Dazil and Greenstar were unique for their high resistant starch content (RS) and lower area under the starch hydrolysis curve (SHAUC) while Proclaim was opposite. SHAUC was positively correlated (p < 0.001) with rapidly digestible starch (RDS) content (r = 0.626) but negatively correlated with RS content (r = −0.635). Principal component analysis showed that the first three principal components accounted for 62.8% of the total variance and the contribution of SHAUC was 33.2%. These results confirm that in vitro SHAUC and a combination of RDS and RS may be predictive of the digestibility profile of cooked lentils.Proximate composition and starch nutritional properties of twenty cooked lentils were assessed to identify unique varieties that could be used in value added foods. Significant variations exist among the lentil varieties ( Lens culinaris) are nutrient-dense foods that are included in dietary recommendations and several clinical practice guidelines [Pulses such as lentils , slowly digestible starch (SDS), and resistant starch (RS) . RDS is Foods with low GI typically contain high levels of SDS and RS and have been recommended for the treatment and prevention of diabetes, heart disease, and related conditions ; as suchThis study was undertaken to define the chemical composition, dietary fiber fractions and starch nutritional properties of current and emerging Canadian lentil varieties, and to identify unique lentil varieties and key factors affecting starch digestion in cooked lentils. Furthermore, dietary fiber fractions including galacto-oligosaccharides (GOS) were also investigated to advance the understanding of their influence on starch digestion. Finally, principal component analysis and cluster analysis were used to assess independent variables that contribute to lentil starch digestion.® Infinity Ultrapure Water System .Twenty lentil varieties of red and green types and with varying seed sizes were obtained from the Saskatoon Crop Development Centre (CDC) in March 2014, and all the analyses were completed in 2015. The names and market classifications of each variety are listed in ® Works, Inc., Wilmington, NC, USA) to pass through a 250 µm sieve. The ground samples were stored in sealed plastic bags at room temperature prior to analysis.Whole lentil seeds (150 g) were rinsed and cooked in a rice cooker  with distilled water (1:3 seed:water ratio) as previously outlined . The cooFreshly cooked lentils were sent to a commercial service provider  for proximate analysis, where standard AOAC  methods for total fat (AOAC 922.06), ash (AOAC 923.03), energy  and protein (AOAC 992.15) were used. Total dietary fiber was analyzed in house (AOAC 991.43) using a commercial kit  with modifications to allow for the analysis of GOS. The filtrate for soluble dietary fiber (SDF) analysis was reduced in volume using a rotary evaporator (Büchi Rotavapor R-215) and diluted with deionized water in order to determine the content of nondigestible GOS by HPLC with an anion-exchange column and a pulsed amperometric detection unit (HPAEC-PAD)  . Raffinog for 12 min. The supernatant (2.7 mL) was transferred to a beaker, and amyloglucosidase (0.3 mL) and invertase (0.2 mL) were added to the solution. This enzyme solution was freshly prepared. For each digestion, 100 mg of cooked lentil sample . NonRS was starch digested within 16 h and RS was starch not hydrolyzed even after 16 h. To differentiate between the RS obtained from Englyst et al.’s method and Megazyme kit, the term “RS-direct” was used for the RS obtained by the direct assay kit.The method of Englyst et al.  with minmple see  above wa−1) was calculated from the integrals of equation t0) to 120 min (ft), where C is the percentage of starch hydrolyzed at time t (min), C∞ is the equilibrium percentage of starch hydrolyzed after 120 min, and k is the kinetic constant. The hydrolysis index (HI) was calculated as the ratio of the area under the hydrolysis curve (0–120 min) of lentil sample and the area of standard material from white bread. The expected glycemic index (eGI) was calculated using equation (eGI = 8.198 + 0.862 × HI) proposed by Granfeldt et al. [The digestion kinetics of the lentil samples were tracked by a nonlinear model established by Goni et al. . The firt et al. .t-test and one-way analysis of variance (ANOVA)) were performed using SAS . When appropriate, the difference amongst means was determined using Tukey’s post hoc test, except that Dunnett’s method was used for multiple comparison of kinetic parameters and eGI of cooked lentils to those of white bread control. Pearson correlation coefficients and principal component analysis (PCA) were performed on centered and standardized data to elucidate the relationships among variables of the chemical and nutritional properties of samples. Cluster analysis (CA) was conducted using furthest neighbor linkage method and with Euclidean distance being the measure of similarity. Dendrogram and K-means plot were drawn to visualize the clusters of lentil samples. Both PCA and CA were conducted by OriginLab 9 . Statistical significance was set at the 5% level of probability.Samples were tested at least in triplicate. Statistical analyses  and lower carbohydrate and total starch content (p < 0.05) than green-type lentils, regardless of seed size. At the same seed size, fat content in red type (1.2%) was significantly higher than that of green type (0.9%) (p < 0.05). Within red-type lentils, the extra-small seeds showed higher ash and protein content (p < 0.001) but lower fat (p < 0.05) and lower carbohydrate (p < 0.01) content than the small seed. This observation was also true for green-type lentils, i.e., small seeds also had higher protein content and lower carbohydrate content (p < 0.05)  . The varp < 0.05). At the same seed size, verbascose in red type (1.1%) was significantly higher than that of green type (0.9%) (p < 0.05). Within red-type lentils, extra-small seeds showed higher SDF and raffinose content but lower verbascose content than small seeds (p < 0.01). Within green-type lentils, small and medium size seeds had significantly lower verbascose content than large size seed (p < 0.05). Interestingly, verbascose was higher in larger seed-size lentils regardless of lentil type  and attributed the difference to higher total flavonol index of red lentils (452.5 μg g−1 db) than that of green lentils (449.9 μg g−1 db). Another study showed that there were exceptions with two green lentils that contained high levels of phenolic compounds [As shown in ompounds . The incp < 0.05, RS content obtained by Megazyme kit (RS-direct) supports the previous observations  and alsoStarch hydrolysis curves were plotted as time vs. percent digestible starch (derived from glucose measurements) taken at different sampling times  during in vitro digestion . The aveC∞, k), SHAUC, HI and eGI for each lentil sample and for the reference food (white bread) are presented in C∞; 0.14 min−1 (Redberry) to 0.33 min−1  for k; 9527 (Redberry) to 10,516 (Proclaim) for SHAUC (mg min mL−1); 84% (Redberry) to 92.7% (Proclaim) for HI; and 81  to 88 (Proclaim) for eGI. The eGI of cooked lentils obtained in this study is slightly higher than the GI of 74 for canned lentil listed in The International Table of Glycemic Index and Glycemic Load Values [The kinetic parameters . Because of collinearity among eGI, HI, and SHAUC, and the fact that SHAUC is a primary outcome of starch digestibility, SHAUC, instead of eGI, was used in multivariate analysis that explored key factors affecting lentil starch digestibility, and in identifying unique lentil varieties.No significant differences were identified in the starch hydrolysis variables among lentil types or seed size; however, these were all significantly different from white bread control (Dunnett p = 0.023) and protein (p = 0.007) and was positively correlated with carbohydrate (p = 0.013) and stachyose (p = 0.025). SDS was negatively correlated with stachyose (p = 0.031). As expected, derived RS significantly correlated with these nutrients, but in an opposite direction to RDS. Of the results obtained using the Megazyme kit, only carbohydrate showed a significant (p < 0.05) correlation with nonRS and RS-direct. In contrast, SHAUC was significantly correlated with more measures of chemical composition and with most dietary fiber components, except SDF and verbascose. SHAUC was not correlated with carbohydrate content or total starch. A recent study with lentils also found that none of the individual starch fractions was significantly associated with total starch and total dietary fiber content [Pearson correlation coefficients between chemical components and nutritional properties of the cooked lentils are presented in  content . As sucheGI, be the primary index used to assess the overall digestion profile of cooked lentils.Results from the Englyst method  showed gPCA provides an overview of the similarities and differences between the measured properties and was used in this study to visualize the variations in chemical components and nutritional properties of the cooked lentils. The results showed that the proportions of eigenvalues of the correlation matrix for the first three principal components were 28.5%, 22.0%, and 12.3%. Two components accounted for 50.5% of the total variance and three components explained 62.8%, indicating that three components could provide a good summary of the data. A biplot in which the points represent lentil varieties and the vectors represent chemical components and nutritional parameters of cooked lentils is presented in C∞, were highly correlated and also highly negatively correlated to RS, suggesting that RDS and RS determined by the indirect method [The directions of the vectors indicate that PC1 explains starch digestion fractions determined by the Englyst method, dietary fibers, and SHAUC; PC2 distinguishes chemical compositions  A; and PCt method  are goodt method .Lentil varieties that are clustered together in the biplot are those that have similar profiles of chemical and nutritional properties. Green lentils are mostly located in the middle to left panel of PC1, unique in lower dietary fiber content, while red lentils are located in the middle to upper panel of PC2 (except Proclaim), showing lower carbohydrate and total starch A. WithinIn other studies, Asterix has been reported to be of low GI . AsterixPearson correlation and PCA were informative and showed that RDS, RS, and SHAUC are reliable indicators to differentiate the chemical compositions and nutritional properties of these twenty lentils. However, the biplot did not show clear separations of lentil varieties corresponding to starch digestibility and/or SHAUC. Therefore, cluster analysis was conducted to group these twenty lentils, specifically focusing on RS and SHAUC.K-means plot corresponding to the RS content and SHAUC values of these twenty cooked lentils are shown in A dendrogram from hierarchical cluster analysis and −1), high RS (10–17%); includes Dazil, Redberry, Imvincible, Greenstar, Cherie, Roxy, and Viceroy;Group 1, low SHAUC (<9825 mg min mL−1), intermediate RS (9–13%); includes Greenland, Kermit, Impala, Impress, Imax, Improve, Imigreen, and Impower;Group 2, intermediate SHAUC , intermediate RS (9–13%); includes Rosie, Asterix, Scarlet, and Impulse; unique in overall higher dietary fiber content;Group 3, high SHAUC  and low RS (<9%); the variety is Proclaim.Group 4, the highest SHAUC . Regardless of lentil type, smaller seed size seems to be associated with higher RS content in cooked lentils. Dietary fiber fractions appeared to have little correlation with starch digestible fractions but were highly correlated with SHAUC. Based on SHAUC and RS content, twenty lentil varieties could be classified into four groups by PCA and CA. The consistency between PCA and CA indicated that multivariate analyses were feasible in the study of lentil characteristics and useful for selecting the appropriate variety for final use suitability.The proximate composition, dietary fiber fractions, and starch nutritional properties were significantly different among the twenty cooked lentil varieties. The differences were more variety- than type-dependent. Generally, cooked red lentils tend to have lower GI values than green lentils. However, Proclaim was unique for its lowest RS while having the highest


