Starch-PPC pastes showed reduced thixotropy and improved stability and gelling properties compared to starch paste. The three types of proanthocyanidins all showed evident inhibitory effects on the digestion and retrogradation of potato starch, including short-term and long-term retrogradation. Among the three, PPC with a lower DP had stronger effects on the starch short-term retrogradation and gelling performance, whereas larger PPC molecules exhibited a greater impact on starch recrystallization and digestive characteristics. The research consequences were conducive to explore the application of functional PPC in starch-based food processing.The effects of three types of polymeric proanthocyanidins (PPC) with different degrees of polymerization (DP), namely PPC1 (DP = 6.39 ± 0.13), PPC2 (DP = 8.21 ± 0.76), and PPC3 (DP = 9.92 ± 0.21), on the physicochemical characteristics and in vitro starch digestibility of potato starch were studied. PPC addition (5%,  Potato starch (PoS) has attracted extensive attention in regard to its physicochemical characteristics, such as digestibility, fine textural properties, high swelling power, and transparency. In addition to producing sacchariferous products, potato starch not only plays a unique role in processing flour products, snacks, and chemical starch, but it also serves as a food additive, including gelling agents and film-forming agents used in dairy products, baked foods, beverages, and other foods ,2. HowevPolymeric proanthocyanidins (PPC) are a kind of polyphenol widely distributed in the plant realm . These cTo date, several papers have focused on the influence of proanthocyanidin on pasting, thermal, rheological, and digestion characteristics of potato starch. Zhang et al.  found thw/w), respectively. All other chemicals were of reagent grade and supplied by Aladdin Co., Ltd. .Polymeric proanthocyanidins  derived from grape seed were purchased from Solarbio Technology Co., Ltd. . Potato starch (PoS) was obtained from Rogate Starch Co., Ltd. , and the contents of lipid, water, ash, and amylose were 0.32%, 9.86%, 0.25%, and 26.61% (v/v/v), and the volume of all solvent mixtures was 600 mL. The grading elutions were named as fraction 1, 2, and 3, respectively. Then, the organic solvents of these fractions were removed by vacuum rotary evaporation and lyophilized to drying. The obtained components were, respectively, labeled PPC1, PPC2, and PPC3.The PPC was fractionated with a Sephadex LH-20 column, according to a means expounded in the literature, with certain modifications ,19. A to2 = 0.9954; the standard curve of catechin molar concentration was y = 0.1756x − 0.0013, R2 = 0.9999). A total of 10 mg of the proanthocyanidin sample thoroughly dissolved in 20 mL methanol solution, which was then diluted 20 times with methanol and acetic acid and, respectively, named as liquid A and liquid B. One milliliter of liquid A and liquid B was applied to measure the content (m) and the amount of substance (n) of proanthocyanidin according to the methods of standard curves of catechin mass concentration and catechin molar concentration, respectively [m refers to the proanthocyanidin content, n is the amount of substance of proanthocyanidin, and M is the relative molecular mass of catechin.The average degrees of polymerization of PPC1, PPC2, and PPC3 were determined by the vanillin-hydrochloric acid method ,21. Brieectively . In thisw/w), and added with 20 mL of distilled water. The slurries of PoS and PoS-PPC samples were characterized using the RVA, in accordance with the description of Li et al. [The pasting characteristics of PoS and PoS-PPC blends were determined using RVA , according to preceding papers . Based oi et al. . The preThe rheological characteristics of PoS and PoS-PPC compounds were analyzed by an MCR 302 rheometer  with a parallel plate measuring system, according to Kong et al.  and Chen−1 and then decreasing from 1000–0 s−1. The obtained curve was fitted with the Power Law equation for regression fitting:σ is the shear stress (Pa). γ is the shear rate (s−1). K is the consistency coefficient (Pa·sn). n refers to the flow behavior index .The changes in shear stress of the samples were determined within the range of increasing shear rate from 0–1000 sAn oscillatory frequency sweep mode (0.2–20 Hz) was conducted at room temperature with 1% strain. The storage modulus (G′) and loss modulus (G″) were obtained.o), peak (Tp), conclusion temperature (Tc), gelatinization (ΔgH), and retrogradation enthalpy (ΔrH). Finally, the retrogradation degree R (ΔrH/ΔgH) was computed on account of previous research [The thermal characteristics of PoS and PoS-PPC samples were measured with DSC  on the basis of He et al.  and Xu eresearch .cA and aA stand for the crystal and amorphous areas, respectively.XRD  was used to determine the crystallinity of PoS and PoS-PPC complexes, based on a previous paper . The scaThe in vitro digestibility of PoS and PoS-PPC compounds was analyzed in accordance with the preceding literature, with some modifications ,30. A toG20 and G120 refer to the contents of glucose generated at 20 and 120 min of the hydrolysis process, respectively. FG is the content of free glucose in samples, and TG is the total amount of starch in the original sample. Here, p < 0.05, the significant difference was set.The data were represented as the means ± standard deviation (SD) conducted in triplicate analyses. Duncan’s test was carried out in SPSS 24.0 statistical software, and at v/v/v) to preparatively separate the proanthocyanidins [On the basis of the difference in the molecular weight, molecular structure, and molecular polarity of proanthocyanidins with different DPs, we reported on the use of such a gradient with acetone/methanol/water solutions . As for all the samples, stress shear hysteresis loops appeared in the shearing process, indicating a thixotropic effect. A