Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody–antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody–antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65,  The majority of epitopes currently analyzed are discontinuous . It has The majority of epitopes are composed of 8–12 aas ,4,5,6. EEpitope structures are often rich in hydrophilic and charged aas, especially Arg, Asp and Glu, and with a lower ratio of aliphatic hydrophobic aas, although several epitopes have been described that depend on hydrophobic aas for stable interactions ,14,15,16The number of aas essential for antibody reactivity varies depending on the type of antibody–antigen interaction. For example, an interaction with a high affinity may experience a 1000-fold reduction in antibody reactivity when substituting an essential aa, whereas a neighbour aa may be substituted without interfering with the affinity, although they are a central part of an epitope structure ,7,9,20. Several assays for epitope characterization exist, but one of the most straightforward methods is to use immunoassays ,10,13,19Knowledge of epitopes related to the glutamic acid decarboxylase (GAD) protein is sparse. GAD is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the conversion of L-glutamic acid to the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and is among others expressed in the pancreatic β-cells ,24,25. IThe two isoforms are divided into three functional domains, an amino-terminal domain, a middle domain where the catalytic site resides (PLP-binding domain), and a carboxy-terminal domain. Roughly, antibodies to GAD67 are primarily associated with stiff-person syndrome (SPS) and autoimmune polyendocrine syndrome type 1, whereas GAD65 is a dominant autoantigen in type 1 diabetes (T1D) ,27,28,29Epitope specificity in GAD-related neurological diseases has been examined only for SPS, indicating that the antibodies are directed against continuous GAD epitopes in all 3 domains, but predominantly against the catalytic region . In contAs epitope characterization of discontinuous epitopes can be rather challenging, no in depth characterization of GAD65 epitopes has been conducted until now. A single study describes reactivity to continuous GAD65 epitopes in patients with slowly progressive T1D. Here, GAD65 antibodies reacted specifically with an N-terminal linear epitope in the membrane-anchoring domain between aas 17–51 . FurtherIn this study, three continuous GAD65 epitopes were thoroughly characterized with emphasis on essential aas necessary for antibody recognition and aa side chain functionality in order to obtain further knowledge of the specific interactions between GAD65 and autoantibodies related to T1D. To identify potential epitopes of two GAD65 antibodies, screening of overlapping resin-bound peptides covering the respective immunogens (aa 4–22 for the mAb GAD65 and aa 353–585 for the pAb GAD65) was conducted by modified enzyme-linked immunosorbent assay (ELISA). To identify the terminal epitope borders of the GAD antibodies, N- and C-terminal truncated resin-bound peptides were screened for antibody reactivity by modified ELISA. Peptides 1 (MASPGSGFWSFGSEDGSGDS), 52 (PSLRTLEDNEERMSRLSKVA) and 55 (GTTMVSYQPLGDKVNFFRMV) were used as templates for the generation of truncated peptides. 8FWSFGSE14. For the pAb, which recognized two continuous epitopes, notable antibody reactivity was found to the N-terminal truncated peptide TLEDNEERMSRLSKV and the C-terminal truncated peptide PSLRTLED c,d, alth515TLED518 and 550LGDKV554. Screening of GAD65 pAb reactivity to truncated analogues of peptide 55 (GTTMVSYQPLGDKVNFFRMV), illustrated that the N-terminal truncated peptide LGDKVNFF and the C-terminal truncated peptide MVSYQPLGDKV were the shortest peptides to be recognized e,f althoTo determine the complete GAD65 epitopes, competitive inhibition assays were conducted using relevant truncated peptide analogues.8FWSFGSE14 constitutes the complete epitope of the mAb. 514RTLED518 constitutes the complete epitope 1 of the pAb. When analyzing antibody reactivity of the pAb to the truncated peptides (TLED-SLRTLEDNEE), containing the central TLED motif, it was observed that the four aas alone were not sufficient to inhibit antibody reactivity to the control peptide (peptide 52) b. The pe549PLGDKVNF556 constitute the second complete continuous epitope of the pAb. Finally, the second continuous epitope of the pAb was determined using truncated peptides (PLGDKV-YQPLGDKVNFFR) c. As seeTo determine the contribution of each aa in the identified epitopes to the specific antibody interaction, competitive inhibition assays were conducted using Ala-substituted peptides. Finally, functionality substituted peptides were screened for antibody reactivity, to determine whether the specific aa contribution to the antibody–antigen interaction relates to side chain functionality or the specific side chain. Various aa-substituted analogues were tested for antibody reactivity in competitive immunoassays. For the mAb, the aas in position 1 and 4 were essential for reactivity, as peptides containing aas of similar functionality in these positions did not inhibit antibody reactivity. Only Ser in position 3 and 6 could be substituted with Thr without influencing antibody reactivity d. PeptidAnalysis of the antibody reactivity of the pAb to the RLTED epitope, revealed that only Asp in position 5 could not be replaced with an aa of similar functionality without interfering with antibody reactivity e. The reTo identify the orientation of the identified epitopes, the crystal structure of GAD65 was examined using RasWin software. Unfortunately, the N-terminal structure of GAD has not been crystalized, thus no structure could be assigned to the mAb epitope. However, given that the epitope is located in the first 20 aas of the proteins, the epitope is most likely localized in a flexible region without any ordered structure, which applies to the majority of terminal epitopes ,34.514RTLED518 (colored in red) was found in a flexible region connecting an α-helix (green) and a β-strand (blue). Unfortunately, the structure of aas 518–519 was not verified in the crystal structure. These aas are represented by red (aa 518) and grey (aa 519) dots. 2 aas at this position are not likely to alter the complete protein structure. The epitopes of the pAb were verified by the crystal structure. Epitope 549PLGDKVNF556) of the pAb is located in a flexible turn connecting two strands. As seen, the essential Leu residues in the epitopes are accessible , lacking these two aas. The specific side chain of Phe8 was essential to the binding of the antibody, as it could not be replaced with Ala or an aa of similar functionality. In contrast, Trp could not be replaced with Ala but to some degree with Phe, indicating that a large hydrophobic aa in this position is sufficient to sustain antibody reactivity. The remaining aas could to some degree be substituted with Ala and still inhibit antibody reactivity. The present study describes epitope characterization of antibodies to GAD65. The mAb recognized a single epitope, whereas the pAb recognized two linear epitopes. It remains to be determined whether the pAb recognized two linear epitopes due to the structural nature of the immunogen applied, as it remains unknown, whether a native or non-native immunogen was used for immunization. The mAb, originally generated to aas 4–22, recognized the aa sequence WSFGSE14 . Especia514RTLED518 and 549PLGDKVNF556. For the RLTED epitope, the aas Thr and Leu inhibited antibody reactivity by approximately 50% when inhibited with Ala, but not when substituted with aas of similar functionality, indicating that these aas contribute to sustain a stable structure. Alternatively, may Leu contribute with hydrophobic interactions, as replacement of Ala to some degree inhibited antibody reactivity, whereas replacement with Ile did not influence antibody reactivity. This remains to be determined. Only Asp appeared to interact directly through the side chain with the antibody, as substitution with Ala reduced antibody reactivity. Asp could, however, to some degree be replaced with Glu, indicating that a negatively charged aa in theory is sufficient to obtain antibody reactivity, whereas the remaining aas most likely contribute to maintain a stable conformation. A similar reactivity pattern was observed for the PLGDKVNF epitope. The majority of the aas could be replaced with Ala or an aa of similar functionality without interfering with antibody reactivity. Only Leu could not be replaced with Ala without reducing antibody reactivity, whereas Leu could be replaced with Ile without interfering with antibody reactivity. The pAb originally directed to aas 353–585, recognized 2 epitopes, the aas As seen for both the mAb and the pAb, peptide backbone and specific aa interactions were essential for antibody binding. These findings are in accordance to previous studies describing that some epitopes have a significant dependency of specific aa side chains in the epitope, whereas other epitopes only make direct contact through a single or two aa side chains ,10,13,19The fact that the mAb seemingly was more dependent on specific aas for antibody recognition, may be explained by the nature of the antibodies. The mAb is more specific for a single epitope, whereas the pAb is less specific for the individual epitope, due to heterogeneity. The pAb represents a collection of antibodies that are directed to the complete fragment, aa 353–585. As a consequence, the pAb has a high overall affinity due to the recognition of multiple epitopes. When analyzing antibody reactivity to the two individual epitopes, the pAb was very tolerant to changes in the primary structure of the epitopes. One explanation to this observation is that the affinity of the pAb to the two epitopes is reduced, which