Tau forms intracellular insoluble aggregates as a neuropathological hallmark of Alzheimer’s disease. Tau is largely unstructured, which complicates the characterization of the tau aggregation process. Recent studies have demonstrated that tau samples two distinct conformational ensembles, each of which contains the soluble and aggregation-prone states of tau. A shift to populate the aggregation-prone ensemble may promote tau fibrillization. However, the mechanism of this ensemble transition remains elusive. In this study, we explored the conformational dynamics of a tau fragment by using paramagnetic relaxation enhancement (PRE) and interference (PRI) NMR experiments. The PRE correlation map showed that tau is composed of segments consisting of residues in correlated motions. Intriguingly, residues forming the β-structures in the heparin-induced tau filament coincide with residues in these segments, suggesting that each segment behaves as a structural unit in fibrillization. PRI data demonstrated that the P301L mutation exclusively alters the transiently formed tau structures by changing the short- and long-range correlated motions among residues. The transient conformations of P301L tau expose the amyloid motif PHF6 to promote tau self-aggregation. We propose the correlated motions among residues within tau determine the population sizes of the conformational ensembles, and perturbing the correlated motions populates the aggregation-prone form. Neurofibrillary inclusions are the hallmark of neurogenerative diseases such as Alzheimer’s disease (AD) as the most common of the tauopathies ,2. TheseThe longest isoform of human tau is 441 residues in length  Figure A. Human 275VQIINK280) and PHF6 (306VQIVYK311) /7, provided a stronger indication of the secondary structure propensities , 1HN-Γ2[X(2)] and 1HN-Γ2[X(1)+X(2)] denote the 1HN-Γ2 rates caused by the single spin-label at residue X(1), at residue X(2), and the simultaneous spin-labels at residues X(1) and X(2). PRI as the cross-correlated paramagnetic relaxation effect on each fined as ,44:(2)ΔHThe PRI correlation map was generated by the same method used to prepare the PRE correlation maps, Equation (1) ,43,44,45In summary, we have revealed that TauF4Δ comprises segments of residues that undergo correlated motions, and the segments constitute the elementary structural units in the heparin-induced tau fibril. The inter-segment correlated motions probably determine the conformational ensembles of TauF4Δ. Tau is a representative member of IDPs. However, tau is not a polymer physics random coil . Instead


Trypanosoma. T. congolense and T. brucei brucei are predominant in Africa; T. evansi and T. vivax in America and Asia. They have in common an extracellular lifestyle and livestock tropism, which provokes huge economic losses in regions where vectors are endemic. There are licensed drugs to treat the infections, but adherence to treatment is poor and appearance of resistances common. Therefore, the availability of a prophylactic vaccine would represent a major breakthrough towards the management and control of the disease. Selection of the most appropriate antigens for its development is a bottleneck step, especially considering the limited resources allocated. Herein we propose a vaccine strategy based on multiple epitopes from multiple antigens to counteract the parasites´ biological complexity. Epitopes were identified by computer-assisted genome-wide screenings, considering sequence conservation criteria, antigens annotation and sub-cellular localization, high binding affinity to antigen presenting molecules, and lack of cross-reactivity to proteins in cattle and other breeding species. We ultimately provide 31 B-cell, 8 CD4 T-cell, and 15 CD8 T-cell epitope sequences from 30 distinct antigens for the prospective design of a genetic ensemble vaccine against the four trypanosome species responsible for African animal trypanosomiasis.African animal trypanosomiasis is caused by vector-transmitted parasites of the genus  Trypanosoma , which are transmitted by insect vectors. Pathogens and vectors co-evolved and their distribution varies geographically. Trypanosoma congolense and T. brucei brucei, the most studied of the four, are transmitted by hematophagous tsetse flies (genus Glossina), and along with mechanically transmitted T. vivax are abundant in Africa [T. evansi is also mechanically transmitted by fly bites and along with T. vivax can be found in America and Asia [African animal trypanosomiasis (AAT) is a debilitating disease that affects wildlife and domestic animals in vast areas of Africa, Latin America, and Asia . Known an Africa . T. evanand Asia . Neverthand Asia . In Afriand Asia .Infections can be treated with the drugs diminazene aceturate and isometamidium chloride, but the appearance of resistances is common ,5. A proTrypanosome spp. (downloaded from TriTrypDB release version 39) [In light of the high biological complexity of the parasites, and the very limited resources dedicated to the development of vaccines for AAT, a reverse vaccinology approach could be the most adequate strategy to follow. Herein we have used the genomic information available on AAT-causing sion 39) , and a sTrypanosome spp. that have been related to AAT from TriTrypDB (release v39) [T. brucei Lister427, T. brucei TREU927, T. congolense IL3000, T. evansi STIB805, and T. vivax Y486.We downloaded the whole annotated proteomes of all ase v39) . We mergTrypanosome spp. protein collection, clustering them with a shared identity >90% [We used CD-HIT, running stand-alone with default settings, to reduce the redundancy of our ity >90% . The resity >90%  to obtaiH) measure [i is the frequency of an amino acid of type i, and M is the number of totally different amino acids (20). H = 0.0 is equivalent to no variation in a given position among the studied protein sequences within the alignment, whereas higher values correspond to higher variation in that position [The Shannon entropy  [T. cruzi-masked proteome file.For the prediction of CD4 T-cell epitopes we used the IEDB recommended method, the MHC II binding predictions tool (/mhcii/) , over th/mhcii/) . We conshttp://tools.iedb.org/mhci/) [For de novo prediction of CD8 T-cell epitopes we used the IEDB recommended method, the MHC I binding predictions tool (g/mhci/) . We ran Bos taurus and Bos indicus proteins. We used the NCBI protein blast suite (BLASTP) with the default parameters, except for the PAM30 Scoring Matrix and an expectation value  of 10,000, as the epitope sequences to blast were shorter than 100 residues in all cases. Following the same procedure, we also blasted the epitopes sequences against the NCBI nr- collection of sheep , goat , and pig  proteins. In order to find out the epitopes identity  to proteins in cows, we blasted them against cow microbiome protein sequences obtained from the Cow Microbiome Project  and agaiThe prediction of patterns related with surface exposure of those antigens with predicted B-cell epitopes was made with SignalP, TargetP, and TMHMM . The posWe used the RaptorX web portal to model the 3D structure of the vaccine ensemble peptide sequence . SolventTrypanosome spp. responsible for AAT to obtain the conserved “proteome”. We departed from the five available sequences at TriTryDB r39  and imposed the strictest conservancy possible: any residue position that was not identical in all five of them would be masked and thus not accounted for in the forthcoming steps  1–3, as determined by [We used Bepipred2.0 to predict B-cell epitopes that were at least 15 residues long. Before applying it to the invariant proteome file we manually selected those antigens in it that were expected to be surface-exposed by the annotation terms . Bepipremined by , or mappMycobacterium bovis epitopes in cows [T-cell epitopes also had to be predicted. We used the IEDB Analysis Resource MHC-II and MHC-I binding tools to respectively predict CD4 and CD8 T-cell epitopes. The MHC-II binding tool at IEDB does not allow to specifically select class II bovine leukocyte antigens for the binding predictions so we used the available HLAs and the IEDB recommended set of tools . Althoug in cows . We applThe most economic scaffold to administer the vaccine ensemble would likely be genetic delivery of the epitopes as plasmid DNA (pDNA). We therefore designed a genetic construct that encompasses the 54 selected epitopes shaped as a “string-of-beads” . LinkersUpstream of the epitope “string-of-beads” there is a cytomegalovirus (CMV) promoter sequence that governs its transcription, whereas at the string´s 3´-end there is a bovine growth hormone (BGH) poly-adenylation (polyA) signal to tag the growing mRNA molecule with a polyA tail. These two adjoining sequences are widely used within mammalian expression plasmids and they have been shown to work in the context of bovine cells, for instance ,28. Bos taurus code and its most likely abundant codons. As can be seen in Bos taurus differs in some residues with respect to that of AAT spp. However, plain reverse translation does not account for the optimal codon usage. This is a complex process where not only the codon usage is important, as it involves many other parameters, such as those facilitating transcription  or those increasing translation efficiency . Thereby, we used an in vitro-translated mRNA (IVT) codon optimization tool [Bos taurus codon usage; [Thinking of a delivery of the vaccine ensemble to cows, the full peptide sequence of the ensemble  was subjion tool  to optimn usage; ) with thn