large hysteresis ring area signifies a strong thixotropic property [K value is related to the starch viscosity, and the larger the K value, the poorer the system mobility and shear stability [K, decreased with the addition of PPC, which suggested that the starch paste thixotropy was reduced and stability was improved by adding PPC, and the effect was more noteworthy for PPC with a lower DP. This finding was in agreement with the BD result of RVA. The same trends of K value and hysteresis ring area of starch added with polysaccharides have been reported before, and these results indicated that the relative stability and uniformity of the starch gel structure was enhanced [The flow characteristic curves of the PoS and PoS-PPC complexes are shown in property . The K vtability . The hysenhanced ,19. n is inversely correlated with the pseudoplasticity degree of the entire system [n value, for PPC1 the most. This meant that the presence of PPC made the entangled starch molecular chains have a greater tendency to be straightened or dispersed under the action of the external force, which was conducive to the flow of starch paste, consequently lowering the viscosity more obviously. The result was unanimous to the change of the RVA test.The value of e system , PPC repThe viscoelastic properties were determined by dynamic rheological analysis . The stoIn summary, starch-PPC pastes had better gelling properties than starch paste, and the lower the degree of PPC polymerization, the more obvious the effect. PPC addition could make PoS more suitable candidates as gelling agents for food industry manufacturing, including soft candies, ice cream, and meat foods production, thereby improving these products’ nutrition, texture, and stability .o, Tp, and Tc) of PoS within 2 °C, indicating that PPC could delay starch gelatinization [The thermodynamic parameters obtained from DSC are shown in nization . The resnization  and Zhennization , who fougH represents the energy required for the melting of the starch crystallization zone, especially for the double helix structure of amylopectin crystals [gH for starch gelatinized with 5% PPC1, PPC2, and PPC3 decreased to 12.17, 8.70, and 8.07 J/g, respectively. PPC has a polyhydroxyl structure. The OH groups of PPC interacted with the side chain of amylopectin and bound to the amorphous area of starch particles to varying degrees. They facilitated the transition from the water-absorbing swelling in the amorphous zone to the crystalline zone for the starch granules, thereby altering the coupling force between the crystalline and amorphous matrix [The gelatinization enthalpy value Δcrystals . Compares matrix . The conrH and R value of starch mainly reflect the recrystallization level of amylopectin and the long-term retrogradation degree of starch [rH and R values of starch declined significantly, indicating that the degree of order and crystallinity of starch decreased during the retrogradation process. On the one hand, the strong hydrophilicity of PPC reduced the content of free or available moisture in the starch-PPC system. On the other hand, PPC associated with starch molecules by intense hydrogen bonding and hydrophobicity interactions and, consequently, played a role in steric exclusion, which all limited the activity of the starch chains and retarded retrogradation [The Δf starch . With thradation . Among tradation , which aThe results of XRD are shown in After adding PPC1, PPC2, and PPC3, the relative crystallinity of potato starch decreased from 17.94% to 16.86%, 15.54%, and 14.44%, respectively. It is speculated that when embedded in the starch-moisture matrix, PPC could have interfered with the rearrangement of starch chains, especially for amylopectins, and a hydrogen-bond interaction might have been the main cause of their mutual effects . The resPoS is often served as a substrate for producing starchy packaging films due to its edibility and biodegradability. However, on account of the shortcomings of PoS in processing, starch film production faces the problems of poor mechanical properties and easy crystallization ,55. Throp < 0.05). PPC3 also showed a prominent effect in the improvement of RS content, with 16.35%. These results indicated that PPC could effectively hinder the digestibility of potato starch, and the inhibition impact was closely associated with the DP of PPC. In general, the replacement of PoS with PPC3 had the strongest suppression of starch digestibility among the three. As presented in For the following probable reasons, on the one hand, PPC could bind to the amino acid residues and non-competitively inhibit the catalysis of α-amylase and α-glucosidase, mainly through hydrophobic interaction, thus reducing the activity of enzymes . As the In this study, we utilized three types of PPC with different DPs  to research the influence of PPC on physicochemical and in vitro digestion properties for better application of PPC in starchy food. PPC enhanced the thermal stability and delayed starch gelatinization of PoS. It pronouncedly affected the elastic properties, instead of the viscous properties, of PoS pasting. The reduced thixotropy indicated that the relative stability and uniformity of the starch gel structure was enhanced, showing better gelling properties. Starch digestion and retrogradation could be restrained by PPCs with different DPs in varying degrees. Among the three, PPCs with a lower DP more evidently influenced starch gelling performance, whereas larger PPC molecules exhibited a greater impact on starch long-term retrogradation and digestive properties. This might be attributed to the different type of starch chains combined with PPCs of different DP. The findings of the present study may render a beneficial recommendation for the application of PPC to modify gelatinization, thermodynamic, rheological, and digestive characteristics of PoS-based foods.