was supported by prolonged developing times and reduced reactivity and ultimately increased tolerance to aa changes. This remains to be verified, e.g., by SPR experiments. As presented in this study, hydrophobic aas were essential for antibody reactivity. Originally, it was emphasized that hydrophilic amino acids are important for antibody–antigen interactions, which conform to that many epitopes are surface-oriented ,4,14. SeStructural analysis of the epitopes of the pAb revealed that the epitopes are located in flexible structures, which is in accordance to the literature ,10,13. TAntibody reactivity to the GAD67 isoform, with a sequence identity of approximately 74%, was determined as well, but the GAD65 antibodies were specific for GAD65 (results not shown), as the epitopes identified were not present in the GAD67 isoform. A similar study has been conducted using the GAD67 isoform as well . Here, tAntibody reactivity to GAD65 epitopes has been very sparsely described, which only identify potential antigenic regions rather than concrete epitope structures. This most likely relates to that the majority of the immunodominant epitopes of GAD65 are discontinuous ,16. This2 resin was purchased from RAPP Polymere GmbH . Alkaline phosphatase (AP)-conjugated anti-mouse IgG and para-nitrophenylphosphate (pNPP) were purchased from Sigma Aldrich . Tris-Tween-NaCl (TTN) buffer , AP substrate buffer , carbonate buffer  were from Statens Serum Institut . Mouse GAD65 (clone no. 144) mAb was from GeneTex (immunogen: aa 4–22 of human GAD65) and rabbit GAD65 (clone no. 2) pAb was from Origene (immunogen: aa 353–585 of human GAD65). Overlapping peptides used for initial scanning and free peptides used for final epitope identification were purchased from Schäfer-N . Tentagel S NH8FWSFGSE14, 514RTLED518 and 549PLGDKVNF556 as templates. Each aa was systematically substituted with Ala or an aa of similar functionality. All peptides are listed in The human GAD65 published sequence (Swiss-Prot: Q05329.1) was used to generate overlapping 20mer peptides, which covered the complete protein. Each peptide contained an overlap of 10 aas to the next peptide. In total, 58 peptides were generated by 9-fluorenylmetyloxycarbonyl solid-phase peptide synthesis. Peptides 1–3 were screened for reactivity to the mAb and peptides 36–58 were screened for reactivity to the pAb. Peptide 2 (aa 11–30), 55 (aa 511–530) and 57 (aa 510–560) were used as templates for generation of N-terminal and C-terminal truncated peptides. Ala- and functionality-substituted peptides were generated using the aa sequences pNPP  diluted in AP substrate buffer. Finally, the buffer was transferred to a Maxisorp microtitre plate  and the absorbance was measured at 405 nm, with background subtraction at 650 nm, on a Thermomax microtitre plate reader .Resin-bound peptides were screened for reactivity as described elsewhere ,10. BriepNPP  diluted in AP substrate buffer and the absorbance was measured at 405 nm, with background subtraction at 650 nm, on a Thermomax microtitre plate reader . Maxisorp microtitre plates were coated with peptides 1, 52 and 55 (1 μg/mL) in carbonate buffer for 2 h at RT. In another plate, peptide analogues (1 mg/mL) and GAD65 antibody  were incubated for 1 h at RT. Next, the peptide-antibody samples were added to the coated microtitre wells and incubated for 1 h at RT on a shaking table, whereafter the wells were rinsed with TTN. Following this, the wells were incubated with AP-conjugated anti-mouse IgG or anti-rabbit IgG  for 1 h at RT on a shaking table. Bound antibodies were quantified using The software Raswin Cn3D 4.1 was used to identify the location and secondary structure of the identified mAb and pAb epitopes in human GAD65 (PDB ID: 2OKK).


Prosthetic feet are spring-like, and their stiffness critically affects the wearer’s stability, comfort, and energetic cost of walking. Despite the importance of stiffness in ambulation, the prescription process often entails testing a limited number of prostheses, which may result in patients receiving a foot with suboptimal mechanics. To understand the resolution with which prostheses should be individually optimized, we sought to characterize below-knee prosthesis users’ psychophysical sensitivity to prosthesis stiffness.We used a novel variable-stiffness ankle prosthesis to measure the repeatability of user-selected preferred stiffness, and implemented a psychophysical experiment to characterize the just noticeable difference of stiffness during locomotion.All eight subjects with below-knee amputation exhibited high repeatability in selecting their Preferred Stiffness (mean coefficient of variation: 14.2 ± 1.7%) and were able to correctly identify a 7.7 ± 1.3% change in ankle stiffness (with 75% accuracy).This high sensitivity suggests prosthetic foot stiffness should be tuned with a high degree of precision on an individual basis. These results also highlight the need for a pairing of new robotic prescription tools and mechanical characterizations of prosthetic feet. Mobility is a key predictor of quality of life for lower-limb amputees . CriticaA common device prescribed to persons with lower limb loss is the modern energy storage and return (ESR) prosthetic foot, which intends to mimic the spring-like behavior of the biological ankle-foot complex during gait. ESR feet deflect during mid-stance, storing energy which is subsequently returned during terminal stance phase to help propel the body forward . The effDespite the importance of stiffness in locomotion, prosthetic manufacturers do not offer quantitative stiffness information—only qualitative scales associated with body weight and activity level—making cross-comparison between feet exceedingly difficult. The prescription process is further hindered by the excessive time required to test each prosthesis, as each tested device must be individually donned, aligned, adapted to, assessed, and doffed. As a result, it is not uncommon for patients to pilot only one or two prostheses; furthermore, the down-time required to switch prostheses hinders direct comparison of their impact on patients.With the advent of computer-controlled active and quasi-passive / semi-active prostheses, new tools for improving the prescription process are on the horizon. Caputo et al. have proposed using a powered prosthesis emulator to adjust mechanical behavior of the prosthesis in real-time, thereby quickly simulating the mechanics of different prosthetic feet . With suThe resolution with which below-knee prosthesis users can discriminate changes in prosthesis stiffness has important implications for the balance of patient and clinician voices in the shared-decision prescription process. If, for instance, patients are highly sensitive to small changes in stiffness, and exhibit high repeatability in their preferences, then clinicians can rely more heavily on patient feedback. This could be particularly true if patient sensitivity to stiffness proves to be of substantially higher resolution than clinicians’ perception of the corresponding gait changes. If, conversely, patients exhibit poor sensitivity, or have difficulty finding or communicating their preference, then prosthetists may need to emphasize alternative metrics for assessment.In this experiment, we sought to determine the sensitivity of below-knee prosthesis users to prosthetic ankle stiffness. Specifically, we assessed 1) the repeatability (variability) of user-selected preferred stiffness, and 2) the difference threshold, or Just Noticeable Difference (JND), for stiffness during locomotion. Additionally, we sought to determine if simple measures, such as self-reported mobility assessment and peripheral vibration sensing, accurately predicted perception of stiffness. Our results provide a rigorous yet patient-centric perspective on the importance of optimizing stiffness on an individual basis, and provide valuable insight into the potential for new prescription methods using robotic devices.To perform this experiment, we used a custom Variable Stiffness Prosthetic Ankle-Foot (VSPA Foot), which has the distinct advantages of providing known torque-angle mechanics, while enabling adjustment of stiffness in real-time, between steps during locomotion . First, The VSPA Foot  was useThe cam profile was designed to create a linear torque-angle relationship at the ankle joint in both plantarflexion and dorsiflexion, providing the mechanics of a simple torsional spring at the ankle joint. Plantarflexion stiffness  was designed to be 33% of dorsiflexion stiffness. This ratio follows work completed by Major et al.  based ondorsiflexion stiffness, as the ankle is primarily spent dorsiflexed from neutral during stance phase. The relationship between dorsiflexion stiffness and slider position is described by a cubic fit to the characterization data  over WiFi, or directly by the subject, using a small dial which could rotate indefinitely and had no absolute reference. The ankle was worn without a shoe to avoid additional compliance and confounding factors [SolFlex crepe, Shore A durometer 50–55, SoleTech, Nahant, MA, US) was epoxied to the bottom of the foot to aid in shock absorption, and a thin tread  was epoxied below that shoe material for traction.The ankle could be commanded to change stiffness by either the experimenter, who could communicate with the onboard computer  were recorded Table . Periphek = 7.5 x Mass (kg), a standardized setting established from preliminary experiments. The prosthetist stopped the treadmill as necessary to adjust the alignment. During this process, the speed of the treadmill was slowly increased by the subject to 1.0 m/s, which was the nominal experiment speed. All experiments were performed at 1.0 m/s, except by Subjects 2, 4, and 6, who felt uncomfortable at this speed and elected to walk at 0.87 m/s. This speed