usage;   and the antibiotic resistance gene for propagation and selection (Kanamycin resistance in this case), we estimated a pDNA length of ~6.3 Kb. Such length can be now fully synthesized in vitro with a cow-optimized codon usage pattern.The pDNA construct will be eventually expressed within cow cells to yield the poly-peptide epitopes. We used RaptorX to obtain a 3D model of what the vaccine ensemble would look alike upon being expressed. Notably, RaptorX also displays solvent accessibility (Acc) per position of all the residues within the submitted ensemble assigning a classification of buried (“B”), medium (“M”), or exposed (“E”) to each of these residues . The retIf we take into account the Acc classification by RaptorX, it appeared that the highest percentage of “B” residues matched to CD4 and CD8 T-cell epitopes , have been proposed . It mustialidase  or immunialidase ,10. Furtiewed in ). It shoiewed in . Some ofiewed in ,34,35 orT. vivax .T. congolense, T. evansi, and T. vivax remains largely unexplored, the majority of the antigens of origin of the predicted epitopes were either uncharacterized proteins or had low annotation scores inferred by homology. However, the antigens from where the B-cell epitope DSLCTLNMVPGVSVYGEKRVEVGATQ; CD4 T-cell epitopes AQIHDIYRMIPPLQVVLVSAT, DGRIGIILMDNITEVQSGQK, and AKNKFFYMYVQELNYLIRF; and CD8 T-cell epitopes MMHAYASRY, FGPWFVEPV, and EHNWMFAEI emerged, had T. brucei brucei homologues counting with medium to high annotation scores and experimental evidence of expression at either the transcriptional and/or protein level. The aforementioned B-cell and first CD4 T-cell epitope sequences, respectively matched to fibrillarin and a putative ATP-dependent RNA helicase. The other two CD4 T-cell epitopes matched to the T. congolense homologue of a well-characterized T. b. brucei mitochondrial ATP synthase subunit alpha (UniProt ref.: Q9GS23). This protein had experimental evidence provided by three different laboratories and it has been shown to be very important for the correct growth of the parasite [T. b. brucei NADH dehydrogenase and proteasome subunit beta type-3; whereas the last one was an uncharacterized T. b. brucei protein with a >90% identity to the T. equiperdum chromosome passenger complex (CPC) protein. The uncharacterized T. b. brucei homologue and one of the ATP synthase subunit alpha had 3D crystal structures, and thus experimental evidence at the protein level.We used publicly available and tailor-made computational methods to identify the epitopes presented in this article. Unfortunately, we did not find any validated AAT epitopes at the IEDB repository that complied with the conservation criteria, so we had to run de novo predictions. Therefore, the next step to take will be to experimentally validate that the predicted epitopes are presented and recognized in the cow immunological context, and that they are capable of eliciting immunogenic responses. Despite being based on predictions, the goodness of currently available tools for this purpose has been acknowledged by several authors and the use of prediction tools is seen as a great aid for the identification of new epitopes ,36,37,38parasite . Each ofTaking into account that (i) mixed infections with various trypanosome species have been reported ; (ii) thT. brucei dampens macrophage pro-inflammatory responses, and both T. brucei and T. congolense can modulate in their favor the T-cell response functionality [+ T-cell responses can provide resistance to T. brucei infection [T. congolense infection [+ T cells specific of a VSG conserved peptide has also been described [Trypanosome parasites responsible of AAT persist in the bloodstream thanks to a series of evolved mechanisms that allow them to escape innate immune controls and survive the successive waves of adaptive immune responses. At the initial phases of the infection, it has been described in mouse experimental models that ionality . Howevernfection . Moreovenfection . These Tnfection . Nonetheescribed . Thereby+ T cells, CD8+ T cells were described to have a parasite-promoting effect in a mouse model of experimental T. brucei infection [+ T cell depletion in trypanosusceptible Boran cattle naturally infected with T. congolense through tsetse bites did not lead to any effect in the parasitemia or anemia, which is the main determinant of survival [+ T cells was observed in T. evansi experimentally infected sheep [+ T cells in AAT infections is still obscure, and the potential usefulness of their inclusion in a vaccine product should not be disregarded. In fact, CD8+ T cells might promote certain level of protection yet to be discovered, since they are exacerbated during the natural infection process [In comparison to the suggested protective role of CD4nfection . But CD8survival . Similared sheep . Thereby process .T. brucei and T. congolense have shown that these trigger a polyclonal B-cell activation, which may contribute to take the immune attention away from relevant antigens, like VSGs (see [T. vivax have been described to disrupt B-cell lymphopoiesis in primary and secondary lymphoid organs, which affects the establishment of pools of memory B cells [+ T cells inducing epitopes and the adjuvant used to exacerbate the exerted response biases it towards a Th1 profile. Whether such an adjuvant is sourced outside the vaccine construct or can be encoded within it or co-immunized in another construct [Nevertheless, being overall extracellular pathogens, the humoral response against AAT trypanosomes is likely to be the most important towards ultimately controlling the infections . In ordeSGs (see  for a re B cells . In sighonstruct , remainsHere we provide a tentative design for a pDNA ensemble, thinking of its likely reduced development and production costs in comparison to other delivery platforms. Following previously described constructs ,28, we hAt present there are five DNA vaccines already approved for veterinary use . RemarkaPotency and longevity of vaccine-exerted immune response will be fundamental and will have to be determined experimentally. In this respect, the questioned poor immunogenicity concerns on pDNA vaccination might be circumvented by including codon optimization and administrating the construct in combination with the appropriate adjuvant. Besides, the pDNA delivery methodology and the prime–boost regime followed can be decisive . BearingDespite there are two drugs available for the treatment of AAT the appearance of resistances is common due to poor attachment to treatment. Besides, the implementation and maintenance of vector control programs, crucial to control the disease, is irregularly performed as they are linked to economic and political stability, which may not be assured in the affected regions. For these reasons, the availability of a vaccine that could prevent the infection would mean a major breakthrough to limit the impact of AAT. Some efforts have been initiated but the biological complexity of the parasites and the very limited funding available for research on the subject has precluded its development.We propose an alternative vaccine approach based on epitopes from a range of different parasite antigens to cope with the parasites antigenic complexity. The epitopes were prioritized in terms of their absolute conservation among all the sequenced genomes of the distinct AAT-causing spp. The main rationale, besides taking advantage of the biological relevance of highly conserved protein motifs for the parasites´ biology, is to ultimately provide a single vaccine ensemble to tackle all ATT-causing trypanosomes. Progressing a single vaccine product would contribute to save developmental costs. Moreover, we propose a pDNA vaccine construct to carry the epitope-based “string-of-beads” because it could be synthetized at a fraction of the cost of producing recombinant protein subunits or virus vectored vaccines. Remarkably, it is also a platform capable of yielding the high amounts of product needed to immunize large animals, which can be lyophilized and suitably used in tropical regions. All epitopes in the ensemble were prioritized in agreement with their lack of identity to any protein sequences present in cows, and in the proteomes of sheep, goat, and pig species, so as to avoid the appearance of cross-reactivity reactions, thus favoring the safety of the vaccine but also likely promoting its immunogenicity.A major limitation of the study is that we did not find experimentally validated conserved AAT epitopes in the IEDB repository database, so we had to rely on de novo predictions of epitopes derived from the computed invariant proteome file. We applied a very conservative threshold and only top qualifying predicted epitopes were selected for progression. Nonetheless, whether they are presented and recognized in the course of an infection will need to be experimentally demonstrated. Appropriate processing is a key feature towards the immunogenicity of the epitopes, and this will have to be thoroughly studied in relation to the delivery of the epitopes in the form of a genetic construct. Furthermore, their optimum ordering and spacing within the ensemble will also require to be adjusted experimentally as well as the selection of the most adequate adjuvant. Thereby, the pDNA ensemble provided here must just be considered as a theoretical scheme, subject to any required reformulations upon validation of the predicted epitopes and the study of their immunogenicity and capacity to induce protective immune responses. Other acknowledged limitations of the reverse vaccinology strategy we have followed are the inability to provide glycan-based epitopes and the absence of conformational B-cell epitopes, since these cannot be isolated from their context and thus we only focused on linear ones.+ T cells against some AAT-causing trypanosomes. Finally, since the functionality of CD8+ T cells is still under debate, we placed them at the end of the string but decided to keep them in the proposed ensemble, as CD8+ T cells may play a role in eliminating infected macrophages and, therefore, freeing the suppressive control they have on AAT immunity. In an attempt to anticipate a vaccine candidate, we placed the B-cell epitope component first as it is expected that an antibody-mediated response will play a major role in controlling free-living AAT infections. In the construct these are immediately followed by CD 4 T-cell epitopes, which will be fundamental for the correct response to all the former, but also because there is evidence of the importance of CD4This work lies at a very preliminary stage of the long vaccine development path. Notwithstanding, one of the advantages of an in silico vaccinology approach is that it can contribute to saving time and resources. Hopefully, the epitopes here selected can reach experimental studies in the near future and prove they can indeed elicit immunogenic responses against infections with AAT trypanosomes.