In recent years, buried bellows have often had safety accidents such as pipeline bursts and ground subsidence due to the lack of adequate mechanical properties and other quality problems. In order to improve the mechanical properties of bellows, fly ash (FA) was used as a reinforced filler in high density polyethylene (HDPE) to develop composites. The FA was surface treated with a silane coupling agent and HDPE-g-maleic anhydride was used as compatibilizer. Dumbbell-shaped samples were prepared via extrusion blending and injection molding. The cross-section morphology, thermal stability and mechanical properties of the composites were studied. It was observed that when 10% modified FA and 5% compatibilizer were added to HDPE, the tensile yield strength and tensile breaking strength of the composites were nearly 30.2% and 40.4% higher than those of pure HDPE, respectively, and the Young’s modulus could reach 1451.07 MPa. In addition, the ring stiffness of the bellows was analyzed using finite element analysis. Compared with a same-diameter bellows fabricated from common commercially available materials, the ring stiffness increased by nearly 23%. The preparation method of FA/HDPE is simple, efficient, and low-cost. It is of great significance for the popularization of high-performance bellows and the high value-added utilization of FA. There have been problems in urban underground pipe networks for a long time, such as insufficient performance of drainage and sewage pipes, uneven product quality, and so on. It is extremely urgent to improve the mechanical properties of underground pipelines. Nowadays, buried large-diameter double-wall bellows are also gradually being upgraded from cement or steel materials to polymer materials. HDPE has become the preferred material for most bellows manufacturers due to its excellent comprehensive mechanical properties . The int2O3 and BaO nano-additives on the mechanical properties of HDPE [2O3 nano-fillers reached 4% and 6% respectively, the increase in Young’s modulus relative to the matrix was 229% and 208% respectively. D. K. Mohan Kumar et al. used HDPE as matrix material and bagasse ash and nano-montmorillonite as reinforcement materials to prepare composites. The results showed that the composites had higher tensile strength and bending strength compared with pure HDPE [With the increasing demand for high-performance bellows and the reduction in the amount of polyethylene raw materials, many people try to add inorganic fillers to HDPE to obtain stronger mechanical properties and cut the use of polyethylene raw materials. There are a large number of studies that report improvements, with an emphasis on the mechanical properties of polyolefins, with the addition of inorganic particles . G. Savi of HDPE . The resAs mentioned above, many people add inorganic fillers to polyolefins to enhance the strength or toughness of the materials. However, these inorganic fillers usually come from mining, which easily damages geology and forests. Therefore, many people currently use solid waste as inorganic reinforcing fillers. For example, there are some people who use FA Cenospheres, modified FA, ultrafine FA or other mineral materials as reinforcement materials, which grant HDPE excellent strength and stiffness. FA is an aluminosilicate rich waste with light weight and high strength obtained from coal-fired power plants, whose disposal causes serious environmental pollution . High vaPipe circumferential bending stiffness refers to the ability of a pipe to resist circumferential deformation, which is referred to as ring stiffness. Ring stiffness is a very important index for bellows, indicating their ability to resist external pressure . Ring stIn this paper, FA was used as an inorganic filler to enhance the strength and stiffness of HDPE. However, it is usually necessary to add a large volume of filler to the polymer to obtain the desired results . Thus, FHDPE ;FA ;3-Amino propyl tri ethoxy silane (APTS) ;Maleic anhydride modified HDPE (HDPE-g-MAH) ;LDPE Absolute ethanol and glacial acetic acid were obtained from Aladdin Biochemical Technology Co., LTD, .The composition of FA used in this article was provided by Shanxi Hujin Coal and Electricity New Materials Co., Ltd. . The company analyzed the mean composition of the FA using X-ray fluorescence analysis in accordance with the GB/T 14563-2008 test standard in the In order to improve the compatibility of the FA and HDPE, the surface of the FA was modified . Silane-In order to distribute FA more evenly within HDPE and prevent it from causing secondary pollution, FA was prepared as a filling masterbatch . A certaThe particulate composites were obtained by mixing HDPE, the FA masterbatch and the HDPE-g-MAH compatibilizer in various proportions at 210~215 °C. Blending was carried out in a micro compounder .Dumb-bell shaped specimens were then molded as per GB/T 1040.2-2006 specifications, using injection molding , at 210~215 °C. The amount of compatibilizer was expressed as a weight percentage of FA. The dumb-bell specimens were then subjected to tensile properties and thermal properties testing.Different techniques were used to test the characteristics of the silane-treated FA and HDPE composites in this study. The first technique was scanning electron microscopy . For morphology observation, the sample was cryogenically fractured in liquid nitrogen. Samples were then covered with gold and examined with the scanning electron microscope at an operating voltage of 10 kV.−1 in a nitrogen atmosphere and a heating rate of 10 °C/min.The second technique was thermo-gravimetric analysis , working at temperatures ranging from 40 °C to 800 °C, a flow rate of 100 mL min−1. FT–IR spectra were used to compare untreated and treated FA.The third technique was Fourier transform infrared spectroscopy (FT–IR). FT–IR spectroscopy was performed using a Bruker Tensor 27 FTIR spectrometer . All data were recorded at room temperature in the spectral range of 450~4000 cmA tensile tester  was used to measure tensile properties. Tensile tests were performed as per GB/T 1040.2-2006. Composite specimens were mounted and subjected to strain at a rate of 5 mm/min until failure occurred. Tensile tests of the composites were carried out at room temperature. Five specimens per batch were tested and the average strength was calculated.The ring stiffness calculation model for the bellows was established using Solidworks software . As shown in In order to simulate the process of testing the stiffness of the bellows ring, two parallel steel plates were placed at the upper and lower ends of the bellows. The cuboids in In this study, the mesh model was established using Ansys Workbench software . Both the steel plates and bellows adopted the method of automatic meshing, which can be used to automatically select the mesh type for the entity and generate the mesh. Then, the