is slightly less than is typically reported for self-selected walking speed, but was selected to ensure stability during trials in which the stiffness was changed between steps. When the prosthetist and subject were content with the alignment, and the subject expressed comfort walking with the prosthesis at the experiment speed, the treadmill was stopped for a short break before Experiment 1.Subjects first walked on the treadmill with their customary prosthesis for several minutes to familiarize themselves with the treadmill and laboratory space. The customary prosthesis was then removed, and the VSPA Foot was fit and aligned to their customary socket by a certified prosthetist. The subjects then walked on the treadmill with the VSPA Foot set to an initial stiffness given by PS), which is the ratio of the standard deviation (unbiased estimation) to the mean.After familiarization and alignment, the subjects walked on the treadmill and were asked to “select the stiffness that you find to be the most comfortable at this walking speed.” To change foot stiffness, subjects were free to rotate the dial at any time during the gait cycle; however, active stiffness adjustments were made only during the swing phase of gait. Subjects were encouraged to explore a range of stiffness levels until the prosthesis felt uncomfortably stiff or uncomfortably soft. After selecting their preferred stiffness (PS), the treadmill was stopped, and the researcher “reseeded” the stiffness by pseudo-randomly increasing or decreasing it by 25%. The subjects repeated the process of selecting a PS for a total of five trials. The process typically took longer (approximately 10 min) for the first trial, and less time in subsequent trials, as the subjects learned the effects of changing stiffness and became more confident in their choices. Subject 2 did not fully understand the instructions and had to be retested on a different day.reference stiffness and a comparison stiffness. The reference stiffness corresponded to each subject’s mean preferred stiffness (PS) and was constant across all trials. The comparison stiffness varied between trials, and was equal to the PS scaled by a value in the set: {0.80, 0.85, 0.90, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20}. This range was informed by preliminary experiments, with the intent of generating both incorrect answers for the smallest changes and very high accuracy for the largest changes. Each comparison stiffness was presented eight times, for a total of 72 trials. The reference stiffness was presented first on half of the trials, and second on the other half. The order of stiffness values was set before the experiment. The trials were pseudo-randomized; to avoid destabilizing the subjects, the trials were randomly re-shuffled until all consecutive stiffness values were within 25% of each other.Stiffness perception was assessed on the same day as Experiment 1, following a short break. Stiffness perception was determined by changing the stiffness of the prosthesis while subjects walked on the treadmill; specifically, we implemented a two-interval forced-choice (2IFC) task and the method of constant stimuli , 18. SubProsthesis stiffness was changed by the experimenter approximately every 6–8 strides, and the change was indicated to subjects by an audible tone. The number of strides was chosen to allow subjects ample time to sense and report the stiffness changes, while minimizing the amount of time spent walking. Subjects only responded after the transition from the first stimulus to the second stimulus . Subjects were allowed to take breaks whenever necessary to avoid fatigue bias. Before the official trials began, subjects were trained with stiffness changes of 15–25%, until subjects reported that they clearly understood the paradigm.P) of trials judged stiffer than the reference was used to calculate the psychometric function, a sigmoidal function which describes the relationship between stiffness change and probability of perceiving that change as an increase in stiffness. Specifically, a logistic function was fit using a maximum likelihood criterion, with lapse rate fixed at 0.02. The lapse rate accounts for trials in which subjects respond independently of stimulus level , and reduces biases on the parameters that determine the psychometric function [P = 0.25 and P = 0.75, denoted X0.25 and X0.75, are used to calculate the threshold of perception. This threshold is commonly termed the Just Noticeable Difference (JND), and is specifically calculated as:For each stiffness value, the proportion (function . The compercent change in stiffness that can reliably (with 75% accuracy) be identified as having been an increase or decrease in stiffness.In this study, the JND represents the smallest At the end of the experiment, subjects were asked to describe the strategies they used to sense changes in stiffness. As mentioned previously, Subject 2 did not originally understand the instructions for selecting preferred stiffness, so their JND trials were performed with a reference stiffness of 571.5 Nm/rad  and preferred stiffness was reassessed on a second day.To assess the sensitivity of ankle kinematics to ankle stiffness, and specifically the magnitude of biomechanical changes associated with the JND, ankle kinematic data were recorded in separate walking trials from the onboard ankle encoder. Subjects walked on the treadmill for 90 s at each of five stiffness levels (order randomized): the mean preferred stiffness modified by 0%, ± 10%, and ± 20%.The kinematic data were recorded at 30 Hz by an onboard 14-bit absolute encoder, zero-lag filtered with a fourth-order, 7.5-Hz Butterworth filter in post-processing, and then up-sampled to 100 Hz with a spline interpolation. An angle of zero corresponded with the average angle during swing. The last 20 of each subject’s strides were segmented and averaged. The kinematics were then averaged across subjects at each of the tested stiffness levels. An estimate of the kinematics at the preferred stiffness ± JND was created with a linear interpolation from the ankle kinematics at the closest measured stiffness levels.PS. Neither JND nor CVPS were normally distributed , therefore standard errors were calculated with bootstrapping .Each fitted psychometric function was ensured to have an acceptable goodness of fit , 19. MeaPS. JNDs were also regressed on PLUS-M T-scores and Vibration sensing score  to see if either simple test may be used to predict perception.To test whether there was a relationship between stiffness perception and consistency of preferred stiffness selection, individual JNDs were linearly regressed on CVPS), which serves as a measure of the subjects’ consistency of selection, was 14.2 ± 1.7%.Subjects’ self-selected preferred stiffness Fig.  ranged fPsychometric functions were fit to the data for all eight subjects Fig. , and indPS), PLUS-M score, and vibration sensing score  positive correlation between CVPS and JND. There was a non-significant (p = 0.25) negative trend between self-reported mobility (PLUS-M score) and JND; removing Subject 2 due to acquiescence bias (see endnote 2), this correlation became significant (p = 0.04). As expected, the dysvascular subjects tended to have worse vibration sensing, but there was not a significant correlation between vibration sensing score and JND (p = 0.85).The individual JNDs were regressed against three predictors: preferred stiffness variability  or elboPS). Specifically, subjects with high repeatability in selecting preferred stiffness could perceive smaller changes in stiffness, suggesting that the ability to consistently select a preferred stiffness may depend largely on the ability to sense it  were likely dependent on learning the sensations and effects of stiffness during the exploration in Experiment 1.The pool of subjects is not a representative sample of all lower-limb amputees, due to the inclusion criteria . It is possible that lower-level ambulators (below the Medicare Classification Level K3) will be less sensitive to stiffness, due to a variety of factors such as less experience walking with a prosthesis, or comorbidities such as peripheral neuropathy or excessive adipose tissue in the residuum.Preferred stiffness is likely dependent on several factors which were controlled in our experiment, but may vary in a clinical setting. It is unknown how changes to alignment affect preferred stiffness; this is a potential avenue for future study. Similarly, we elected not to use a shoe on the prosthetic foot to facilitate complete control of stiffness adjustments, but shoes can add substantial series compliance , which wDue to the mechanics of the VSPA-Foot, prosthesis plantarflexion and dorsiflexion stiffness could not be changed independently. We made the assumption that subjects would be most sensitive to dorsiflexion stiffness, and thus deemed dorsiflexion stiffness the nominal stiffness in the experiments. Feedback from subjects regarding their strategy confirmed this, but it is possible that we would have received different results with independent variation in stiffness or a different ratio.Below-knee amputees were able to sense a 7.7% difference in prosthetic ankle stiffness, and demonstrated high repeatability in selecting their preferred stiffness (coefficient of variability of 14.2%). This information substantiates the argument that that new tools are needed to allow patients and prosthetists to quickly locate an optimal stiffness based on shared decisions, and that standardized systems for measuring and reporting prosthesis stiffness should be adopted by manufacturers, researchers, and clinicians. Additionally, these results should inform future studies investigating the effects of prosthesis stiffness, providing a weight-normalized reference stiffness and a feasible range of stiffness levels to test.