The aim of this study was to characterize the influence of Konjac glucomannan (KGM) on the functional and structural properties of wheat starch. Results showed that KGM significantly decreased the starch hydrolysis rate, with a lower level of rapidly digestible starch (RDS), and a higher content of slowly digestible starch (SDS). Besides, KGM decreased the content of leached amylose, while enhanced the swelling power, water‐holding capacity, freeze‐thaw stability, and paste clarity of wheat starch, which indicated a good improvement on the functional properties. Differential scanning calorimetry (DSC) and X‐ray diffraction patterns (XRD) manifested that the addition of KGM disrupted the original crystalline structures of wheat starch, which may result in the increased hydrolysis rate of starch. Interestingly, this did not consist with the decreased starch hydrolysis rate showed in the study. Moreover, FTIR results showed the existence of the interaction between KGM and starch. The morphological characterization demonstrated that the addition of KGM contributed to a more compact structure of freeze‐dried wheat starch. And KGM inhibited the expansion of starch granules and formed a barrier around the gelatinized starch. Therefore, the barrier around the starch granules and the interaction between KGM and starch were considered to be the important reasons that affected the starch digestibility. Konjac glucomannan decreased the starch hydrolysis rate.Konjac glucomannan changed the chemical structures of wheat starch.Konjac glucomannan formed a physical barrier around starch. Starch from wheat, α‐amylase Type IX‐A from human saliva , pepsin from porcine gastric mucosa (activity 456 U/mg), amyloglucosidase from aspergillus niger (activity 30–60 units/mg protein), amylose from potato, and 3,5‐Dinitrosalicylic acid and fluorescein 5(6)‐isothiocyanate were purchased from Sigma‐Aldrich Co.2.2Starch digestibility was measured by Englyst, Kingman, and Cummings  with som2.32KI solution. The standard curve was made with amylose as a standard substance . The content of amylose leached during gelatinization was obtained by the iodine colorimetry with I2.4The swelling test was determined based on a reported method  of starch was according to Nawab, Alam, and Hasnain . About 02.6m—The weight of starch paste[g]; m—the weight of starch paste precipitation[g].Freeze‐thaw stability was measured using Wattanachant’ method  was dissolved and mixed with distilled water in a preweighted aluminum pot and placed at 4°C for 12 hr until the water was completely in equilibrium with the sample. The condition was set to 10°C/min speed heat from 20 ℃ to 120 ℃ in DSC instrument, as a comparison with an empty plate.2.9The diffraction patterns of freeze‐dried starch samples were determined by X‐ray diffractometer  running at 40 kV and 40 mA. Samples were scanned in the range from 10 to 55° (2θ)  was dissolved in 40 ml of deionized water and stir well and then pasted on 95°C for 20 min. After cooling to room temperature, freeze‐dried starch samples were obtained by vacuum freeze‐drying and pulverized through 100 mesh sieve. The FTIR spectra of four freeze‐dried starch samples were tested at 4,000 ~ 400 cm2.11Particle size analysis of freeze‐dried wheat starch samples was evaluated by Malvern laser particle size analyzer . The results were expressed in terms of the volume distributions of freeze‐dried wheat starch. The mean particle size parameters were reported as D (volume‐weighted mean) and D (surface‐weighted mean). Each sample was performed in three replicates.2.12Freeze‐dried wheat starch samples were cut into cubes, and morphological properties of starch/ KGM samples were obtained by a scanning electron microscopy  at a voltage of 15 kV. The starch sample was adhered to double‐sided coated carbon tape and fixed on the aluminum frame and then coated with gold–palladium. A meaningful freeze‐dried starch sample images were obtained at 1,200 × magnification.2.13The internal structure of starch granules was investigated by using confocal laser scanning microscopy (CLSM) . The significant differences (p < .05) were evaluated and displayed by different letters.Data in our study were presented as means ± 33.1The effects of Konjac glucomannan (KGM) on starch in vitro digestion were investigated. As shown in the Figure The addition of KGM had a positive effect on the content of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). As shown in the Figure 3.2As shown in the Figure Compared with native wheat starch, the addition of KGM significantly improved the swelling power from 11.73 to 17.66 g/g Figure , and the3.3Freeze‐thaw stability is detected to understand the ability of frozen wheat starch with KGM addition to retaining their shape and texture after thawing. As shown in the Figure Paste clarity determines the appearance and use of starch products . During gelatinization, starch granules persist as highly swollen fragile forms, commonly termed granule “ghosts” (Debet & Gidley, 4Konjac glucomannan significantly decreased the starch hydrolysis rate, reduced the content of RDS, and increased the content of SDS and RS and thus slowed down the release of glucose from starch and reduced postprandial blood sugar. The lower level of leached amylose suggested that the KGM may have formed a barrier around the starch, which prevented the diffusion of amylose during the gelatinization. And the leached amylose would interact with KGM that led to the formation of KGM–amylose complex. Due to the strong hydrophilic ability, KGM increased the swelling power and water‐holding capacity of the KGM–starch mixtures. Besides, KGM was identified to enhanced freeze‐thaw stability and paste clarity of wheat starch. All these findings demonstrated that KGM improved the functional properties of starch. It was confirmed by the results of the chemical structures and morphologies characterization, the barrier around the starch granules and the interaction between the leached amylose and KGM were responsible for the reduced starch digestibility.There is none potential conflict between authors and others that bias our work.This study does not involve any human or animal testing.


Dear Reader,Archives of Gynecology and Obstetrics has successfully improved the quality of the journal during the last years. The journal receives currently about 2,000 submissions per year from all over the world. Outstanding editors, associate editors, editorial board members, and reviewers have greatly contributed to the successful development of the journal. In addition, the members of the editorial office did a great job in handling the manuscripts. The quality of the scientific work improved a lot. All this led to an increase of the impact factor which is now 2.283.The Editorial Board of Archives of Gynecology and Obstetrics have a long and outstanding history. In this year, we celebrate the 150th anniversary. The journal was founded in 1870 as“Archiv für Gynäkologie”. Since 1922, it is the official journal of the German Society of Gynecology and Obstetrics . In the review by M. David and A. D. Ebert, “Fair to all interests and impartial towards all schools of thought in honour of the 150th anniversary of the foundation of the Archiv/Archives”, published in this issue, the history of Archives of Gynecology and Obstetrics is summarized. After the American Journal of Obstetrics and Gynecology, our journal is the second oldest journal in this field. You are invited to submit manuscripts for publication in the different sections. We appreciate your suggestions leading to further improvement of the journal's quality.


P = 0.03). Metatarsophalangeal joint passive stiffness in sitting and standing positions was 23% higher in the basketball group than the runner and other sports athlete groups (P < 0.01). However, there was no significant group differences in metatarsophalangeal joint passive stiffness and vertical stiffness. Significant correlations among all stiffness variables were determined (P < 0.05). These findings indicate that ankle and metatarsophalangeal joint passive stiffness, rather than vertical leg stiffness, would be in relation to types of sports participation. Ankle and toe strengthening exercises could improve basketball players' performance and prevent injury.While individual ankle and metatarsophalangeal joint stiffness is related to training intensity and sport performances, sport athletes may develop specific passive joint stiffness among the spectrum from endurance to powerful types of sports. The objective of this study examined whether marathon runners, basketball players, and other sports athletes would demonstrate distinct passive ankle and metatarsophalangeal joint stiffness as well as vertical stiffness. Fifteen marathon runners, nineteen basketball players, and seventeen other sports athletes performed both joint stiffness measurement and single-leg hopping tests. We used a computerized dynamometer to control foot alignment and speed for passive ankle and metatarsophalangeal joint stiffness measurements. We calculated vertical stiffness by body deceleration and body mass displacement during hopping on the force platform. One-way ANOVA was performed to identify the group differences. Bivariate correlation test was also performed among ankle, metatarsophalangeal, and vertical stiffness. The basketball group displayed 13% higher ankle passive stiffness than the other sports players group ( Ankle and metatarsophalangeal (MTP) joint passive stiffness is extensively assessed for various sport populations in both clinical and sport settings. The ankle and MTP joint passive stiffness is related to body balance and propulsion for locomotion , 2. The The MTP joint would allow the forefoot to generate forces by pushing against the ground during propulsion of walking and running . StefanyTo date, most of the commercially available dynamometers , which have been used to determine strength and stiffness of the ankle joint in controlled speed and displacement , have liSince passive stiffness can be increased with training  and moveTo date, vertical stiffness (Kvert) has been predominately applied to describe the overall mechanical behaviour/efficiency of the lower extremities , 24 as Kt-tests revealed that there were no significant differences of age, body height, body mass, and playing experience of sports between athlete groups  at a sampling frequency of 1,000 Hz for the left and right feet, respectively . The part-tests were then performed if significant athlete group effect was determined in the ANOVA. Pearson product correlation was also performed to analyse the correlation among ankle stiffness, MTP stiffness (sitting and standing), and vertical leg stiffness. The correlation (r2) was classified as little/no (>0.0 to ≤0.25), fair (>0.25 to ≤0.50), moderate to good (>0.50 to ≤0.75), and good to excellent (>0.75 to ≤1.0). All statistical analyses were conducted using SPSS version 19.0 , and the significance level was set at 0.05.A customised MATLAB  code was applied to process all ankle stiffness, MTP stiffness, and vertical stiffness (Kvert) variables. The left and right data of each participant were averaged for further analysis. A one-way ANOVA was performed on each variable to examine if there was any significant difference between athlete groups. Additional independent-sample P < 0.01 for all comparisons) but not for Kvert (P=0.39). Post hoc tests indicated that both sitting and standing MTP joint passive stiffness of the basketball group were higher than the runner (P < 0.05) and the other sports athlete groups (P < 0.01)   .P < 0.01 for all comparisons, The correlation results revealed the significant correlations among all stiffness variables  and other sports athletes (sitting 25% versus standing 24%). A plausible explanation would be that the basketball players would require stronger toe and ankle strength as indicated by passive joint stiffness for various powerful jumping  and streAnother possible explanation would be related to the spectrum between powerful and endurance types of performances. Distant runners may, however, require optimal toe and ankle strength/stiffness to attain the moderate speed for a long running distance –8. A preThe vertical stiffness (Kvert) has been commonly used to describe sports performance of different populations as it can describe the overall mechanical behaviour/efficiency of the lower extremities . HoweverThe previous ankle and MTP passive stiffness measurements are still largely depended on manual skills as the previous devices did not have good control on alignment, angular speed, and displacement during torque measurements . In the There are some experimental limitations when interpreting our results. Firstly, a single male university athlete group was recruited in this study, and it is not generalizable to other groups. Different genders, playing levels, and positions may have shown remarkable differences in jumping intensity and frequency in match plays . SecondlBasketball athletes displayed higher ankle and MTP joint passive stiffness compared with distant runners and other sports athletes, suggesting that mechanical properties of joint stiffness are in relation to sport intensity between powerful- and endurance-trained athletes. Ankle and toe strength exercises could be implemented to develop sufficient ankle and MTP joint passive stiffness that is required in basketball.