mesh size of the bellows was set. The principle of setting the bellows mesh size was to select the smallest possible mesh unit size that the computer could run.The ring stiffness test is described in detail in ISO 9969-94. The basic principle is to place the bellows between two parallel plates and compress it at a constant speed to obtain the curve of the relationship between force and ring stiffness. The ring stiffness is calculated from the reaction force when the pipe produces a deformation of 0.03 times the diameter.The static structure analysis was performed using the Mechanical APDL solver in Ansys Workbench software (Version 2020 R2). All bellows were subjected to the same boundary conditions and displacement loads. As shown in −1 in −1 was enhanced after modification. This may be due to the increase in O-Si-O bond content on the surface of the FA or the more stable existence of O-Si-O bonds within FA modified by silane. The absorption peak at 1097 cm−1 corresponds to the Si-O single bond’s stretching vibration, which is also enhanced in the spectrum of treated FA. This indicates that many Si-O bonds were added to the surface of the modified FA. This phenomenon is consistent with the results reported by T. T. Wu et al. [−1 is the stretching vibration region of N-H single bonds. The change in absorption peak strength at 3439 cm−1 indicates that the aminopropyl in the silane coupling agent was successfully attached to the surface of the FA.u et al. . In addiThe amount of FA added is one of the important factors affecting the mechanical properties of composites. In this study, FA/HDPE composites were prepared by adding different amounts of FA masterbatch to HDPE. As shown in With increasing addition of FA masterbatch, the change trend of the Young’s modulus of the composites was similar to that of the tensile strength, but the elongation at break of the composites decreased significantly. After adding 10% FA masterbatch, the elongation at break of the composite material significantly decreased from 500% to 325.55%, a decrease of approximately 34.9%. This is because the rigid FA cannot be stretched together with HDPE, so that the elongation at break of the composite material significantly decreased. This phenomenon generally occurs in most composites, where there is always a tradeoff between tensile strength and elongation at break . With fuIt can be seen from According to Improving the ring stiffness of large diameter HDPE bellows is a difficult problem, impeding further popularization of the use of large diameter PE pipe. Improving the ring stiffness of large diameter HDPE bellows is key to improving the quality of pipe . In ordeIn this paper, high-strength FA was used as an inorganic filler to enhance the mechanical properties of HDPE. When the added quantity of FA masterbatch increased from 0 to 15%, the tensile strength and Young’s modulus of the composites increased at first and then decreased, and the elongation at break decreased continuously with the addition of FA masterbatch, which is consistent with the results of Y. Huang et al. . In contIt is obvious from In a study by M. V. Deepthi et al. , HDPE-g-In order to verify whether the ring stiffness of HDPE composites bellows was improved, finite element analysis was used to simulate and calculate the ring stiffness of the bellows. In addition, the ring stiffness of a bellows made from a common commercially available material was also analyzed and calculated, and the ring stiffness of the two was compared. The main tested parameter of the two materials was Young’s modulus. Compared with the ring stiffness of the common material bellows, the ring stiffness of the composite bellows increased by approximately 23%. This shows that the composites are anticipated to be more useful as the preparation material for bellows. The HDPE composites prepared in this paper not only improved the strength and stiffness of the composites, but also alleviated the problem of greatly reduced toughness caused by FA added into HDPE. The utilization of FA not only reduced the preparation cost of large diameter HDPE bellows, but also achieved the reuse of solid waste materials.2. The steel plates in the pipe are generally connected by welding, but quality is difficult to guarantee and water leakage is prone to occurring. Due to the complicated preparation process, its production speed is relatively slow. Pure HDPE bellows is also now common and its ring stiffness is generally 4~8 KN/m2; its strength has gradually failed to meet increasing needs. In this paper, a high-strength and high-rigidity FA/HDPE composite material was prepared. Compared with the above-mentioned types of bellows, the preparation process was simple and the cost was low. For common bellows diameters, the ring stiffness could reach 10.6~18.4 KN/m2. Combining the performance advantages of stiffness and low cost of use, FA/HDPE is a potential high-performance buried bellows material.At present, common polyethylene bellows usually mix plastic and steel or optimize the wave shape and increase the number of inner ribs to enhance the strength and rigidity of the pipe. The ring stiffness of this bellows was generally 8~12.5 KN/m(1)The tensile strength and Young’s modulus of the composite increased at first and then decreased with the increase in FA masterbatch content, and the elongation at break decreased with the addition of FA. The existence of LDPE in the FA masterbatch could alleviate the decreases in strength, stiffness and elongation at break caused by excessive addition of FA. When 10% modified FA masterbatch was added to HDPE, the tensile strength of the composite material reached the maximum. Compared with pure HDPE, the tensile yield strength of 10% FA masterbatch/90% HDPE composites was increased by approximately 24.5%, and the tensile breaking strength was increased by approximately 30.7%.(2)A small amount of compatibilizer could enhance the tensile strength of the composite, but too much compatibilizer would reduce the tensile strength of the composite. The addition of compatibilizer to FA masterbatch/HDPE composite could make FA and HDPE have a stronger interaction. After adding the compatibilizer to the FA masterbatch/HDPE composite materials, the elongation at break of the composite materials would increase, while the rigidity would decrease. When 5% compatibilizer was added to the 10% FA masterbatch/HDPE composite, the comprehensive mechanical properties of the composite were the best. Compared with the 10% FA masterbatch/HDPE composite, the tensile yield strength, tensile breaking strength and elongation at break of the composite increased by 4.6%, 7.4% and 9.1%, respectively.(3)The ring stiffness of bellows made of 10% modified FA masterbatch/5% HDPE-MAH/85% HDPE composite was calculated by finite element analysis, and it was found that the bellows made of this composite could achieve higher ring stiffness. Compared with the common diameter bellows made from common commercially available materials, the ring stiffness increased by approximately 23%. Therefore, this composite is expected to be a material for the preparation of large diameter HDPE bellows.In this study, modified FA was prepared into a masterbatch as a reinforcing filler, and a series of FA/HDPE composite materials were prepared. The influence of the added amounts of FA masterbatch and compatibilizer on the mechanical properties of composite materials was studied. Further studies included cross-sectional scanning electron microscope images and thermal stability of the composite materials. In order to study whether the composite materials were suitable for preparing large-diameter bellows, the ring stiffness of common diameter bellows made of FA/HDPE composites was simulated and calculated using finite element analysis. Based on the experimental results and observations, the following conclusions can be drawn:


Cancers had significantly greater mean stiffness indices and lower mean homogeneity of stiffness than benign masses (p < 0.05). SoftVue stiffness imaging demonstrated small stiff masses, mainly due to cancers, amongst predominantly soft breast tissues. Quantitative stiffness mapping of the whole breast and underlying masses may have implications for screening of women with dense breasts, cancer risk evaluations, chemoprevention and treatment monitoring.We evaluated whole breast stiffness imaging by SoftVue ultrasound tomography (UST), extracted from the bulk modulus, to volumetrically map differences in breast tissues and masses. A total 206 women with either palpable or mammographically/sonographically visible masses underwent UST scanning prior to biopsy as part of a prospective, HIPAA-compliant multicenter cohort study. The volumetric data sets comprised 298 masses  in 239 breasts. All breast tissues were segmented into six categories, using sound speed to separate fat from fibroglandular tissues, and then subgrouped by stiffness into soft, intermediate and hard components. Ninety percent of women had mammographically dense breasts but only 11.2% of their total breast volume showed hard components while 69% of fibroglandular tissues were softer. All smaller masses (<1.5 cm) showed a greater percentage of hard components than their corresponding larger masses ( Elasticity assessment became part of the 5th edition of Breast Imaging Reporting and Data System (BI-RADS) for handheld ultrasound (US) in 2013, created under associated features of masses as soft, intermediate or hard . Data from the other arm of the multi-center study using UST for dense breast screening as an adjunct to mammography  are not reported here and could not be accessed while under FDA review of Pre-Market Approval (PMA). Patients in ARM2 were eligible to receive SoftVue imaging as part of their clinical visit for evaluation of a palpable or mammographic abnormality. Informed consent was thus obtained from all women within this observational cohort study whereby their main inclusion criterion was their willingness to participate with a SoftVue scan during their clinical visit. Notable exclusion criteria were age <18 years, body weight >350 pounds , inability to give informed consent, inability to lie prone on the UST table, and any open sores or wounds on the breast precluding immersion into the UST water bath. Water within the SoftVue scanning tank is exchanged between patients and sanitized with ProTex . For this study, all patients were included between UST scan dates 4/2017–10/2018 for this consecutive data set, using the same version of the SoftVue unit and associated reconstruction algorithms across all centers of the trial.All identified masses were biopsy-confirmed by subsequent or prior histology, unless considered as a characteristic cyst by hand-held ultrasound criteria. All complicated cysts underwent aspiration with cytologic confirmation. Some women had more than one mass in each or both breasts. Masses were grouped into the main categories of cancer, fibroadenoma, cysts and other benign , then separated according to size (≤1.5 cm and >1.5 cm diameter), which also matched the set-point of 1.5 cm for post-processing using a high pass spatial filter . A SoftVue scan is operator-independent and covers the entire volume of the breast, up to but not including the axilla. The patient lies prone on a table that houses a water bath in which the breast is pendant during scanning. A ring-shaped sensor surrounds the breast inside the water bath and scans the whole breast from nipple to chest wall  in approximately two minutes, providing a stack of 2.5 mm thick coronal images  and just recently received PMA as an adjunct for dense breast screening  are tied to an external standard, the pixel values of the derived surrogate can be used as a consistent reference for comparing relative differences in the bulk modulus between patients. Furthermore, during UST image processing, pixel values are calculated and displayed on a color scale, designed to map the full range of stiffness values found in a given breast, optimized for relative stiffness differences in that breast. In this study, we chose a range of colors that are segmented into groups as noted below, emphasizing that the stiffness parameter is displayed as quantified pixel values that can be seen on a relative color scale and reproduced from patient to patient.Sound speed images use quantitative pixel values  that can be separated into the tissue categories of fibroglandular  or fatty by the use of K-means clustering techniques that classified all pixels as either white or black  ,16,17,18Correlations between sound speed and stiffness values were evaluated to assess their independence, especially given that stiffness is derived in part from incorporating sound speed values. The total volume of each tissue component was determined for each patient and then averaged across the patient cohort. Similarly, the percentage of each such component was also determined.Mass boundaries were hand-traced by a radiologist with extensive UST experience (PL) and over 20 years as a MQSA-certified breast radiologist, using MIM software to generate regions of interest (ROIs) surrounding all margins of detected masses. Mass margin contours were traced on the single most representative coronal image using a combination of sound speed and reflection image stacks. Careful note was made of these underlying margins to clearly separate tumor from peritumoral regions which have also been documented in quantitative analyses of mass locations . Minor eThe average stiffness index of each mass ROI was defined on a scale of 0 to 1, based on the pixel values of stiffness. As noted from segmentation using K means clustering, the pixel values were distributed to the three stiffness levels that roughly correspond to the range of colors