the second epitope (VGVNPWAITLAVSLAV) had an ABCpred score of 0.60 and antigenicity score of 1.45.A total of 34 B-cell epitopes were initially identified. Upon antigenicity analysis, 14 antigenic B-cell epitopes were detected. There were 11 epitopes that were determined to be non-allergenic and non-toxic. Once overlapped with the 11 CD8+ epitopes interacting with HLA-A*02:06 ranged from -132.37 to -93.27 kCal/mol , less than 20 hours  and less than 10 hours . Based on the generated Gibbs-Helmholtz curve for the first construct, the Δ Hm, Δ Cp, Tm and Δ Gr values were 23.60 kCal/mol, 0.72 kcal/(mol K), 61.30° C, and 1.1 kCal/mol, respectively  value was estimated to be 10.30, and the instability index was 27.10. The aliphatic index and the GRAVY value of the first construct were determined to be 89.30 and -0.06, respectively. This indicates that the first construct is basic, stable, thermostable and hydrophilic. The extinction coefficient was estimated to be 118830-1 cm-1 at 280 nm measured in water, assuming all cysteine residues are reduced and 119080-1 cm-1 at 280 nm measured in water, assuming all pairs of cysteine residues form cystines. Vaccine construct two consisted of 7659 atoms with a molecular weight of 54.51 kDa. The second vaccine construct contained 36 negatively charged residues and 48 positively charged residues. The pI value, instability index, aliphatic index and GRAVY value for the second construct were 9.58, 42.05, 78.45 and -0.13, respectively. The second construct is also basic and hydrophilic; however, it was unstable and less thermostable than the first construct. The extinction coefficient was estimated to be 124330M-1 cm-1 at 280 nm measured in water, assuming all cysteine residues are reduced and 124580-1 cm-1 at 280 nm measured in water, assuming all pairs of cysteine residues form cystines. The N-terminal amino acid was considered to be methionine for both constructs. The half-life of both vaccine constructs was approximately 30 hours  plasmid amounted to 6998 bp, while the second vaccine construct and the plasmid amounted to 6922 bp   , malarias (CHPV) , cutaneos (CHPV) , tubercus (CHPV)  and SARSs (CHPV) . Two in-t for BU , 34. Botulcerans , 34. Naihttps://tcdb.org) classified 16 families and 89 subfamilies within the MFS (The source of the T-cell and B-cell epitopes are MFS transporter proteins. The MFS is the largest and most diverse superfamily of transmembrane secondary carriers . They se the MFS . Only se the MFS . However the MFS . The epi+ epitopes and the cytokine-inducing capabilities for the CD4+ epitopes is promising. The TH type 1 (TH1) response is suggested to be critical in eradicating M. ulcerans infection  and CC chemokine ligand (CCL) 2 was found to be dependent on mainly TLR2 and, to an extent, TLR4 in primary human keratinocytes  with the following accession numbers: GCA_022374915.1 - in-silico work on the vaccine with assistance from VA and LM. TI wrote the manuscript while LM, VA, AA, MA and MO edited the manuscript. MA and MO supervised the work. All authors contributed to the article and approved the submitted version.TI, MA, and AA conceived the idea. TI conducted the This work is based on the research supported wholly by the National Research Foundation of South Africa (Grant Number 141385), as a bursary award for the first author.The authors wish to acknowledge the Centre for High-Performance Computing, South Africa, for the computer programs and facilities used for this project.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.


At present, there are hardly any studies about the effect of inulin (IN) on the physicochemical properties and structures of different crystalline starches. In this study, three different crystalline starches  were compounded with natural IN, and its pasting, retrogradation, and structural characteristics were investigated. Then, the potential mechanism of interaction between IN and starch was studied. The results showed that there were some differences in the effects of IN on the three different crystalline starch. Pasting experiments showed that the addition of IN not only increased pasting viscosity but also decreased the values of setback and breakdown. For wheat starch and pea starch, IN reduced their peak viscosity from 2,515 cP, 3,035 cP to 2,131 cP and 2,793 cP, respectively. Retrogradation experiment dates demonstrated that IN delayed gelatinization of all three starches. IN could reduce the enthalpy of gelatinization and retrogradation to varying degrees and inhibit the retrogradation of starch. Among them, it had a better inhibitory effect on potato starch. The addition of IN reduced the retrogradation rate of potato starch from 38.45 to 30.14%. Fourier-transform infrared spectroscopy and interaction force experiments results showed that IN interacted with amylose through hydrogen bonding and observed the presence of electrostatic force in the complexed system. Based on the above, experimental results speculate that the mechanism of interaction between IN and three crystalline starches was the same, and the difference in physicochemical properties was mainly