In this study, downregulating the expression of japonica-type SSIIa in Nipponbare endosperm resulted in either shrunken or opaque grains with an elevated proportion of A-type starch granules. Shrunken seeds had severely reduced starch content and could not be maintained in succeeding generations. In comparison, the opaque grain morphology was the result of weaker down-regulation of SSIIa which led to an elevated proportion of short-chain amylopectin (DP 6-12) and a concomitant reduction in the proportion of medium-chain amylopectin (DP 13-36). The peak gelatinization temperature of starch and the estimated glycemic score of cooked grain as measured by the starch hydrolysis index were significantly reduced. These results highlight the important role of medium-chain amylopectin in influencing the functional properties of rice grains, including its digestibility. The structural, regulatory and nutritional implications of down-regulated japonica-type SSIIa in rice endosperm are discussed.The enzyme starch synthase IIa (SSIIa) in cereals has catalytic and regulatory roles during the synthesis of amylopectin that influences the functional properties of the grain. Rice endosperm SSIIa is more active in  SSIIa) codes for a major starch synthase enzyme isoform involved in the elongation of short-chain amylopectin in the cereal endosperm, which is important in distinguishing starch properties , alk, gel(t) and SS2-3 gene in other studies] was originally mapped to the alk locus located on chromosome 6  of rice, an essential trait in predicting cooking and eating qualities (Starch synthases (SS) play a critical role during starch biosynthesis, elongating glucan chains by the addition of glucose from the substrate ADP-glucose . These eoperties . In ricemosome 6  and is hmosome 6 . SSIIa aualities .indica line Kasalath as the canonical protein sequence, previous research has demonstrated that substitution of either Valine-737 with Methionine , or Leucine-781 with Phenylalanine  consequently lead to a reduction in specific activity of less than 10% compared to that of the indica sequence  is common among japonica rice lines such as Nipponbare because its SSIIa is weakly active, making the enzyme less efficient in catalyzing the elongation of short amylopectin chains, which leads to low GT  in Nipponbare by indica SSIIa (active) from Kasalath produced indica L-type amylopectin  located along the SSIIa coding gene have been linked with varietal differences in GT due to variations in amylopectin chain length distribution (CLD) . Using tsequence . Substito low GT . In conty active . This relopectin .japonica types (haplotypes 3 and 4) have similar levels of SSIIa in the soluble phase but reduced levels in the starch associated protein fraction compared to those belonging to indica types (haplotypes 1 and 2)  compared to japonica types  . Furtherca types  due to t of rice . Additio of rice . Additio of rice . Clearly of rice . All thejaponica line Kinmaze was identified completely devoid of SSIIa expression. This SSIIa null mutant, generated using N-methyl-N-nitrosourea, had a 4% increased amylose content, and a reduction of about 6°C in the gelatinization temperature  and a different technique (RNA silencing), are in broad agreement with the results obtained by In rice, an SSIIa mutant from the perature . The 4% tibility . It willjaponica rice (Nipponbare cultivar) using a technique effective in partially downregulating the expression of SBEIIb in rice endosperm (japonica rice SSIIa based on Nipponbare reference genome. The PCR fragment was cloned in pGEM-T Easy and subcloned into pBX17 and pVec8 using previously published methods (Agrobacterium tumefaciens AGL1 as previously described (A hairpin RNA (hp-SSIIa) was constructed to downregulate the expression of SSIIa in the rice endosperm of ndosperm . Total R methods . The gen methods . The gen methods . The Nipescribed . Tissue escribed .Regenerated transformed plants and negative controls were grown in pots partially submerged in water-filled tanks to simulate irrigated conditions inside a biosafety glasshouse. The temperature was maintained at 22°C during the night for 8 h and 29°C during the day time for 16 h of natural light. The actual average daily temperature was observed to be 26.5 ± 3.5°C and is consistent with our previous study . InitialRNA was extracted from grains at mid-development (15 dpa) using NucleoSpin RNA Plant (Macherey-Nagel). A total of 5 μg RNA template was used to synthesize cDNA using SuperScript III reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was done in a Rotor-Gene 6000 (Corbett) using 100 ng cDNA templates amplified utilizing previously published SSI and SSIIa primer pairs . The reaSSIIa in wild-type rice grains, which was used to screen for SSIIa-downregulated lines. A total of 100 μg protein, quantified using Coomassie Protein Assay Reagent (Bio-Rad), was resolved in 4–10% precast gradient gels (Invitrogen). Total proteins were extracted from 5 to 20 dpa developing grains using a previously published method to track SSIIa expression across several developmental time points. In addition, soluble, granule-associated and granule-bound proteins from starch granules of mature grains (32 dpa) were also extracted as previously described  were extracted as previously described . This deescribed . For graescribed . The immOnly grains that have the hairpin RNA insert detected by PCR and downregulated SSIIa as detected by western blot as described above, were selected and subsequently characterized. Mature panicles of transgenic plants were harvested and dried at 37°C overnight. The opaque seeds were carefully threshed by hands, and then dehulled and polished by machine using standard methods. Shrunken seeds were also manually threshed and dehulled but they were not polished because they shatter into powder after passing through the milling machine. Consequently, all succeeding starch structural and functional analyses were done in polished (white) and wholemeal flours (brown) for opaque lines and only in wholemeal flours for shrunken lines. Photomicrographs of whole rice grain samples were obtained using either a LEITZ M8 stereomicroscope or by scanning in Image Scanner III . Some seeds were set aside for planting in subsequent generations.Peak GT from rice flour was measured by differential scanning calorimetry . Total sin vitro starch hydrolysis index (HI) method which mimics the oral and gastrointestinal phases of carbohydrate assimilation in humans  content and estimated glycemic score (EGS) of freshly cooked polished rice grains were estimated using an n humans . The HI lunteers . A totallunteers . Becauselunteers .N(X) (Determination of chain length distribution (CLD) of debranched amylopectin by fluorescence-assisted capillary electrophoresis (FACE) was performed based on a previous method . MoleculN(X) . For SECN(X) . A waxy N(X) . The folN(X) .13C cross polarization/magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) experiments were performed at a 13C frequency of 75.46 MHz on a Bruker MSL-300 spectrometer also as previously described  under variable-pressure mode. Images of starch granules were taken with a back-scattered electron detector. Starch granules were also isolated and viewed under a polarized light microscope after iodine staining to check for birefringence. Granule size distribution (by volume) of the starch slurries was determined using a laser diffraction particle size analyzer . The percentage of small starch granules was determined using a cut-off diameter of 1.9 μm (refer to the results section). The diameter of large starch granules (>1.9 μm) was calculated as the diameter of starch granules at the peak of large starch granules. Characterization of starch crystallinity by x-ray diffraction (XRD) was carried out on a Panalytical X’Pert Pro diffractometer using the crystal defect method based on our previous publication . Solid-sescribed .T-test) were done using GraphPad Prism Version 6. The standard error of the mean (SEM) was used to representing error values. Statistical significance was defined at least as P < 0.05.Three biological replicates from at least two independent transformed lines were used for every analysis whenever applicable during the screening of hp-SSIIa-shr and hp-SSIIa-op. Mature grains of hp-SSIIa-op were used for whole grain and starch analyses for five generations (T1 to T5), planted and harvested at different seasons to assess stability and replicability of traits. Gene and protein expression profiling was performed on later generations (T3 to T5). A comparison of results was done using Nipponbare negative segregant as control. Statistical analyses  harbored the hygromycin resistance gene based on marker screening by PCR. A total of 5 out of 31 lines (16%) of hp-SSIIa were selected at T1 generation based on corroborative results from the seed appearance  and PCR SSIIa expression in Nipponbare rice endosperm are the only ones reported in this study.A total of 23 independent transformants were obtained that harbored artificial microRNAs targeted toward SSIIa (ami-SSIIa). Three ami-SSIIa lines cloned using the osa-miR528 backbone using the pWBVec8 expression vector were selected at T1 generation based on grain appearance and PCR screening. However, the lines could not be maintained beyond T2 generations. Consequently, the results for hairpin RNA downregulation of The starch granules of hp-SSIIa-shr T1 seeds were severely distorted during development compared with the controls. The granules were rounded and had lost their compound structure . FurtherIn contrast to the phenotypic consequence observed in hp-SSIIa-shr seeds, most of the starch granules of hp-SSIIa-op are rounded but they have maintained their compound structure . The dis13C CP/MAS NMR also did not vary significantly from that of Nipponbare , while that of hp-SSIIa-op had no reduction compared to polished Nipponbare control. The peak gelatinization temperature of our rice lines are quite high compared to other reference values for Nipponbare in literature, which is 66.5°C in P-value = 0.0320). Lastly, the resistant starch content of the hp-SSIIa-op lines was unaltered while the predicted glycemic index by in vitro hydrolysis index was significantly reduced . The hp-SSIIa-shr lines were not included in the estimation of resistant starch content and in vitro hydrolysis index due to sample size limitations and also because they were not amenable to polishing.The chain-length distribution (CLD) characterization of debranched amylopectin from selected seeds based on appearance, western blot and PCR screening of hp-SSIIa-op opaque lines  and hp-SSSIIa downregulation by hairpin RNA silencing. Western blot detection of granule-bound proteins of SS5 revealed that SSIIa was undetectable (indica control (IR64) was very high. As expected, the amount of GBSSI was higher in IR64 compared to Nipponbare and SS5, while SSI appeared to be similar in the granule-bound proteins of the three lines tested. SSIIa was not detected in the soluble protein fraction from flour samples of mature desiccated grains of the three lines tested  confirmed that the level of SSIIa in the total protein extract was partially down-regulated compared