on the stiffness image . Each mass thus also had an associated color pattern, in addition to their average stiffness index. The corresponding stiffness distributions were then determined for each major mass types of cancers, fibroadenomas and cysts, across the patient cohort to assess any differences. The stiffness properties of the masses were compared with the corresponding whole-breast values to identify any associations of mass type with the six tissue components.Spatial filtering is a relatively common post-processing step that can emphasize or deemphasize structures based on their size . A high t-test. Mass stiffness indices, derived from average pixel values, were also assessed for texture differences  by testing the single higher-order statistical feature of homogeneity. This provided some insight to future uses of more complete radiomics ]  . FrequenThe average age for participants in this study was 48.9 years . The retrieved data sets represent 239 individual breasts from 206 diagnostic patients with 298 masses, including cancer and benign masses, as noted in The association of stiffness vs sound speed values was negligible (correlation coefficient of 0.0048) suggesting that the stiffness property of tissues is independent of its sound speed component.The averages associated with the six tissue components, totaling 737 cc, are summarized in Images of sound speed, reflection and stiffness are shown in Examples of filtered and unfiltered images of a mammographically occult cancer are shown in p = 0.001). For the filtered images, small cancers were stiffer than small fibroadenomas . Only smaller cancers were significantly altered by spatial filtering, increasing their hard component by 23.1%, from pixel averages of 61.5% and 30.8% for unfiltered hard and intermediate components, to 84.6% and 11.5%, respectively (p < 0.001).Unfiltered and filtered stiffness distributions were separated according to mass size and type, as shown in p = 0.00036 versus p = 0.080; bold in upper p = 0.037 versus p = 0.127; bold in lower p = 0.035).Quantitative stiffness values of large and small masses, as displayed by the unfiltered and spatially filtered algorithms, are shown in Additional examples of spatially filtered stiffness images are shown in Considering benign masses first, simple cysts had a soft appearance (blue-black color) with little or no internal stiffness, regardless of size a. SmalleCancers displayed visual characteristics in To the best of our knowledge, this study represents the first assessment of whole breast stiffness by ultrasound tomography, which combined the transmission properties of attenuation and sound speed to create a unique surrogate of bulk modulus. SoftVue stiffness imaging in the coronal plane can be volumetrically assessed and compared throughout the breast and between patients, including underlying masses that are readily localized in volumetric formats and/or the thumbnail images in axial and sagittal planes. Other investigators using FDA-cleared UST  have not employed stiffness or attenuation, limiting their clinical evaluations to the use of reflection and sound speed for characterizing breast density ,10 and dIn related work, direct stiffness measurements of resected breast specimens have been described as a tissue spectrum, with progressive increases in overall mass stiffness, extending from the softest benign solid tissues to the hardest types of different cancers . Surgicaλ/2  cannot be achieved and is typically > λ. With UST, data are amenable to a wave equation solution which accounts for refractive and diffractive effects and allows the reconstruction algorithm to approach the theoretical limit of λ/2. For example, the speed of sound algorithm utilizes the portion of the pulse spectrum near 1 MHz which corresponds to a wavelength of 1.5 mm and, therefore, a λ/2 of 0.75 mm which is the coronal plane resolution of the SoftVue system. At 1 MHz, a conventional system would have a resolution of several millimeters. SoftVue’s stiffness fusion imaging thus provided submillimeter resolution of stiffness patterns within common breast masses throughout the breast that can be used by current breast radiologists assessing qualitative visible differences, as well as quantitative tissue analyses that help substantiate different mass appearances.SoftVue demonstrated the ability to achieve 0.75 mm resolution in the coronal plane despite the low operating frequency of 1–3 MHz. In conventional US approaches, images are produced by assuming that acoustic waves travel as rays. Refraction and diffraction are not taken into account. Consequently, the resolution limit of As expected, fatty tissue was found to be universally soft while 69% of fibroglandular tissues were also relatively soft . The ~2%From a simple visualization perspective, the average mass size of 1.1 mL and average breast volume of 737 mL suggest that visualization of underlying masses is a volumetric balance of relative stiffness. Namely, small cancers are mostly stiff and could either be readily seen amongst a predominantly soft background, since 9% (66 mL) of hard fibroglandular tissue would be scattered throughout that volume. Or, a cancer could be potentially obscured if it was also partially embedded in a larger grouping of hard fibroglandular tissue, such as in The unfiltered properties of the small mass group yielded statistically significant discrimination of cysts from cancers and cysts from fibroadenomas, but not cancers from fibroadenomas . The filIn the case of the larger mass group, the situation was reversed. The spatially filtered data did not yield statistical significance for cancers versus fibroadenomas but the unfiltered data did. This result can also be attributed to the action of the spatial filter which, in addition to suppressing the image rendering of stiffness data from adjacent larger fibroglandular structures , also suppressed the stiffness data within the larger cancers, thereby diluting their intrinsic contrast. In other words, larger cancers don’t require spatial filtering to improve their already high relative image contrast, such that the spatial filter removed ~15% hard components and often left remaining hard components within the margins of larger cancers. It is uncertain whether spatial filtering of large cancers may thus represent an imaging correlate of the fibrotic peri-tumoral region surrounding some larger cancers. In any case, it can be concluded that cancer stiffness appears to decrease with increasing size. A similar but weaker trend is apparent for the benign masses in The differences noted between smaller and larger masses and the filtered and un-filtered mass images can be explained in part by the physics of the UST imaging process. In the case of the non-filtered cancer images, we note a trend of decreasing stiffness with increasing mass size . This trOur study demonstrated the