related to the ratio of amylose to amylopectin in different crystalline starches. These findings could enrich the theoretical system of the IN with starch compound system and provide a solid theoretical basis for further applications. Starch was a macromolecular carbohydrate synthesized by natural plants through photosynthesis, which played an indispensable role in the human diet and industrial applications. Currently, starch is mainly obtained from high-starch-based crops, such as wheat, corn, and potatoes. Natural plant starches are classified into A, B, and C starches according to their X-ray diffraction (XRD) patterns . Most ceHowever, natural starches have great limitations in food processing due to their thermal instability, pH sensitivity, and retrogradation phenomenon . TherefoAt present, IN has become a research hotspot in the food field. Luo et al.  found th® HSI, Belgium).The WS, PoS, and PeS were purchased from Henan Enmiao Food Co., Ltd. . Amylose contents of WS, PoS, and PeS were 31.64%, 24.02%, and 48.82%, respectively, which were obtained according to the method by Doblado-Maldonado, Gomand, Goderwas, and Delcour . The IN Inulin was mixed with starch in the ratio of 1:1 (g/g). Then, 2.5-g IN was dissolved in 25-ml distilled water and stirred for 30 min. Then, the same weight of starch (dry base) was added and stirred again for 30 min to obtain IN-starch suspension.The pasting properties of the IN-starch compound systems were measured by a Rapid Visco-Analyzer . The prepared IN-starch suspension was poured into a special aluminum for RVA and measured by the procedure of Liu et al. . First, 0), peak temperature (TP), final temperature (TC), and gelatinization enthalpy (ΔH) of the samples were recorded.The differential scanning calorimeter  was used to determine the thermal and retrogradation properties of the samples. Approximately 2.5 mg of starch was weighed into an aluminum crucible, and then, 5 μl of IN solution  was added, sealed, and equilibrated for 12 h at room temperature. The sample was heated by a program that went from 20 to 120°C at a rate of 10°C/min, with a hermetically sealed empty aluminum pan as a reference. The initial temperature (T0r), peak temperature (TPr), final temperature (TCr) and enthalpy of retrogradation (ΔHr) of the samples were recorded.The pasted sample trays were stored at 4°C for 7 days. Then, the same procedure was followed to determine the retrogradation parameters. The initial temperature . The testing conditions were a scanning area of 5-35°; a scanning speed of 4°/min; and a sampling step width of 0.02°.−1; the resolution was 4 cm−1.The molecular structure of IN-starch compound systems was analyzed by an FT-IR Spectrometer  according to Shi et al. . The samThe interaction force between IN and starch was determined according to the method of Ren et al.  with sliIn vitro starch digestion was analyzed according to the method described by Yan et al. . FG was the amount of glucose hydrolyzed without enzyme addition, FG = 0.where Gp < 0.05) with IBM SPSS Statistics 26.0 Software Program . The results were expressed as the mean values ± standard deviations. For all figures, Origin Pro software  was used.All the experiments were conducted in triplicate. An analysis of variance (ANOVA) was performed by Duncan's test , followed by PeS (10.90 J/g), and then WS (10.07 J/g), reflecting a higher degree of crystallinity of amylopectin in PoS. The addition of IN decreased the ΔHg values of WS, PoS, and PeS. The decrease in ΔHg may be attributed to several reasons. On the one hand, IN competed with starch for the water needed for gelatinization, resulting in incomplete starch gelatinization and ΔHg decrease. On the other hand, IN was a small molecule weight polysaccharide that could easily insert into starch molecules upon heating, leading to weakened stability of part of the crystalline region, thus causing a decrease in ΔHg , followed by PeS (4.41 J/g) and WS (2.48 J/g). This difference was related to the ratio of long-chain branches to short-chain branches of amylopectin among different starches  was the ratio of ΔHch chain . Besidesch chain .The gelatinized WS showed diffraction peaks at 17° and 20°. The IN-WS compound system had not changed the position of the peaks, while it enhanced the diffraction intensity at 17°. The diffraction peaks of the gelatinized PoS appeared at 17° and 22°, and the addition of IN enhanced the diffraction intensity at 17° and 22°. Similar to gelatinized PoS, the same changes were observed at 17° and 22°. The peak shown near 17° for starch was characteristic of amylose . The add−1 was ascribed to stretching vibration absorption of associated hydroxyl groups between polymers, which may be intramolecular hydrogen bonds within the same molecule or intermolecular hydrogen bonds between adjacent molecules , the Natural Science Foundation of Henan Province (212300410297), the Science and Technology Basic Research Program of Henan Province (202102110302), and the Doctoral Research Foundation of Zhengzhou University of Light Industry (2020BSJJ015).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.