There are plenty of publications indicating that in the management of small and medium size hole (less than 400 µm), use of long-lasting gas and face-down position is not always required; however, it may be necessary for the treatment of large holes. Ocriplasmin and expansile gas had been reported to be successful for management of small- and medium-sized holes and vitreomacular attachment.Macular hole has been believed to be a disorder of vitreomacular interface, which forms as a result of abnormal vitreous traction from incomplete vitreous detachment. However, our recent studies demonstrated that dynamic forces, caused by mobile posterior cortical vitreous with fluid currents, exist already at early stages of macular hole development. Therefore, in eyes with flexible vitreous, the contributions of tractional forces due to vitreous shrinkage are unlikely. These facts indicate that in the development of idiopathic macular holes, there is a greater contribution of dynamic forces than has been previously reported. This review also evaluates the recent findings in the assessment of the idiopathic macular holes and the recent therapeutic strategies for optimal management. Inner limiting membrane is considered to improve anatomical closure rate; however, it is still questionable if peeling is necessary in holes less than 250  Macular hole is a retinal defect located in the centre of the fovea, causing significant vision impairment . Knapp iThere are two types of macular holes which can be observed: idiopathic macular holes (IMH) , which iDevelopment of MH with retinal detachment is a specific complication of high myopia with posterior staphyloma  .MHs can resolve, persist stable, or progress to full-thickness macular holes. According to Gass, in case if complete posterior vitreous detachment develops, the fovea can return to normal, or if Müller cell cone is stripped from the retinal surface, a lamellar hole may develop .In the general population, the prevalence of MHs was reported to be around 3.3 per 1,000 people . Until 1µm), medium (>250 µm and ≤400 µm), and large (>400 µm) [μm and 450 μm MH, and in sense of planning surgery, 400 μm is not very practical [μm is a much better marker to divide medium and large macular holes, based on their results with 90% success in standard full-thickness macular hole (FTMH) vitrectomy involving internal limiting membrane (ILM) peel and gas tamponade on medium MH between 250 and 650 μm [μm) is less successful, and such techniques as ILM flaps and retinal expansion technique for macular hole apposition (RETMA) should be considered for this matter [μm are excluded from stage 3 compared to 1988 classification [In the MH formation, an important role is on the vitreous traction . Gass cl>400 µm) . Howeverractical . Accordid 650 μm . They nos matter . Also, ification .μm), medium (>250 to ≤400 μm), and large macular hole (>400 μm) [Recent publication of results of European Eye Epidemiology (E3) consortium to standardize epidemiological studies proposed a spectral-domain optical coherence tomography- (SD-OCT-) based classification for macular diseases, where MHs are subclassified as small (<250 >400 μm) . A detai>400 μm) . Chun et>400 μm)  into 2 t>400 μm) . MH subd>400 μm) . Preoper>400 μm) .µm without VMA [Some holes, especially without vitreomacular attachment (VMA), may close spontaneously. Spontaneous resolution of such holes was reported in a range from 2.7% to 8.6% of cases –17. Somehout VMA . But usuhout VMA , 20 had hout VMA .µm and 25 µm [SD-OCT provides precise measurements of MH dimensions, and contemporary equipment allows to obtain images with the resolution between 10 nd 25 µm . OCT imaMeasurements of base diameter (BD) and minimum linear diameter (MLD), hole form factor, macular hole index, and tractional hole index are described for MH evaluation , 24.In 2012, Mori et al. published the results of wide-angle montaged images of SD-OCT in patients with macular hole. They described two patterns of posterior vitreous configuration, “smooth or wavy” vitreous surfaces. Posterior vitreous cortex had a smooth curvature at the onset of separation, and with progressive separation, posterior vitreous folds increased. This finding indicates redundancy progression of posterior cortical vitreous through the process of MH formation. This “wavy” interface implies vitreous mobility. The mass and movement of the vitreous represents the potential force to act on the retina. In addition, granular hyperreflection was observed in 50% to 60% of eyes with stage 1 or 2 holes in the peripheral vitreous and in 33% of eyes with stage 3 or 4 holes, and also they described areas of peripheral double-layered retinoschisis in the peripheral retina adherent to posterior vitreous cortex .There has been a controversy in the origin of vitreous traction in the pathogenesis of MH formation. Guyer and Green  and JohnAdditionally, need to mention, even though IVTS group provided in 2013 definitions of lamellar MH and macular pseudohole based on B-scan OCT image findings , recent The most important prediction for successful MH surgery is preoperative visual acuity (VA). The better the preoperative VA is, the higher the rates of visual gain and anatomical closure , 32. ShoVitrectomy for closure of MH is reported to have high success (85%–100%) . JacksonP < 0.001).Eckardt et al. in 1997  was the μm. Free flap ILM is used in patients with persistent MH hole after previous surgery, where a free patch of peripheral peeled ILM is placed over or in the MH [Nowadays, ILM peeling becomes a routine technique for MH surgery for most surgeons. For staining the ILM, such adjuvants as indocyanine green , triamcin the MH .As an alternative to ILM peeling ILM, abrasion had been proposed, in order to thin the ILM and loosen its adhesion to the underlying retina while still stimulating glial cell activation . HoweverFluid-air exchange with subsequent gas exchange was usually carried out, after vitrectomy and ILM peeling . Gas tamDifferent isovolumetric gas concentrations used by surgeons and total duration of gas filling usually range from 2 to 11 weeks  [Closure rate of 90% with SF6 and 91% with C3F8 was reported in a retrospective cohort study with face-down positioning postoperatively . In a prMaintaining the face-down positioning with gas tamponade was reported to be useful in the MH closure; however, it is not comfortable for patients and can be associated with complications, such as back pain or ulnar nerve palsies . HoweverOCT in the early postoperative period is reported to be a useful tool to determine whether MH is closed even in gas-filled eyes. Swept-source optical coherence tomography (SS-OCT) is known to have longer wavelength than spectral domain OCT and has the ability of a higher penetration through the opaque media, thus resulting in high-quality, high-resolution images in gas-filled eyes , 59. Theµg) demonstrated a better resolution of macular adhesion (26.5 vs. 10.1% in the placebo group) as well as an increased rate of nonsurgical macular hole closure (40.6 vs. 10.6% in the placebo group) in a multicentre, randomized, double-blind, phase III trials [Ocriplasmin is a truncated form of human plasmin with proteolytic activity for vitreoretinal interface components including fibronectin and laminin; it was approved for the treatment of symptomatic VMA, including association of VMA with MH of <400 , 15. A sI trials . OcriplaI trials . OcriplaI trials .27 G surgery is a technique using instruments with a diameter of instrument ∼0.35 mm. However, visual outcome and closure results were reported to be comparable for narrow-gauge surgery vs 20 G . SakagucCurrent treatment options for MH management discussed in this review allow to achieve high rate of macular closure and improve visual recovery. Use of repeated OCT is useful for confirmation that correct tactic had been chosen particular for expansile gas or ocriplasmin usage. For the patients with small or medium holes, significantly better results are expected by application of ocriplasmin, however, with lower closure rate that surgery. Holes without VMA vitrectomy is usually the only possible treatment with the choice to perform ILM peeling and uses a short-term or long-term gas or prone posture.


Dioscoreae opposita Thunb. cv. Tiegun were isolated by aqueous steeping (SBS), enzyme extraction (EBS), and alkaline extraction (ABS) methods, respectively. The physicochemical, mineral composition, thermal and morphological characteristics of these starches were investigated. The starch granules were oval, spherical and kidney-shaped and its crystal type is a mixture of A-type and B-type patterns. The starches having larger average granule size showed more amylose and phosphorus contents than those with smaller average granule size. Differential scanning calorimetry (DSC) showed that the SBS had an endothermic transition ranging from 65.8 °C to 76.3 °C with an enthalpy of 2.0 J/g. The endothermic transitions of ABS and EBS showed the regions of 67.9 °C to 73.0 °C, and 66.8 °C to 82.0 °C, respectively. The gelationization enthalpies of ABS and EBS were 13.8 and 11.5 J/g, respectively. Additionally, ABS presented greater clarity in comparison with EBS and SBS. Pasting properties indicated that ABS had the highest peak viscosity, breakdown, but SBS had the lowest trough, final viscosity, setback, and pasting temperature. Generally, ABS and EBS could be used as food thickener or frozen food additives. SBS and EBS were potential technological alternatives in quality preservation of frozen starch-based products and other industrial applications. Starches from the bulbils of  Dioscoreae opposita Thunb of varieties of cultivars are planted in China, particularly in Henan Province, for medical and food purposes. Among them, the most sought is Dioscoreae opposita Thunb. cv. Tiegun, which is commonly named “Huai Shan Yao” in China. This cultivar is edible and has been routinely used to treat diarrhoea, diabetes and asthma for over 2000 years  , peak [pT], and conclusion [cT]), gelatinization temperature range (c−ToT), and gelatinization enthalpy (DH) of three different starches are exhibited in The DSC thermograms of samples showed typical glass transitions of a semi-crystalline biopolymer. The transition temperatures  [DSC showed that SBS had an endothermic transition ranging from 65.8 °C to 76.3 °C with an enthalpy of 2.0 J/g. The endothermic transitions of ABS and EBS showed the regions of 67.9 °C to 73.0 °C, and 66.8 °C to 82.0 °C, respectively. The gelationization enthalpies of ABS and EBS were 13.8 and 11.5 J/g, respectively. The gelatinization temperature for bulbils starches of 72.9 °C)  and lowe72.9 °C) . The dif72.9 °C) . The hig72.9 °C) . Hence, 72.9 °C) .p < 0.05). Among them, SBS had the lowest enthalpy of gelatinization (2.0 J/g) while ABS had the highest enthalpy (176.4 ± 0.23 J/g). High gelatinization enthalpy of ABS might be due to its high crystallinity, high proportion of long chains and great average chain length [The gelatinization is indicative of the amount of energy to disrupt double helices and causes the loss of molecular order. The ordered structures may be formed by long chains. The greater the amount of ordered structures required, the more thermal energy to break the crystalline order . Gelatinn length . In addiThe swelling powers were similar as the temperature increased from 55 °C to 65 °C, indicating that starch granules did not swell at temperature below 65 °C, and this is consistent with the DSC result. Comparing with the values obtained at 65 °C, once the temperature of 95 °C was reached, the swelling powers of starches increased by approximately 13.0 (g/g), 14.0 (g/g) and 15.9 (g/g) for SBS, EBS and ABS. The effect of temperature on starch swelling power is attributed to the greater vibration of the molecules at a higher temperature, causing the combination of starch with the water molecule to form hydrogen bonds. The results illustrated that starches swelled quickly from 75 °C to 85 °C, and the swelling power of ABS was lower than that of SBS and EBS. Additionally, the swelling powers of SBS, EBS and ABS at 95 °C were lower than normal potato (1159 g/g) and normal corn (22 g/g), but higher than high amylose corn (6.3 g/g) . The lowThe results showed that the solubilities of EBS, ABS and SBS increased slightly with the increase in temperature from 55 °C to 65 °C, whereas they increased significantly from 0.4% to 12.9%, 0.1% to 12.4%, 1.0% to 15.1% for SBS, EBS and ABS in the range of 65 °C to 95 °C, respectively. Since the solubility of the sample could be affected by the amylose content, amylose-rich starch has lower solubility even after long heating periods . The solThe clarity of starch gels was expressed as light transmittance by measuring the absorbance at 640 nm after fixed intervals of storage period. It is important in food and industrial applications as it affects product quality, in particular its aesthetic attractiveness. ABS presented greater clarity in comparison with EBS and SBS . The greFreeze-thaw stability is an important property, which could be used to evaluate the ability of starch to withstand undesirable physical changes during freezing and thawing. The freeze-thaw stability during freeze-thaw cycles  was evaluated with the percentage of syneresis and is exhibited in A starch sample is freeze-thaw stable if it releases little or no exudate when subjected to freeze-thaw cycles. From the results of 3% and 5% starch samples , the synPasting, which occurs after the gelatinization process, implicates granules swell in water to form a gel . The pasPasting temperature is a measure of the temperature at which a starch starts to thicken. The ability of starches to withstand heating at high temperature and shear stress is an important factor in many processes. EBS exhibited a higher pasting temperature (85.1 °C) than SBS (83.6 °C) and ABS (84.4 °C), in accordance with the gelatinization temperatures obtained by DSC . The pasDioscorea opposite Thunb. cv. Tiegun were collected from Wen County, Jiaozuo City, Henan Province of China. The morphological differences among Dioscorea opposite Thunb. cv. Tiegun and other varieties are visible in Fresh bulbils of The fresh bulbils were first washed with clean water and then pulped in a commercial kitchen blender . The slurry was filtered using suction, and the large granules were retained and pulped again. The slurries were collected and washed with deionized water. After depositing, the supernatant was discarded and the upper non-white layer was physically scraped off. The remaining white layer was re-suspended in deionized water, followed by centrifugation at 4000 rpm for 15 min. The re-suspended procedure was repeated seven or eight times and the total duration was 10 h. The final starch product was dried in an oven  at 45 °C for 12 h. The dried starch was sieved using a 200-mesh sieve and collected and named as SBS. The yield was then calculated as the percentage of the fresh bulbils weight.The fresh bulbuls were washed, chopped into small pieces, then dried in an oven against spoilage at 45 °C for 24 h and stored in a desiccator at room temperature before use. Dried material of 100 g was soaked in 400 mL deionized water solution containing alkaline proteinase at a concentration of 100 mg at a pH of 10 (pH of the mixtures was adjusted using 0.1 M NaOH). The mixture was then incubated at 45 °C for 150 min and subsequently at 4 °C refrigerator for 3 h. The upper supernatant was discarded and the bottom starch layer was then pulped with a blender. The slurry was washed with deionized water and stirred for 3 h. After depositing, the upper layer was discarded and the upper non-white layer was physically scraped off. The remaining white layer was re-suspended in deionized water. The procedure was typically repeated seven or eight times until the upper supernatant turned clear and the starch layer exhibited a lightly white color appearance. The total duration was 10 h. The final starch product was dried in an oven at 45 °C for 12 h and then sieved with a 200-mesh sieve and collected and named as EBS. The yield was then calculated as the percentage of the dried bulbils weight.w/v) concentration with a solid to liquid ratio of 1: 4, and the final pH was 13.80. The mixture was left at 35 °C for 3 h, then, in 4 °C refrigerator for 3 h. The top supernatant was discarded and the bottom starch layer was collected and pulped with a blender. A slurry product was obtained and then washed with deionized water, followed by stirring for 3 h. After depositing, the upper layer was discarded, and the upper non-white layer was physically scraped off. In addition, the remaining white layer was collected and re-suspended in deionized water and then subjected to stirring and depositing as described above. The procedure was repeated seven or eight times until the top supernatant layer turned clear and the starch layer exhibited a lightly white color; the total duration was 10 h. The final starch product was then dried at 45 °C for 12 h and sieved with a 200-mesh sieve and collected and named as ABS. The yield was calculated as the percentage of the dried bulbils weight.As described above, for sodium hydroxide extraction of starch, fresh bulbuls were first washed and chopped into small pieces and then dried against spoilage at 45 °C for 24 h and stored in a desiccator at room temperature before use. One-hundred grams of the dry materials was mixed with NaOH solution of 0.2%  . The amy−1 with a resolution of 4 cm−1.FT-IR analyses of SBS, EBS and ABS were carried out on a Nicolet iN10 FT-IR spectrophotometer  equipped with a liquid nitrogen-cooled MCT detector. The spectra were recorded from 4000 to 650 cm1158 represents the transmittance of C–O–C stretching vibration absorption at 1158 cm−1, and T2931 indicates the transmittance of C–CH2–C stretching vibration absorption at 2931 cm−1.The infrared crystalline index was calculated using the following formula:The morphological characteristics of SBS, EBS and ABS were examined by scanning electron microscopy (SEM) . The samples were first coated with a thin gold layer and placed on the substrate. The corresponding images were then observed at a voltage of 5.0 kV with 500- and 1000-fold magnification under high vacuum.The thermal behaviors of samples were measured using a differential scanning calorimeter Q100  with nitrogen as purge gas. A starch sample of 3 mg was placed onto a DSC pan and 12 μL of distilled water was then added through a micro-syringe. An empty pan was used as a control and reference. The samples were scanned at a temperature range from 25 to 110 °C at a heating rate of 5 °C/min. The enthalpy variation (ΔH), onset temperature (To) and conclusion temperature (Tc) were calculated accordingly.α radiation (λ=1.54 Å) and a linear focus at 40 kV and 15 mA. Measurements were performed in symmetric or powder geometry (θ-2θ) with a sweep from 2 to 40°, step size of 0.01 and rate of 3°/min [The X-ray diffraction patterns of starch samples were collected using an X-ray diffractometer  with Cu Kf 3°/min . The swelling power and solubility of SBS, EBS and ABS were measured according to the method described by Leach et al. with slight modifications . SamplesStarch paste clarity was measured using the method reported by Craig et al . Tubes cFreeze-thaw stability was investigated using a method reported by Hoover et al.  with a sw/w). The mixture was stirred at 960 rpm for 10 s until a state of homogenous dispersion was reached. The sample was then subjected to rotation at 160 rpm. Samples were kept at 50 °C for 1 min, then heated from 50 °C to 95 °C at a rate of 12 °C/min, kept at 95 °C for 2.5 min, followed by cooling from 95 °C to 50 °C at a rate of 12 °C/min and holding at 50 °C for 0.5 min. The total analysis time was 12.5 min. Peak viscosity, trough, breakdown, final viscosity, setback, and pasting temperature were recorded [Pasting properties of three different bulbil starches were evaluated using Rapid Visco Analyzer (RVA) . Dry starches were mixed with distilled water to a concentration of 10%  indicating the higher stability of the starch crystallites and more heating was required to swell the granules, which could be a potential alternative during the cooking process in the industries. SBS and EBS displayed lower syneresis and better freeze-thaw stability in contrast to the corresponding ABS during the first three circles. These characteristics made SBS and EBS potential technological alternatives in quality preservation of frozen starch-based products and other industrial applications. Furthermore, ABS and EBS had the higher peak viscosities, final viscosities, setbacks and were suitable for use as food thickeners or frozen food additives. ABS had the best transparency and could be used for making medicine.The bulbils starches isolated by three methods in this work presented distinctive chemical, mineral composition, and functional properties, which indicated that they could be the promising alternatives in food and other industries. Starch isolated from bulbils of In conclusion, different isolation methods induced changes in the morphology of starch granules and chemical composition, which led to distinct functional properties. In the practical application process, people could choose the proper bulbil starch obtained by different extraction methods according to different characteristics.