independence of stiffness from sound speed and adds new information to the interpretation of the whole breast and tissue properties of common masses. Previous work has shown strong correlations between the higher values of whole-breast average UST sound speed and mammographic density, which are representative of the underlying fibroglandular tissue ,10,12. IThe three common breast masses also showed quantitative differences that provide insight to their visual appearances. These will continue to be refined for diagnostic characterization, but also apply to further evaluation of masses, such as cancer response to neo-adjuvant chemotherapy . SoftVueWeaknesses: a limitation of the stiffness measurement used in this study is the use of a surrogate instead of the actual bulk modulus. Consequently, the stiffness measurement was not calibrated against an external standard and expressed in absolute units. Nevertheless, the bulk modulus surrogate represents a relative estimate of stiffness that can be quantitatively compared between patient groups and may mitigate current elastographic artifacts noted with cysts . AdditioFor future screening uses, thorough evaluation of post-processing techniques for whole breast stiffness imaging, such as spatial filtering, is needed before mass characterization benefits can be translated from this initial clinical series to screening populations or studies of mass detection. SoftVue stiffness analyses that need further detail include: comparisons with quantified phantoms, detailed histologic correlates from resection studies, 2D elastographic comparisons with SoftVue, performance differences with post-processing techniques, and stiffness threshold adjustments to its sound speed and attenuation components. Larger studies of SoftVue stiffness imaging may better characterize cancer subtypes and benign findings. While it is encouraging that UST shows promise in ongoing studies of risk evaluation , chemoprp < 0.001). Cancers had greater mean stiffness indices and lower mean homogeneity of stiffness than benign masses (p < 0.05). A common finding was the presence of small stiffer masses, mainly due to cancers, amongst predominantly soft breast tissues. SoftVue stiffness imaging using a surrogate of bulk modulus may become a valuable tool for breast cancer detection, risk factor assessments and/or monitoring of chemoprevention or neoadjuvant chemotherapy treatments.We used ultrasound tomography (UST) to volumetrically map stiffness differences in whole breast tissues and common masses. 90% of women had mammographically dense breasts but only 11% of total breast volumes showed hard components, while 69% of fibroglandular tissues were softer. All small cancers (<1.5 cm) showed greater percentage of hard components compared to large cancers (


Among them, good outcomes have traditionally been obtained using preserved scleral grafts. However, scleral patch grafts have complications such as graft retraction, thinning, dehiscence, and necrosis. Furthermore, to promote epithelial healing, scleral patch grafting must be accompanied by procedures such as amniotic membrane transplantation (AMT) or grafting using conjunctival flaps or autografts. Recently, acellular preserved human corneas have been used in various ophthalmic surgeries, with emerging evidence supporting its use for treating scleral defects as an option that does not require AMT or conjunctival autografting. We investigated whether corneal patch grafting would show wound healing and tectonic success rate outcomes comparable to those of existing techniques.: Three patients presented with intractable ocular pain. Slit-lamp examination showed marked scleral thinning at the nasal side.: Scleral thinning progressed with conservative treatment; microbial staining and culturing were performed. Infectious or non-infectious scleritis was diagnosed according to slit-lamp examination and microbial culture results.: A preserved corneal lamellar patch was grafted at the scleral thinning area.All patients achieved tectonic success with reduction of inflammation following corneal patch grafting. Two patients achieved complete re-epithelialization within 7 days, while 25 days were required for the third patient. No patients experienced graft thinning, rejection, or infection.: Our report suggests the feasibility of using acellular preserved human cornea patch grafts to reinforce inflammatory scleral defects and obtain successful outcomes in terms of wound healing. This technique shows a comparable tectonic success rate and superior effect on scleral defect healing without the need for adjunctive AMT or conjunctival autografting. To prevent these adverse events, scleral reinforcement is performed to cover the thinned sclera using different materials, including allogeneic sclera, amniotic membrane, fascia lata, pericardium, periosteum, and perichondrium.–5 Among them, good outcomes have traditionally been obtained using preserved scleral grafts. However, these are associated with complications such as graft retraction, thinning, dehiscence, and necrosis. Furthermore, scleral graft transplantation must be accompanied by amniotic membrane transplantation (AMT) to promote epithelization over the scleral or conjunctival graft,,8 which increases surgical time. Acellular preserved human corneal tissue can be transplanted to the scleral defect area without the need for AMT or conjunctival grafting, which has been shown to reduce inflammation and accelerate wound healing in rabbits. Compared with the sclera, the cornea has a basement membrane and collagen fibrils that are arranged compactly, which is expected to be beneficial for wound healing and graft survival.Infectious or noninfectious scleral necrosis with calcium deposition could occur after pterygium removal, trauma, idiopathic systemic vasculitis, or retinal detachment surgery.We report on 3 cases of infectious or noninfectious scleral defects with successful transplantation of acellular preserved corneal lamellar grafts followed by rapid wound healing and inflammation reduction. This technique seems to have an effect on scleral graft healing comparable or superior to that of adjunctive AMT or conjunctival autograft.22.1A 74-year-old female underwent right-eye pterygium surgery approximately 13 years ago. She presented to our ophthalmology department with a six-month history of right ocular pain  was transplanted. One week postoperatively, with topical application of 0.5% moxifloxacin , the graft surface was fully epithelized and her BCVA was 20/20 , 2.5% fortified vancomycin, 5% ceftazidime, and 1% voriconazole, as well as oral administration of 200 mg voriconazole twice daily and 400 mg moxifloxacin once daily was performed.An 82-year-old male visited our hospital with a three-month history of severe pain and decreased visual acuity in the left eye Table . At the Five weeks later, although the infection