to their corresponding developmental stages of Nipponbare control, as well as compared to the 15 dpa IR64 used as a reference band . The amojaponica-type SSIIa resulted in distinct seed and starch granule morphologies, elevation in the proportion of short-chain amylopectin, and the reduction of peak gelatinization temperature and estimated glycemic score of cooked grains. Nipponbare is a japonica variety with an SSIIa belonging to haplotype 3 , a near-isogenic line of Nipponbare with introgressed indica SSIIa (Haplotype 1) from Kasalath  were shown to have severely distorted starch granules whose formation appeared to have been aborted early during grain development. These lines also showed reduced total starch content and lower proportions of amylopectin chains of DP 12–36. These results suggest severely reduced activity of other enzymes involved in amylopectin biosynthesis upon reduction of SSIIa protein, and will be the focus of future studies. Such pleiotropic effects are consistent with the generally accepted idea of functional multi-enzyme complexes carrying out starch biosynthesis in cereals, which can be disrupted in the complete absence of SSIIa. Interestingly, the effects of down-regulation of SSIIa may be different in different genetic backgrounds, or under different environmental or growth conditions. A similar study showed field-grown plants lacking SSIIa able to flower and set seed . The stuSBEIIb transgenic mutants in rice, which shifted from A to C to B-type crystalline polymorph and is accompanied by chalky to opaque grain phenotypes (The NMR data showed very similar levels of molecular order (double helix and single helix contents) between isolated granules from Nipponbare and the two hp-SSIIa lines tested. Double helical molecular order arises from non-enzymic inter-twining of adjacent glucan chains in the branched amylopectin structure, provided they are longer than about six residues . It cannenotypes .SSIIa in japonica line, Nipponbare did not produce high amylose and high resistant starch phenotype as occurs in barley  lead to a 4% elevation of amylose content in the japonica background  was significantly reduced from 85 to 63 GI units (or 25%) in two hp-SSIIa-op lines (SS3 and SS5) based on biologically replicated assays. This reduction was still not as much as the reduction in glycemic index estimate for the ami-SBEIIb down-regulated lines (44 ± 1). Nonetheless, it is still noteworthy that the slight reduction in medium-chain amylopectin content in the hp-SSIIa-op line was linked with a lower glycemic index estimate even though this was not accompanied by an increase in amylose chains or long-chain amylopectin as was previously observed in the SBEIIb mutant line, IR36ae  in the SSIIa down-regulated lines is associated with a reduction in the glycemic index estimates. Thus, in addition to increasing amylose content and increasing the proportion of long-chain amylopectin, a reduction in the proportion of medium chains of amylopectin is also associated with the reduction of GI. Lastly, this study also supports the data of In summary, down-regulating the expression of All datasets generated for this study are included in the article/VB conducted all the genetic engineering, functional genomic validation, gene and protein expression, and starch biochemistry experiments. JL conducted the protein expression experiment of developing grains. ZL determined the size distribution of the starch granules. MG determined the crystallinity and x-ray diffraction patterns of starch granules. AB determined the estimated glycemic score of transgenic and control lines. SR assisted in rice tissue culture and transformation experiments. MG, IT, MF, SJ, and SR provided technical supervision, intellectual guidance, and multi-disciplinary expertise during the conduct of all experiments. VB wrote the manuscript with the assistance of all co-authors, especially IT and SR who helped in the several revisions of the manuscript.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.


Stiffness is a valuable indicator of the functional capabilities of muscle-tendon-fascia. Twenty healthy subjects participated in this study in which the passive elastic properties of the medial gastrocnemius (MG), lateral gastrocnemius (LG), soleus muscles (SOL), Achilles tendon  and plantar fascia (PF) were quantified when their knee was fully extended or flexed to 90° using shear wave elastography at 25° of dorsiflexion (DF25°), 0°  of flexion, and 50° of plantar flexion (PF50°) of the ankle joint. The stiffnesses of the AT, MG, LG, SOL and the fascia with the knee fully extended were significantly higher than those with the knee flexed to 90° (p < 0.05), while the stiffness of the PF showed the opposite relationship (p < 0.05). When the knee was fully extended, the stiffness was higher in the LG than in the MG at PF50° and 0° (p < 0.01), and it was higher in the MG than in the LG at DF25° (p = 0.009). Nevertheless, regardless of the knee angle, the stiffness decreased from AT3cm > AT0cm > AT6cm at PF50° and 0° (p < 0.001), while the stiffness decreased from AT0cm > AT3cm > AT6cm at DF25°. Regardless of the knee and ankle angles, the stiffness of the PF increased in a proximal-to-distal direction (p < 0.001). These insights can be used to gain a more intuitive understanding of the relationships between the elastic properties of the muscle-tendon unit and its function. In the clinic, the type of treatment administered depends in part on the region and intensity of pain. For example, eccentric exercises are more effective in the treatment of mid-substance tendinopathy than in the treatment of insertional tendinopathy1. However, because of the complexity of the anatomy and mechanics of the AT and plantar fascia (PF), it can be challenging to identify the region and severity of the damage. In terms of anatomy, the AT, which is the conjoined tendon of the medial gastrocnemius (MG), the lateral gastrocnemius (LG), and the soleus (SOL), is an important bridge for walking, running and jumping in daily life, and each of the contributing muscles exhibit unique structural features3. The twisted structure of the AT is formed by the MG tendons, LG tendons and SOL tendons. These muscle tendons rotate as they descend but do not run parallel to each other4. In addition, the superficial layer of the AT is formed by the MG tendons, while the deep layer consists of the LG tendons and SOL tendons5. Furthermore, the LG is shorter and smaller than the MG, so the MG and LG differ in terms of muscle strength and their contribution to the AT6. In other words, these regional structural features  have great inherent variability, which may affect the position and severity of damage in an individual6. Therefore, it is necessary to further understand the relationship between muscle-tendon-fascia anatomy and mechanical properties. This knowledge may greatly improve our ability to distinguish normal regional changes in muscle-tendon-fascia biomechanical properties from those arising due to damage.Achilles tendon (AT) injuries and plantar fasciitis can cause chronic pain and impairment7, while the lifetime prevalence of plantar fasciitis is approximately 10% in the general population8. Pain caused by Achilles tendinitis and plantar fasciitis can reduce the use intensity of the affected foot, decrease the flexibility of muscle/tendon, and ultimately, lead to muscle/tendon contractures11. Muscle/tendon contractures are the most obvious symptoms and are characterized by decreased muscle lengths/tendon lengths and increased passive stiffness values. They can lead to reduced joint ranges of motion (ROM) during walking and running12. Traditional metrics used to assess passive mechanical properties include the passive ROM and the global joint torques generated by motion resistance. However, the passive ROM cannot be used to distinguish the different structures crossing the joint, such as the skin, joint capsule, synergistic muscles, tendons, nerves and fasciae. Therefore, an investigation of the intermuscle and intratendon/PF distribution of passive stiffness in vivo would provide more information on the muscle-tendon-fascia biomechanics, allowing clinicians to optimize treatment strategies by, for example, targeting stiffer locations.The passive mechanical properties of muscle/tendon/fascia play an important role in the completion of each movement. It has been reported that the incidence rate of Achilles tendinitis is 2.35‰ in the general population13. The passive elastic forces of muscles and tendons and stretch forces determine the stiffness of individual muscle-tendon-fascia units. Stiffness is one of the most difficult indicators to quantify, although it is a commonly used parameter in clinical examinations of muscles and tendons. In addition, soft tissue is dynamic, so the stiffness also changes. For instance, the stiffness of muscles can change during contractions, while that of passive tissues such as tendons can change due to strain stiffening. Thus, accurate assessments of the stiffness of soft tissues across their entire functional range are essential for determining the normal stiffness level and improving their physiological function.Stiffness, one of the properties of soft tissue, is defined as the ability to resist external forces or deformations from an initial shapeet al.14 used MRE to examine the anisotropic properties of muscles, and Orner et al.15 used MyotonPRO to quantify AT stiffness. However, MRE is not frequently used to quantify stiffness because the equipment is expensive and not portable, and whether there is an effect of tight skin on MyotonPRO measurements has not yet been determined. Shear wave elastography (SWE) is a noninvasive technique used to assess the stiffness of soft tissues. It can be used to quantify the stiffness of a regional area using its relationship with shear wave speed: E = 3ρv2 16. Many studies have reported the use of SWE to assess the stiffness of skeletal muscles, tendons and PF. For example, our previous studies have shown that SWE is a valid and reliable tool for quantifying the stiffness of the gastrocnemius and AT. The intraclass correlation coefficient was 0.74 to 0.9517. In addition, Eby et al. also demonstrated a positive significant correlation between muscle stiffness measurements obtained by SWE and stiffness measurements obtained by a material testing system18. Furthermore, SWE was used not only to quantify the stiffness of physiological soft tissues, such as the gastrocnemius, patellar tendon, PF and AT, but also to quantify the stiffness of pathological soft tissues, such as the muscles of patients with spastic cerebral palsy or poststroke spasms and the PF of patients with plantar fasciitis23. In summary, SWE can be used to monitor variations in the stiffness of muscle-tendon-fascia units at different knee and ankle joint angles.Currently, magnetic resonance elastography (MRE) and MyotonPRO devices are used to quantitatively assess the stiffness of soft tissues. Green The aim of this study was to characterize the effects of different ankle joint angles with the knee fully extended or flexed to 90° on the stiffness of the MG, LG, SOL, AT and PF.The present study protocol was approved by