Starch films are developed as the biodegradable packaging materials to replace the petroleum-based materials in recent years. Thus, it is extremely beneficial to improve the hydrophobicity and mechanical strength of starch films, through a novel approach of co-cross-linking in neutral conditions, with glyoxal and AZC. In this work, systematic studies have been conducted to assess the performance of the co-cross-linked starch along with the control starch and starch cross-linked by glyoxal or AZC alone. Results showed that the co-cross-linked starch films exhibited significantly improved hydrophobicity and strength and the wet stress reached 1.53 MPa, compared to the control, glyoxal or AZC cross-linked starch films. More interestingly, the co-cross-linked film also demonstrated excellent UV shielding capacity and transmittance at visible wavelength range. The reaction mechanism was revealed based on the findings from UV, FT-IR and NMR spectra. This work established an innovative approach to improving the performance of starch film in neutral conditions for packaging applications. In paCross-linking can link two polymer chains by bi-functional reagents, by reducing the water-binding capacity and enhancing the stress-transferring ability. As a popular approach, the formed covalent linkages among the molecular chains can efficiently limit the movement of polymer chains and improve the performance –12. In tHowever, a greener and economically more efficient approach is expected with enhanced environmental awareness in order to avoid the release and utilization of the hazardous chemicals. Glyoxal is a bi-aldehyde cross-linking agent that has been approved by the FDA for use in the food packaging industry and can be linked to starch molecular chains on two ends. Besides, the relatively low temperature of the cross-linking process induced by glyoxal is an overwhelming advantage over the carboxylic cross-linking agents which often need temperatures as high as 170°C –23. FurtConsidering the versatility of glyoxal, a novel co-cross-linking system based on glyoxal was developed in this work. Ammonium zirconium carbonate (AZC) is a water-soluble inorganic salt that has been widely used to improve the water resistance of coated papers or enhance the performance of the xylan, galactoglucomannan and starch films via the hydrogen linkages between Zr and hydroxyl groups –28. Besi3 might also improve the UV shielding ability of co-cross-linked starch film, which can avoid the oxidation of lipid and prolong the shelf life of packaged food. Hence, it is significant to systematically study the strength, hydrophobic properties and transmittance of starch films produced via this co-cross-linking system.In our work, we found the addition of glyoxal can limit the self-polymerization of AZC in aqueous solution  was first revealed with UV–vis, FT-IR spectroscopy and 2.2.1.Soluble starch (MW 342) and AZC with ZrO content of 18% were purchased from Guoyao Chemical Co., Ltd., China. Glyoxal water solution (40%) was kindly provided by Nanjing Chemical Industry Group (China). All chemicals were used without any further purification.2.2.2.2.1.The 10% control starch solutions were prepared by dissolving 10 g soluble starch into 90 g water through heating at 90°C for 2 h. The Gly–AZC starch solution was obtained through first heating at 60°C for 1 h and then heating at 90°C for another 1 h. The dosages of glyoxal and AZC were 10% (wt) and 5% (wt) to the dry starch, respectively. For comparison, the starch solution added by glyoxal or AZC alone was also prepared under the same condition, at a dosage of 15%. The as-prepared starch solution was poured into the hydrophobic Petri dishes and oven-dried at 40°C for 2 h. Before characterization, the films were kept at 23°C and a relative humidity (RH) of 50% for 2 days and then peeled off. The grammage of films was determined based on the amount of dry starch (g) over the unit area coated. The starch granules for the characterizations of FT-IR, NMR, TGA and XRD were obtained through grinding the starch samples using a solid grinder  for 5 min after the as-prepared starch solutions were freeze-dried at −80°C and 15 Pa for 48 h.2.2.2.The water contact angle of starch film was measured by the sessile drop method using a contact angle analyser  at 25°C and 50% RH with 4 µl of deionized water . The rep−1. The specific tensile stress and specific Young's modulus were calculated accordingly [For the testing of mechanical properties, the starch films were cut into 5 mm × 30 mm rectangular strips. Afterwards, their tensile stress and Young's modulus were measured at room temperature using a UTM6502 universal testing machine  equipped with a 500 N load cell and pneumatic grips. The cross-head speed during tensile testing was 5 mm minordingly .The water stability tests were conducted as follows. The starch films were immersed in deionized water and stirred for 5 min (600 rpm) at 25°C and 60°C, respectively. Then the photographs were taken to determine the water stability of starch films in water.M1. Then the films were soaked in deionized water for 7 min and the swollen films were wiped gently using Kimwipes every minute and weighed as M2. The swelling ratio of starch films in water was calculated as follows:The swelling ratio of control and cross-linked starch film in water was also determined. Firstly, the cross-linked starch films were conditioned at 105°C for 4 h to remove the moisture and weighted as terature –34.−1, at a resolution of 0.5 cm−1. 13C NMR of the starch granules dissolved in deuterium peroxide (D2O) were recorded on a Varian Unity 400 spectrometer operated at 400 MHz.The 650 FT-IR spectrometer (Tianjin GangDong Sci & Tech. D) was used on the control starch, glyoxal cross-linked starch, AZC cross-linked starch and Gly–AZC starch granules after grinding with KBr by co-adding over 16 scans, capturing spectra between 500 and 4000 cm−1.The thermal properties of the starch were examined using an SDT Q600 TGA System thermogravimetric analyser . The sample weight was 1–10 mg and heated from 30°C to 800°C under the nitrogen atmosphere at a heating rate of 10°C/min and a nitrogen flow rate of 100 ml minθ range from 5° to 50° with a scan step time of 30 s.The Ultima X-ray diffractometer  was used to determine the crystallinity of starch samples over a 2The surface of starch granules and the cross-sections of the starch films were observed using a Quanta 200 Environmental scanning electron microscope  after gold sputtering according to the reference . The sta3.3.1.a. When the molar ratio rises to 2 : 1, the solution becomes clear and transparent without any precipitation. This can be attributed to the fact that the increased glyoxal dosage efficiently limits the self-polymerization of AZC through the Schiff-base reaction with AZC.The adsorption of the as-prepared mixture solution of glyoxal and AZC was recorded with the UV–vis spectra, and the results are presented in figure 1As the molar ratio increases, an obviously enhanced adsorption intensity appears at 282 nm due to the newly produced Schiff-base structure (O = C–C = N), indicating the successful reaction between the aldehyde and amino groups. As the glyoxal dosage further increases, the maximum adsorption wavelength shifts to 288 nm and the solution becomes dark yellow. It is confirmed that the Schiff-base reaction between glyoxal and AZC successfully occurs, and the nitrogen element in the AZC can be retained. Besides, the exhibited prominent UV adsorbing ability of glyoxal–AZC solution might be beneficial for improving the UV shielding capacity of the co-cross-linked starch films.b illustrates the effect of reaction time on the UV–vis spectra of the mixture solution of glyoxal and AZC. As the reaction proceeds, the solution colour changes from light yellow to dark yellow; and enhanced absorbance intensity is observed, indicating an increased amount of Schiff-base structure. The turbidity phenomenon is not observed due to the polymerization of AZC, which indicates that the free ‘Zr’ in AZC have the chance to form the hydrogen linkage with the hydroxyl groups of starch. Thus, this result is extremely beneficial for increasing the cross-linking density of starch and improving its stress-transferring ability.Figure 13.2.13C NMR of the starch samples were collected, and the results are shown in figures −1 in the AZC sample becomes broader compared with the other samples, which can be attributed to the re-association of hydroxyls caused by the hydrogen linkage between Zr and hydroxyl groups [−1 can be ascribed to the stretching vibration of hydrogen bonds [−1 is decreased, whereas the intensity at 1020 cm−1 is enhanced in Glyoxal and Gly–AZC samples. The adsorption peak at 1740 cm−1 in the Gly–AZC sample is attributed to the stretching vibration of O = C–C = N, indicating the successful Schiff-base reaction between glyoxal and NH3.To further confirm the co-cross-linking of starch induced by glyoxal and AZC, FT-IR spectra and l groups . The adsen bonds . As repo13C NMR are given in figure 3a, the signal for the C6-position of the starch backbone is observed at 60.2 ppm. The signals of C2-5 of the anhydroglucose appear between 69.3 ppm and 77.0 ppm. The signals at 99.6 ppm and 76.6 ppm are assigned to the C-1 and C-4, respectively. In figure 3b, the new signal of C-9 appeared at 177.5 ppm confirms that NH3 has been successfully covalently bonded to the glyoxal through the Schiff-base reaction. The signals of C-8 and C-7 at 95.4 ppm and 90.6 ppm are observed due to the acetalization between glyoxal and the hydroxyl groups of starch.The chemical shifts and assignments of 3.3.