Acinetobacter species are gram-negative coccobacilli ubiquitous in nature and widely distributed in the environment. Acinetobacter baumannii is a bacteria commonly seen in the hospital setting, responsible for causing a wide range of bloodstream infections, urinary tract infections, secondary meningitis, infective endocarditis, and wound infections, and is the cause of outbreaks mainly due to its antimicrobial resistance patterns. The use of broad-spectrum antibiotic coverage with carbapenems is essential in the hospital setting. Therefore, carbapenem-resistant Acinetobacter baumannii (CRAB) poses as a very challenging pathogen. Acinetobacter radioresistens, a rare species in comparison to the more prevalent Acinetobacter baumannii, is an underestimated agent in causing nosocomial infections and also is a potential disseminator of resistance genes. It is also resistant to gamma radiation at 4-8 times higher than other Acinetobacter spp. and is the source of the class D OXA-23 carbapenemase that can confer carbapenem resistance. Therefore, immediate and precise identification of A. radioresistens is crucial for the clinical management of multidrug-resistant bacteremia. Acinetobacter baumannii due to its increasing prevalence as a hospital-acquired infection that is multi-resistant to drug treatment [Although this strain was taxonomically described in 1986 after being first isolated from soil and cotton, research has primarily focused on reatment ,3.Acinetobacter radioresistens. This lesser-discussed member of the Acinetobacter family found on the human skin is considered to be a regular part of the human flora; however, rarely is it discovered to be the cause of bacteremia or pneumonia in patients [A. radioresistens bacteremia in an immunocompetent host, identified through the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF).As of 2020, a literature search revealed only seven case reports describing eight patients across the world infected with patients . Here, w2) of 94% on 2 L/minute nasal cannula oxygen. On physical examination, the patient had bilaterally decreased breath sounds and an irregular rhythm at 120 beats per minute (bpm).An 83-year-old Caucasian male was brought into an emergency department of a community hospital in Chicago, IL, with suspected community-acquired pneumonia (CAP), following a rapid onset fever and altered mental status. Later in the day, the patient had one episode of emesis and aspirated on his phlegm. The patient had no recent hospitalizations and had been vaccinated for COVID-19. The patient’s past medical history was significant for congestive heart failure (CHF) with preserved left ventricular ejection fraction, atrial fibrillation on Eliquis, and coronary artery disease (CAD) status post coronary artery bypass graft (CABG) in 2016. On presentation, the patient was febrile at 39.0°C, hypotensive at 91/49 mmHg, and tachycardic in the 110s, with an oxygen saturation  testing for nucleic acid amplification test (NAAT) of SARS-CoV-2 (COVID-19) yielded negative results. All four vials showed the presence of a gram-negative bacillus-shaped organism later identified as Acinetobacter radioresistens. Further analysis revealed pan-susceptibility to carbapenems, cephalosporins, aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Urinalysis and urine cultures yielded no significant results, and the patient was started on empiric treatment with IV ceftriaxone 2 g and IV azithromycin 500 mg daily and was admitted for further management. Surveillance blood cultures were negative. Following the resolution of infiltrates seen on day two of admission  with pan-susceptibility [The first case reported an immunocompromised patient with human immunodeficiency virus (HIV) with the combination of neutropenia who survived, while seven isolated cases of immunocompetent individuals, of which four had community-acquired pneumonia, showed resolution on antibiotic therapy ,7. A simtibility .A. radioresistens was a cause of bacteremia and CAP identified through the use of MALDI-TOF mass spectrometry , which demonstrated pan-susceptibility to all antibiotics tested . Using MALDI-TOF, this strain of the most fastidious growing bacteria has been able to be quickly and accurately identified through mass spectrometry [In our case, trometry . Unique trometry . In additrometry . blaOXA-23 gene and were possibly correlated with favorable course of the disease. Although Acinetobacterradioresistens has been isolated on the human skin, the bacteria had successfully managed to colonize within the patients’ blood. Its lack of sterilization by gamma radiation and UV irradiation makes it highly survivable within the hospital environment and can potentiate infection. The cause of bacteremia still remains uncertain among the reported cases of CAP in 2019 throughout the world; however, we can place emphasis on the importance of familiarizing providers to A. radioresistens and its expression of resistance genes.The isolate from our patient was susceptible to all β-lactams, including carbapenems, which suggest a weak expression of theAcinetobacter radioresistens is the progenitor of the blaOXA-23 gene, a class D carbapenemase that causes resistance in A. baumannii. Our isolate was susceptible to all β-lactams, including carbapenems, which suggest a weak expression of the blaOXA-23 gene; however, it must not be ruled out and requires precise identification of this organism such as MALDI-TOF MS. According to the World Health Organization, the Acinetobacter species poses to be one of the greatest threats due to the emerging threat of resistance due to the blaOXA-23 oxacillinase gene constitutionally expressed at low levels; therefore, prompt identification and treatment are necessary. In conclusion, with data available on only a handful of studies, our study suggests that Acinetobacter radioresistens remains a highly competent organism that does not seem to be restricted to the hospital setting and that at any time can develop the challenging issue of pan-drug resistance. Therefore, the increasing emergence of this bacterium in the community setting is possible and warrants further studies focusing on the prevalence of this pathogen. A broad approach to antimicrobial drug therapy and the surveillance of resistance genes is imperative to treatment.