the Human Subjects Ethics Committee of Luoyang Orthopedics Hospital of Henan Province (KY2019-001-01) and conformed to the Declaration of Helsinki. Before participation, all participants were fully informed of the aim and experimental procedures of the study and signed the informed consent form.Twenty healthy males were recruited in this study. The mean age, height, and body mass were 21.3 ± 0.86 (19–23) years, 171.25 ± 4.99 (162–181) cm, and 61.02 ± 5.6 (50–75) kg, respectively. The inclusion criteria were that all participants were healthy and did not have a history of ongoing neuromuscular diseases or musculoskeletal injuries specific to the ankle or knee joints over the previous six months. The exclusion criteria included pain in the PF or AT and an anomaly on ultrasound.17. For the MG, LG and SOL, the size of the regions of interest (ROIs) had to be set to 10*10 mm, and the diameters of the Q-box of muscle and muscle fascia were 5 mm and 1 mm, respectively17. For the PF, the three quantification areas were located between the calcaneal insertion of the PF and 1 cm distal to its insertion, and the Q-box diameter was determined by the thickness of the fascia, which was defined as the distance between the superior and inferior borders of the PF23.An ultrasound SWE system  with a 40-mm linear array transducer  was used in this study. The settings of the SWE system were set as follows: the instrument was set to the musculoskeletal mode. The frequency was 4~15 MHz. The SWE mode was the penetration mode. The opacity was 85%. The elastic modulus range for the muscles and AT was 0~800 kPa, while the elastic modulus range for the PF was 0~300 kPa. The depth of the B-scan was 3.0 cm. For the AT, the Q-box diameter was defined by the thickness of the tendon, which was the distance between the superior and inferior borders of the AT24. Participants were asked to wear loose-fitting clothes. During testing, each participant was instructed to lay in aprone-lying position with their feet relaxed and hanging over the lower edge of the treatment bed, the hip and knee joints fully extended, and the upper limbs placed alongside the body24. The ankle joint was fixed using a customized and movable ankle foot orthosis at a designated angle25. The measurement sites for the AT were 0 cm (AT0cm), 3 cm (AT3cm) and 6 cm (AT6cm) proximal to the calcaneal tuberosity26, while those for the MG and LG were defined as the proximal 30% of the lower leg length17. The SOL was measured in the distal half of the SOL for visualization of the distal SOL fascicles alone27. The PF was assessed at 3 successive levels: between the first and second metatarsal bone, on the anterior edge of the inferior calcaneal border and in the posterior section of the PF28. The length was measured with a tape measure, and a black pen was used to mark the location of the measurement site. The stiffness was measured with the SWE transducer positioned on the marks on the skin for the MG, LG, SOL, AT and PF with the ankle joint positioned at 25° of dorsiflexion (DF25°), in a neutral position (0°), or at 50° of plantar flexion (PF50°) and the knee fully extended or flexed to 90°. In addition, to reduce experimental errors, the participants were explicitly asked to keep their lower extremities fully relaxed throughout testing and to refrain from high-intensity exercise for 48 hours before testing28.The dominant leg was identified by asking the participant to kick a ball (kicking preference)29  was obtained from the system and stored for off-line analysis. Each angle was measured at 1-min intervals. Three measurements were performed for each location. The mean of the elastic moduli from all 3 images was used for further analyses.Before the measurements, the participants were asked to relax for 5 min to ensure that the triceps surae was relaxed. During testing, sufficient ultrasound gel was applied between the skin and the transducer to facilitate the transmission of ultrasound waves. The midpoint of the transducer was placed perpendicular to the skin’s surface, where we had previously made a mark, with light pressure. Then, SWE was initiated to examine the shear modulus of the muscle (with clear visibility of the muscle fascicles and aponeuroses) or the tendon/PF 29 Fig. . During 30. Cohen’s d values less than 0.2, 0.2–0.5, and greater than 0.8 correspond to small, medium, and large effects, respectively. The statistical significance level was set to be p < 0.05 (α = 0.05).Statistical analysis was performed using SPSS Version 19.0 . All data are presented as the mean ± standard deviation. The Kolmogorov-Smirnov (KS) test was used to determine whether all stiffness data had a normal distribution. The distributions consistently passed the KS test. For the stiffness data, separate 3-way analyses of variance (ANOVAs) (knee angle×ankle angle×muscle/tendon/fascia) with repeated measures were performed. If there was a knee angle×ankle angle×muscle/tendon/fascia interaction, the main effects were investigated, and a Bonferroni correction was performed. One-way repeated measures ANOVA with Bonferroni’s post hoc test was used to determine the differences in stiffness between the ankle positions or between muscle/tendon/fascia units. In addition, the effect size was also calculated using Cohen’s dThe relationships between muscle-tendon-fascia stiffness and passive knee angle are shown in Tables The relationships between stiffness and passive ankle angles are shown in Figs. There were significant differences among the 3 ankle joint angles in terms of the stiffness of the SOL, MG fascia, LG, LG fascia, SOL fascia, and PF-pro with the knee flexed to 90° (p < 0.05), and no significant difference was found in the stiffness of the MG, PF-mid, or PF-dis. For the 3 regions of the AT, the stiffness increased with increasing ankle dorsiflexion (p < 0.05).Additional analysis showed a significant difference between the MG and LG with the knee fully extended. The stiffness was higher in the LG than in the MG at PF50° and 0° (p < 0.01) and in the MG than in the LG at DF25° (p = 0.009). However, no significant difference was found when the knee was flexed to 90°. For the three different regions of the AT, regardless of the knee angle, the stiffness decreased from AT3cm to AT0cm to AT6cm at PF50° and 0° (p < 0.001), while the stiffness decreased from AT0cm to AT3cm to AT6cm at DF25°. For the 3 regions of the PF, regardless of the knee and ankle angles, the stiffness increased in a proximal-to-distal direction (p < 0.001).The main findings of the present study were that 1) the stiffness of the AT, MG, LG, SOL and fascia with the knee fully extended was significantly higher than that with the knee flexed to 90°, while the stiffness of the PF showed the opposite relationship. 2) When the knee was flexed to 90°, a significant increase in PF stiffness was observed at 0° and DF25°. 3) When the knee was fully extended, the stiffness was higher in the LG than in the MG at PF50° and higher in the MG than in the LG at DF25°. 4) Regardless of the knee position, the stiffness decreased from AT3cm to AT0cm to AT6cm at PF50° and 0°, while the stiffness decreased from AT0cm to AT3cm to AT6cm at DF25°. 5) For the 3 regions of the PF, regardless of the knee and ankle positions, the stiffness increased in a proximal-to-distal direction.35. For example, Hug et al. found that the shear modulus of the MG and AT with the knee fully extended is larger than that with the knee flexed to 90°32, and Le et al. found that the shear modulus of the MG, LG and SOL with the knee fully extended is larger than that with the knee flexed at 90°33. When sufficient mechanical stress is imposed on the lower leg, passive movement of the joint can significantly change the stiffness of the muscle-tendon-fascia unit35. The innovative aspect of the present study is that a quantitative analysis of different positions of the muscle fascia and PF. According to our findings, muscle-tendon-fascia units exhibit different stiffness changes at different angles of the knee joint. In addition, we found that when the ankle was passively dorsiflexed from 0° to 25°, the stiffness of the PF with the knee fully extended was less than that with the knee flexed to 90° and that there was no difference at PF50°. A possible explanation for this result is that when the knee is flexed to 90°, passively dorsiflexing the ankle affects the PF, while the force is shared by the PF and AT when the knee is fully extended. Therefore, we suggest that individuals fully extend their knee when they want to fully stretch their calf muscles and AT and to flex the knee when they want to fully stretch the PF. In addition, the changes in the synergistic muscles, internal tendons and internal fascia associated with different stiffnesses in the lower leg between the experiments  may be associated with the anatomical configuration of the muscles, tendons and fascia  and their passive force-length relationships31.The results of this study showed high variability in the passive stiffness of muscles, tendons and fascia when the knee was fully extended or flexed at 90°. Compared to the stiffness when the knee was flexed to 90°, the stiffness of the AT, MG, LG, SOL and fascia with the knee fully extended were significantly higher. Our results are consistent with those recently reportedet al.32 and Le et al.33. These authors found that the shear moduli of muscles and tendons increase with passive ankle dorsiflexion. Cheng et al. found that the strain of PF increases as the dorsiflexion angle increases36. The increased stiffness observed in the muscle-tendon-fascia was expected and might be explained by the stretching effect. This phenomenon seems to be physiologically plausible since anatomical characteristics indicate that the muscle-tendon-fascia unit becomes significantly stretched with an increasing ankle angle. In addition, the relatively higher stiffness of the AT can be explained by fascia or pulleys. The AT has a relatively short slack length, so it can be stretched before the muscle and its fascia are stretched33. Furthermore, the present study showed that only the stiffness of the PF-pro increased with increasing ankle dorsiflexion. This result explains why plantar fasciitis tends to occur at the proximal end of the PF. Changes in PF stiffness can affect the regulation of heel motion and control as well as joint stability. In theory, an increase in PF stiffness can cause a decreases in the capacity for PF to resist external loading and PF flexibility. At this time, if excessive and repetitive tensile forces are imposed onto the proximal end of the PF, microtrauma can develop, which may cause plantar fasciitis36. In summary, a warm-up  is necessary before individuals perform functional activities such as running and jumping. In this way, the muscle-tendon-fascia can be activated first, thereby reducing the risk of injury caused by large explosive forces or external forces during exercise.We found that the stiffness of muscles, ATs and PF increased with increasing ankle dorsiflexion. Our findings concerning the stiffness estimation using SWE are in agreement with those reported in the studies of Hug 37, but the MG was also longer than the LG, allowing it to extend