θ value of 17.06° and 22.14° and a weak peak at near 2θ value of 19.7°. Compared with control starch, the AZC sample shows an obviously decreased intensity of reflection at 2θ = 19.7°, indicating the loss of ordered structure in starch. It is indicated that the AZC cross-linking with the starch molecules brings a significant reduction to the crystallinity of starch. This might be attributed to the decreased amount of the inter- and intra-molecular hydrogen bonds in the starch. By contrast, decreased crystallinity is not observed in the Gly–AZC sample which is further confirmation that the addition of glyoxal can efficiently limit the self-polymerization of AZC.The X-ray diffraction patterns for both control and cross-linked starch granules are shown in 3.4.a, the control and cross-linked starch samples present two distinct stages. The first weight loss occurring between 100°C and 190°C is attributed to the loss of free and combined water in the starch granules. The cross-linking gives an obvious increase in the hydrophobicity of starch granules, and thus a lower mass loss of water is seen in the cross-linked starch samples.Thermal properties of the control and cross-linked starches are characterized by thermogravimetric analysis (TGA). As shown in figure 5b). Compared with the control starch, the intensity of cross-linked starch  at the maximum degradation temperature is much lower. Besides, the maximum weight loss (MWS) peak in the AZC and Gly–AZC samples appeared at 282°C and 291°C, respectively, in comparison to that (311°C) of control starch. It seems that the aggregation of AZC created by its self-polymerization might destroy the ordered structure of the starch chain, thus resulting in the decreased maximum decomposition temperature (MDT); while the addition of glyoxal can limit the self-polymerization of AZC to some extent, thus giving a higher MDT than the AZC cross-linked starch. The thermal decomposition peak appearing in the Glyoxal sample at 218°C might be ascribed to the degradation of the volatile structure in the glyoxal cross-linked starch.In the second process, the initial decomposition temperature of the starch samples after cross-linking begins at 190°C; by contrast, the control starch is still stable even at around 300°C. This phenomenon might be related to the loose structure of starch due to cross-linking, which is accorded with the decreased crystallinity . Howeverfigure 5a). As seen, the residual contents in the AZC, Glyoxal and Gly–AZC samples at 600°C reach as high as 39, 23 and 20%, respectively, which is much higher than that of the control sample (6%). The results are accorded with the decreased decomposition weight loss at the corresponding MDT (b), indicating the improved thermal stability after cross-linking.The cross-linking gives rise to the starch mass remaining after 600°C, as compared to the control starch a. As seeding MDT b, indica3.5.a. Swelling does occur after the diffusion of water within the starch polymer network. The cross-linking can lead to the formation of a compact and rigid network, which limits the absorption and diffusion of water molecules [figure 7a, it can be seen that the swelling ratios of the Glyoxal, AZC and Gly–AZC samples are decreased obviously compared with the Control sample, particularly in the first 2 min. Specifically, Glyoxal sample keeps intact shape until 5 min, while the AZC and Gly–AZC samples do not disintegrate even after 7 min. Meanwhile, the swelling ratio of Gly–AZC decreases obviously, compared with the other samples, indicating the formation of a highly cross-linked structure in the network induced by the novel co-cross-linking system consisting of glyoxal and AZC.Starch is an inherently hydrophilic polymer that can easily absorb moisture due to the presence of hydroxyl groups; but this feature weakens its mechanical properties. The swelling ratio of cross-linked starch films with different cross-linkers in water is shown in figure 7olecules . From fib, the Control sample almost dissolves in water and presents a transparent state. The Glyoxal sample swells and dissolves partly, without sufficient wet-strength to keep its shape intact. By contrast, the AZC and Gly–AZC film is insoluble in water, suggesting the efficient strengthening of starch polymer networks caused by the cross-linking. The AZC presents a compact and intact shape without any sign of swelling and disintegration. Its wet tensile strength reaches to 0.66 MPa and represents the satisfied cross-linking performance. This is more prominent in the Gly–AZC sample: the film keeps its original shape without any sign of swelling and the wet tensile stress reaches to 1.53 MPa, indicating that the co-cross-linking induces efficient linkages with hydroxyl groups of starch, and helps form a dense network that can limit the penetration of water molecules into the inner parts of starch films.The above phenomenon can be clearly illustrated from the optical images of these four samples impregnated in water for 2 h at 25°C. As seen in figure 7c,d, the initial water contact angle of control starch film (Control sample) is only 42.04° and decreases to 34.06° within the recording time (180 s), exhibiting a highly hydrophilic nature. The glyoxal cross-linked starch film almost doesn't improve the hydrophobicity compared with the control starch, and the initial water contact angle is only 50.91° and decreases to 41.27° at 11 s, then becomes stable until 180 s. Therefore, the cross-linking through glyoxal alone does not contribute to the improved surface hydrophobicity of starch. In contrast, the AZC starch film presents excellent hydrophobic properties with the initial CA of 99.45°. Besides, the water contact angle remains high and still over 90° (92.37°) at 180 s. The starch film co-cross-linked by glyoxal and AZC also exhibits improved surface hydrophobicity, compared with the Control sample.The surface hydrophobic properties of the cross-linked starch films are illustrated through the contact angle (CA) measurements. As presented in figure 73.6.The transmittance and optical properties of the control and cross-linked starch films are exhibited in a depicts the light transmittance through the films as a function of wavelength. As seen, the transmittance at 600 nm is around 80% for these starch films except for the AZC sample. This might be attributed to the increased starch granule size due to the self-polymerization of AZC. For the Gly–AZC sample, the transmittance at 300 nm shows an obvious decrease, compared with other starch films. This is accorded with the result of UV–vis spectra in Figure 8σ) and the specific tensile stress (σ/ρ) of starch films are presented in figure 8b, where ρ is the density. The (σ/ρ) of Glyoxal film is 7.37 kN m kg−1 and increases by 152.24% compared with the Control sample. In AZC, it only increases by 108.22%. The difference might be attributed to the decreased amount of linking bonds in starch network cross-linked by AZC compared with the Glyoxal sample, as well as the relatively weaker hydrogen linkage in the AZC sample against the covalent bonding in the Glyoxal sample. For the Gly–AZC sample, the specific tensile stress (6.53 KN m kg−1) increases by 123.63%. Clearly, the strengthening effect of glyoxal is better than that of AZC at the same dosage. In fact, glyoxal has a lower molecular weight and a higher number of reactive groups that can form the covalent bonds with the hydroxyl groups of starch in comparison with AZC. Moreover, the weight ratio of glyoxal to AZC is also closely related to the strength performance of starch film cross-linked by the current novel co-cross-linking system, which has also been studied in subsequent works.Tensile stress  takes the density (ρ) variation into account. The E/ρ of Gly–AZC increases by 137.92% compared with the Control film. The increase ratio in the dual-cross-linking system is not as high as the Glyoxal sample but is higher than the AZC sample.The relatively high modulus (E) of cross-linked starch films is shown in figure 83.7.a–d). As shown, the control starch granules are individual or separated with diameters of around 150 µm–300 µm . However, the Glyoxal, AZC and Gly–AZC starch present a flaky shape, which is dramatically different to the un-cross-linked control starch granules . Electronic supplementary material, figure S2 shows the cross-section images of starch films and there appear ‘hills and valleys’ in the fracture of Control and AZC film samples; while the cross-sections of the Glyoxal and Gly–AZC samples exhibit relatively smooth surfaces, indicating the increased rigidity after the co-cross-linking . This change in the cross-section morphology after co-cross-linking can also be observed in figure 9c,d.The SEM and AFM images of starch are shown in figure 9From the AFM images, it is clear that the surface of Gly–AZC starch film is more homogeneous and the height is only 14 nm, while the roughness of the control films is relatively higher. As seen in electronic supplementary material, figures S3c and S3d, the addition of glyoxal in Glyoxal film give a much more homogeneous surface than that of AZC film. This indicates that the addition of glyoxal efficiently limits the self-polymerization of AZC when co-cross-linked with starch and contributes to the formation of a uniform morphology in starch films.4.E/ρ of Gly–AZC increased by 137.92%, compared with the Control. The wet tensile stress reached to 1.53 MPa and the initial CA was 71.05°. Moreover, the excellent transmittance at visible wavelength range and UV shielding capacity were observed.The addition of glyoxal and AZC significantly improved the hydrophobicity and strength of the co-cross-linked starch. The 13C NMR confirmed the presence of Schiff-base reaction in the co-cross-linking system. XRD and TG analysis showed that the addition of glyoxal avoided the weakened crystallinity structure and the decreased MDT of co-cross-linked starch to some extent, via limiting the self-polymerization of AZC. SEM and AFM images indicated that a rigid and uniform starch film was formed after the co-cross-linking.The UV, FT-IR spectra and In this assessing work, a starch film with enhanced hydrophobicity, strength and excellent transmittance at the visible light range and UV shielding ability was obtained via a novel co-cross-linking system. Based on the experimental findings, the following conclusions were achieved:


Starches of four legume varieties grown in China were evaluated for composition, granule structure, turbidity, swelling power, solubility, and thermal and pasting properties. The similar granule shapes, surface fissures, polarized crosses, inner structure characteristics, and granule sizes could be observed among all legume varieties through a variety of microscopy techniques such as light microscopy, scanning electron microscopy, and confocal laser scanning microscopy. Amylose contents were in the range of 30.61%–33.55%. All of the starch varieties showed C‐type X‐ray pattern, but exhibited different relative crystallinity percentage. Significant differences were observed among starch varieties in swelling power, solubility, and light transmittance. Thermal analysis and pasting profile of legume starches showed that all the varieties’ differences are probably due to variation in amylose content. The thermal and pasting parameters of starches were evaluated using differential scanning calorimeter and Rapid Visco‐Analyser, respectively, and significant differences were observed in individual pasting and thermal parameters. The present study can be used for identifying differences between legume varieties for starch structural and physicochemical characteristics and could provide guidance to possible industries for their end use. As important plant resources, there are about 16,000–19,000 species and approximately 750 genera in leguminosae family , growth rings (120–400 nm), blocklets (20–500 nm), amorphous and crystalline lamellae (9 nm), and AP and AM chains  and 70%–80% amylopectin (AM) in normal starch granule: Amylose is a linear chain of an hydroglucose units linked by a‐V. angularis L.; Figure 1), baiyue bean , Faba bean , and chickpea , which could commonly be found in China, were collected from their major producing region. Starches were isolated from these four kinds of beans, and their structural and physicochemical properties were investigated and compared. The results are expected to provide useful information for the applications of these starches in food and nonfood industries.Physicochemical and structural properties of starches determine their applications in food and nonfood industries. In this study, four kinds of legumes including red adzuki bean  and sodium cyanoborohydride were purchased from Sigma Chemical . All other chemicals and reagents used in experiments were of analytical grade.2.2g for 15 min, and then, the upper non‐white layer was scraped off. Starch was then collected and dried in an oven at 40°C for 12 hr. Then, the starches were smashed by universal mill grindings (100 mesh) and kept in a container at room temperature for further study.To isolate starches from four kinds of legumes, we adopt the method described by Singh, Kaur, Sandhu, and Guraya , with pr2.3The starch, protein, lipid, and ash content were performed in terms of the standard AACC methods 76–13, 46–30, 30–25, and 08–01, respectively AACC, . The amy2.42.4.12O, V/V) was prepared, and starch granules were suspended in it. Then, an aliquot of starch slurry was transferred onto a glass slide, and a coverslip was placed on the top of the sample for microscopic examination. A polarizing light microscope  with a × 40 objective was used to observe birefringence of the starch granules. The microscope was also used without the polarizing filter to observe the appearance of the sample.A 1:1 glycerol solution  at an accelerating voltage of 20 kV. Starch samples which were dispersed evenly with a rubber suction bulb were mounted on an SEM stub with double‐sided adhesive tape and coated with gold in an argon atmosphere.2.4.3The microstructure of starch granules was viewed by confocal laser scanning microscopy. The observation of each starch sample was conducted according to the reported procedure. Starch granules (10 mg) were suspended in 15 μl of freshly made APTS, and 15 μl of 1 M sodium cyanoborohydride solution was added. The reaction mixture was incubated at 30°C for 15–18 hr, and then, the granules were thoroughly washed five times with 1 ml of distilled water. The stained starch granules were then suspended in 20 μl of glycerol/water mixture  and then visualized using a Digital Eclipse C1plus CLSM system equipped with He/Ne and Ar lasers , and a stand for fixed fluorescent cell samples was used for the detection of the fluorescence signal from dye‐stained starch granules  measurements were performed using an X‐ray diffractometer  with the following operating conditions: radiation source, CuKa; angle of diffraction scanned from 5 to 60°; step size, 0.02; and step time, 2 s. In order to avoid the negative effects of relative humidity on relative crystallinity, the starch samples were equilibrated in a 100% relative humidity chamber for 48 hr at room temperature in the first place.2.6Swelling power (SP) and solubility (S) were measured following the method of Leach, McCowen, and Schoch  at 50, 62.7The light transmittance of starches from different legume varieties was performed according to previous method . Starch (3 mg) was put into the aluminum DSC pan and distilled water (12 μl) was added with a microsyringe and then sealed hermetically, reweighed, and allowed it to stand overnight at room temperature in order to attain an even distribution of water before DSC analysis. Subsequently, the samples were heated from 30 to 120°C at 10°C/min. Meanwhile, an empty pan was used as reference for all measurements. During the scans, the space surrounding the sample chamber was flushed with dry nitrogen to avoid condensation.2.9Pasting properties of starches were evaluated using a Rapid Visco‐Analyser (RVA) model Master . Firstly, starch  and deionized water (25.0 g) were put in the RVA canister, and the slurry was then manually homogenized using the plastic paddle to avoid lump formation before the RVA run. The starch slurry was heated from 50 to 95°C at the rate of 12°C/min, maintained at 95°C for 2.5 min, and then cooled to 50°C at the same rate. The speed of the paddle was 960 rpm for the first 10 s and then 160 rpm for the remainder of the experiment. Pasting temperature, peak viscosity (PV), breakdown viscosity, setback viscosity, and final viscosity were calculated.2.10SD. Duncan's least significant test was used to compare means at the 5% significance level. All statistical computations and analyses were conducted using SPSS 16.0 to compare Simple Pearson's correlation and regression for Windows.All the experiments were performed at least in triplicate and experimental data also were analyzed using analysis of variance (ANOVA) and expressed as mean value ± 33.1The chemical composition of legume starches is presented in Table The amylose content of different legume starches varied from 30.61% to 33.55%, and the highest amylose content was found in red adzuki starch, whereas the lowest amylose content was found in faba bean starch. These values were similar with the values previously reported for black gram . Among all starches, red adzuki bean and baiyue bean starch had the largest angular granules, with the granule diameter ranged from 20 to 40 μm, while the chickpea and faba bean showed a relatively smaller diameter with the granule size ranged between 10 and 30 μm . These observations are mostly in agreement with several other legume starch granule shapes and size , light microscope (LM), polarized light microscope (PLM), and confocal laser scanning microscope (CLSM), and the micrographs are shown in Figure 3–d3). The fissures structure could also be observed on mung bean starch under light microscope , indicating the high degree of molecular orientation within starch granules. The “maltese cross” under PLM suggests the radial arrangement of amylopectin crystallites within the granule at right angles to the surface with their single reducing end group toward the hilum, and the hilum is the original growing point of the starch granule , indicating their similar amylose molecular content as provided in Table Confocal laser scanning microscopy images were used to reveal the distribution of amylose and amylopectin in starch granules and to characterize growth ring, channels, and pores of starch granules. The CLSM optical images of various legume starch granules stained with APTS are shown in Figure 5–d4), and more channels are observed in chickpea starch . The amylopectin molecules are prominently located in the semi‐crystalline growth rings, while amylose has been accepted to be mainly located within the amorphous zones of the two distinct granule domains  of starch samples are shown in Figure s Figure .The position of strong diffraction peaks and relatively crystallinity degree of different legume starches are presented in Table 3.4The swelling power and solubility of the legume starches were investigated over the temperature range of 50–90°C, and the results are presented in Table Swelling power is a measure of the ability of the starch to hydrate under specific conditions such as temperature and water content, and the greater swelling capacity of the starch granules is, the weaker of the binding forces  of the starch pastes of the starches from the different legume varieties for a period of 3 days is shown in Table. The rapid decrease in clarity during the early storage may be due to the aggregation and crystallization of amylose, and then, the decrease rate in clarity during the rest of the storage could be attributed to the leached amylose and amylopectin chains that lead to the development of the functional zone which scatters a significant amount of light , peak temperature (Tp), conclusion transition temperature (Tc), gelatinization temperature range (ΔTr) and gelatinization enthalpy (ΔH) on a dry starch basis are presented in Table To of legume starches ranged from 58.73 to 61.96°C, Tp in the range of 64.07–68.35°C, and Tc in the range of 71.30–78.99°C, and the chickpea starch showed the lowest To, Tp, and Tc values among various legume starches  as shown in Table Tr is related to the heterogeneity of the starch granule sizes and the distribution of amylose and amylopectin, which produce a semi‐crystalline arrangement inside the granule  in PV, trough viscosity (TV), breakdown (BD), final viscosity (FV), setback (SB), peaking time (PT), and pasting temperature (GT) between the starches (Table Pasting viscosity profiles of starches isolated from various legumes are illustrated in Figure es Table . Changeses Table . Peak vies Table . The difTrough viscosity was the highest for red adzuki bean starch and the lowest for baiyue bean starch, and BD viscosity was the highest for baiyue bean starch and the lowest for chickpea starch. TV has been reported to be influenced by the extent of amylose leaching, amylose–lipid complex formation, friction between swollen granules, granule swelling, and competition between leached amylose and remaining granules for free water (Olkku & Rha, The FV value varied from 2573 cP to 4764 cP, and the highest FV was recorded for chickpea starch and the lowest for baiyue bean starch. FV can show the ability of starch to form a viscous paste, which is largely influenced by the retrogradation tendency of the soluble amylose during cooling stage (Olkku & Rha, 4Structural and physicochemical properties of starches from red adzuki bean, chickpea, faba bean, and baiyue bean starch were characterized, and a high variability among various legume starches properties was found. There were minor differences in morphology characteristic properties among various legume starches. The X‐ray diffraction patterns of all legume starches showed C‐type pattern with relative crystallinity degree varied among the varieties. The swelling power, solubility, light transmittance, and gelatinization parameter values of legume starches varied among the varieties. Pasting properties significantly varied and are useful for assessing the cooking quality of legume starches. Differences in physicochemical properties were influenced by the differences in morphology, composition, and structure of various legume starches. The findings would be useful in selecting legume variety or legume starch for specific food and ingredient applications, and also for further investigations on fine structure of starch granules and modifications targeting food ingredient applications.