more distally in the calf9. Second, the MG and LG have the same function in plantar flexion, but they have different contribution levels because the MG provides more than 70% of the force3. Third, the twisted structure of the AT is formed by MG tendons, LG tendons and SOL tendons. These muscle tendons rotate as they descend but do not run parallel to each other4. The torsion on the LG is significantly larger than that on the MG, and the rotation angle of the MG (28.17 ± 15.15°) is approximately one-fifth of that of the LG (135.98 ± 33.58°)2. Fourth, the smallest component of the AT insertion into the calcaneal bone is the LG (14.4 mm), and the largest is the MG (28.3 mm). The mean width of the footprint of the MG is approximately 2 times that of the LG5. Moreover, a previous study showed that the slack angle decreases significantly in the following order: LG > MG > AT37. Therefore, given all of the above factors, it is most likely that tension created during passive ankle dorsiflexion acts on the MG.We report new findings for the MG and LG. When the knee was fully extended, the increase in stiffness of the MG and LG during passive ankle dorsiflexion and the percentage change in the MG were larger than those of the LG, especially at DF25°. This result means that passive movement of the ankle joint with the knee fully extended has the greatest effect on the MG. This result can be explained by anatomical factors. First, not only were the cross-sectional area (CSA) and volume of the MG larger than those of the LG38. In our study, SOL stiffness was measured at the distal region of the SOL, which is near the musculotendinous junction and is exposed to higher stresses than other regions of the SOL, which may result in increased stiffness39. In addition, the Cohen’s d and percentage change in the SOL were smaller than those in the MG and LG, likely because the MG and LG, along with the plantaris, cross the knee joint, while the origin of the SOL is located below the knee. Therefore, when the knee was fully extended, the MG and LG were stretched during passive ankle dorsiflexion. The extent that the SOL was stretched was smaller than those of the MG and LG. Additionally, the SOL is a postural muscle that is composed mainly of slow-twitch type I fibers, while the MG and LG consist mainly of fast-twitch fibers. In summary, regardless of the knee angle, the changes in SOL stiffness were smaller than those in the MG and LG.We also found that regardless of whether the knee was fully extended or flexed to 90°, the stiffness of the SOL was higher than those of the MG and LG, which may be related to the locations at which the measurements were taken and interactions between contractile and connective tissue elementset al.28, who used MyotonPRO for their assessment. Hirata et al.35 also found that the stiffness of the MG was higher than that of the LG at DF25°. Moreover, Akagi and Takahashi40 found that the stiffness in the LG (33.5 ± 6.3 kPa) was higher than that in the MG (27.6 ± 7.3 kPa) at 30° of plantar flexion. Moreover, a previous study showed that the slack angle decreases significantly in the following order: LG > MG > AT35. Thus, stiffness in the MG might be decrease to a greater extent during plantar flexion than during dorsiflexion4. In other words, when the knee is fully extended, the slack angle of the LG is more dorsiflexed than that of the MG, and the slack angle of the MG is more plantar-flexed than that of the LG. This relationship may help explain why the MG is stiffer than the LG at DF25°. Muscle fiber type is another factor that may affect muscle stiffness because the stiffness of type I muscle fibers is higher than that of type II muscle fibers42. In addition, perimysium and endomysium can affect muscle stiffness43. Considering the above factors, it is reasonable that the stiffness in the LG was higher than that in the MG at PF50°, whereas that in the MG was higher than that in the LG at DF25°. According to the results of the present study, we speculate that the probability of MG injury is higher than that of LG injury. Moreover, we also suggest relaxing stiff muscles, such as the MG, after exercise. Additional studies need to be conducted in the future to confirm this hypothesis and suggestion.We found that when the knee was fully extended, the increase in stiffness at PF50° was larger in the LG than in the MG and that no significant difference was observed at 0°, while the stiffness was higher in the MG than in the LG at DF25°. However, the same relationship did not hold when the knee was flexed to 90°. This result is consistent with that of Huang 44. In addition, findings from previous studies have shown that the AT had an inherently high stiffness. This result may be explained by the AT having a relatively small CSA yet experiencing inhomogeneous mechanical loading during daily activities, making the tendon susceptible to microtears and the type of gene expression that promotes type I collagen synthesis45. The AT has high viscoelasticity and mechanical strength, enabling it to respond to altered mechanical loading conditions46. Thus, if the AT is too stiff, it cannot absorb sufficient energy, and thus, there is an increased risk of injury. In addition, stiff tendons cannot transmit forces to neighboring tendons or muscles quickly and effectively and thus are more likely to be injured47. According to the results of this study, AT stiffness increases with increasing ankle dorsiflexion; when the stiffness is high, the risk of AT injuries or tears increases with increasing ankle dorsiflexion angles. According to the distribution of blood vessels, AT3cm is one of the areas with the least perfusion48. As mentioned above, combined with the lack of vascular perfusion and the high sensitivity of the AT3cm during ankle dorsiflexion, we speculate that the injury risk at AT3cm is high and that injuries in this area heal poorly. In addition, the stiffness decreased from AT0cm to AT3cm to AT6cm at DF25°. The reason for this result was that at ankle angles approaching the maximum range of motion, the magnitude of myotendinous junction displacement has reached a maximum, and the relative magnitude of tendon elongation has reached a minimum50. Therefore, more energy must be absorbed by the AT, and AT0cm is close to the calcaneus, so it incurs the largest component of the passive tensile force. Therefore, AT0cm has the highest stiffness at DF25° compared with other angles.Regardless of the knee angle, stiffness decreased from AT3cm to AT0cm to AT6cm at PF50° and 0°, while the stiffness decreased from AT0cm to AT3cm to AT6cm at DF25°. The main function of the AT is to act as a mechanical buffer to absorb energy during plantar flexion of the ankle and prevent soft tissue injury resulting from a sudden increase in external or internal forcesInterestingly, we found that the angles of flexion and extension of the knee and ankle joints have a large effect on the stiffness of the MG, LG and AT; however, the PF and SOL are only greatly affected by the ankle joint angle. This seems to be physiologically plausible since anatomical studies indicate that the MG, LG and AT, along with the plantaris, cross the knee joint, while the origin of the PF is located below the calcaneal tuberosity, and the origin of the SOL is located below the knee. Therefore, when the knee is fully extended, the MG, LG and AT are stretched, as are the SOL and PF, which do not act on the knee joint.As the results from the present study apply only to young participants who are healthy and free of injury or disease, additional studies need to be conducted in the future to quantify the stiffness distribution in patients who present with muscle contractures or tendon/PF injuries due to musculoskeletal or neuronal conditions. At present, the locations of muscle contractures or tendon/PF injuries and PF stiffness are difficult to quantify. Clinically, manual examinations only provide subjective and overall outcomes and do not provide specific values for each region. The design and protocol used in the present study may be applied in studies in patients to identify the locations that actually incur injuries to better prescribe targeted treatments, such as stretching, myofascial release and shock wave therapy. Moreover, if similar results are confirmed among patients, SWE measurements should be used for treatment efficiency and follow-up assessment purposes.51. Only male subjects were recruited in this study. In our pilot experiment, we found that the myofascial thickness of females was less than 1 mm, while males was larger than 1 mm. Therefore, we only recruited males in this study, and the results of the present study can not be generalized to female subjects. We need to develop more accurate algorithms and more diversified Q-boxes to meet the needs of future studies.The SWE technique can be used to identify biomechanical properties of muscles and tendons, but several technical parameters that influence the measurements still need to be addressed. First, it should be noted that the SWE technique is based on the assumption that the tissues being imaged are homogenous, isotropic and purely elastic. However, tissues do not always meet these criteria, and further consideration of the effectiveness of the algorithm is necessary for the use of the SWE technique in muscles, tendons and fascial stiffness assessments. Therefore, the angle between the orientation of the fibers and the probe influences the accuracy of the measurements. In this study, the midpoint of the transducer was placed perpendicular to the muscle/tendon on the skin’s surface, where we had previously made a mark, with light pressure, ensuring that the transducer was aligned with the longitudinal axis of the muscles/tendons to minimize the anisotropic effect on the measurement results. In addition, given that the muscles, tendons and fascia are not homogenous, isotropic or purely elastic, the present study only reported the Young’s modulus. In future studies, we will use two units of measurement (m/s and kPa). Second, SWE cannot be used to measure deep tissues. The stiffness of the SOL belly could not be assessed because its location was too deep. Therefore, the distal portion of the SOL was used for the measurements. Another limitation of our study is that EMG was not used to monitor muscle activity and ensure that the muscles did not contract during the tests. However, all volunteers were asked to remain relaxed, and no signs of muscle contractions were visible on the B-mode images. Therefore, we are confident that all volunteers remained in a relaxed state and followed the verbal instructions. The architecture and size of the muscle-tendon-fascia were not assessed in the present study. The force-generating capacity of muscles depends on their CSA and pennation angle. Although the shear modulus may not be affected by the anatomical CSA and pennation angle of the muscle, future studies still need to consider these parameters in patientsAnkle dorsiflexion induces nonhomogeneous changes in tensile stress between the main lower limb muscles and within the tendon/PF. This study provides a valuable reference for studies in which individual muscle/tendon/PF contributions are estimated.