was partially controlled, scleral necrosis had worsened compared to the initial visit. The defect size was 5 × 8 mm with iris prolapse at the inferonasal limbus that extended to the scleral defect Fig. G. After 3–5,12 We used an acellular preserved human cornea patch graft to manage scleral thinning with inflammation and obtained successful wound healing and inflammation reduction outcomes. In a retrospective report of 13 cases of necrotizing scleritis, scleral thinning, or dehiscence by Sangwan et al, the tectonic success rate was 76.9% and 3 complications were noted . In addition, re-epithelization occurred after an average of 3 to 4 weeks. Oh et al reported that the scleral graft remained intact and no recurrence was observed in 8 patients with noninfectious scleromalacia. Re-epithelization was observed within 7 days in 7 of those patients; 6 weeks were required for re-epithelization in the remaining patient. Kim et al reported a high success rate of grafting using autologous perichondrium with AMT in 17 out of 18 eyes (94.4%) at 6 months postoperatively. However, their surgical technique necessitates harvesting the perichondrium from the tragus prior to AMT, which increases surgical time.Surgical management of infectious or noninfectious scleritis with severe thinning remains challenging, and scleral wall reinforcement has been attempted using various materials. In Case 3, epithelialization took a relatively long time (25 days). Considering that it was a case of fungal infection with a poor prognosis, this could be regarded as a relatively good outcome. The patient's decreased visual acuity was due to corneal opacity and iris synechiae obscuring the pupil.A literature review reveals that our tectonic success rate and re-epithelialization time were comparable to those previously reported in studies of scleral grafting Table ; moreove,14–17 However, they used corneal grafts from fresh corneas or donated whole corneas after Descemet stripping endothelial keratoplasty. Two reports of successful tectonic outcome using acellular corneal graft in patients with scleritis following strabismus surgery have been published.,19 The rate of epithelialization was mentioned in only one study. Taken together, this study supports the evidence that preserved corneal lamellar grafting may be feasible for scleral defects.Several studies of acellular or cellular corneal graft use for scleral reinforcement using various surgical techniques have been published Table ; some re,21 In addition, evidence suggests that acellular preserved corneal grafting is effective for reducing inflammation and promoting wound healing in rabbits.No study directly compares the efficacy of corneal and scleral grafts for treating scleral defects. However, we can infer and compare the effects based on studies in patients who underwent glaucoma valve surgery with corneal grafting. These studies showed comparable tectonic results between corneal and scleral grafts, and they found that surgeons used corneal grafts 3.8 times more frequently than scleral grafts.This study was limited by the small number of patients and short follow-up duration. Nevertheless, it provides evidence that acellular preserved corneal grafting promotes wound healing, has comparable tectonic success rates to those of traditionally used techniques, and may have the advantage of a shorter surgical time in the treatment of scleral defects. Therefore, surgeons should consider acellular preserved cornea grafting as an option for the treatment of infectious or noninfectious scleral defects.Conceptualization: Mee Kum Kim.Data curation: Mee Kum Kim.Formal analysis: Young Ho Jung.Methodology: Mee Kum Kim.Supervision: Mee Kum Kim.Validation: Mee Kum Kim.Visualization: Young Ho Jung, Chang Ho Yoon.Writing – original draft: Young Ho Jung, Mee Kum Kim.Writing – review & editing: Young Ho Jung, Chang Ho Yoon, Mee Kum Kim.


J Cancer 2019; 10(26):6511-6525) has been retracted due to concerns on multiple image duplications as identified by PubPeer users. The corresponding author has been contacted by the journal, and the authors expressed apology for their negligence and mistakes in this paper.This article (IL-6 Promotes Cancer Stemness and Oncogenicity in U2OS and MG-63 Osteosarcoma Cells by Upregulating the OPN-STAT3 Pathway. Chuan Zhang, Kun Ma, Wu-Yin Li.


Resistant starch (RS) type 2-high-amylose corn starch (HACS) was subjected to simultaneous hydrothermal  and microwave  treatment and zein  to improve its resistance to enzymolysis. Scanning electron microscopy (SEM) highlighted the aggregation and adhesion of the composite. The average particle size of the composite (27.65 μm) was exceeded that of both the HACS (12.52 μm) and the hydrothermal and microwave treated HACS (hydro-micro-HACS) (12.68 μm). The X-ray diffraction results revealed that the hydro-micro-HACS and composite remained B-type, while their crystallinity significantly decreased to 16.98% and 12.11%, respectively. The viscosity of the hydro-micro-HACS and composite at 50 °C was 25.41% and 35.36% lower than that of HACS. The differential scanning calorimetry (DSC) results demonstrated that the composite displayed a new endothermic peak at 95.79 °C, while the weight loss rate and decomposition temperature were 7.61% and 2.39% lower than HACS, respectively. The RS content in HACS, the hydro-micro-HACS, and composite was 47.12%, 57.28%, and 62.74%, respectively. In conclusion, hydrothermal and microwave treatment combined with zein provide an efficient physical strategy to enhance the RS type 2-HACS. The increase in consumer demand for healthy food products has led to new food processing and composition requirements. The protein–starch composite can be used as biological material in the food industry , the medAlthough resistant starch (RS), also known as anti-enzymatic starch, cannot be digested and absorbed in the small intestine, it can be fermented by the microbial flora in the colon. It presents beneficial physiological effects , such asZein is a yellow powder belonging to a class of prolamins and is applied in coatings, films, fibers, plastics, adhesives, and inks . Zein caHACS granules are mostly round or oval, while a few are rod-shaped with smooth surfaces A. The hip > 0.05) (p < 0.05) (No significant differences were evident between the redness (a*) values of the three samples ( > 0.05) . The HAC < 0.05) . This exp > 0.05). Luo et al. reported that microwave treatment did not alter the sizes and shapes of the granules in normal maize, waxy maize, and amylomaize V starches [p < 0.05), which was consistent with the scanning electron micrographs. The encapsulation of the protein around starch granules causes agglomeration, increasing their average particle sizes [The average particle sizes in HACS, hydro-micro-HACS, and the composite were 12.52, 12.68, and 27.65 μm, respectively . Hydrothstarches , while tle sizes . The X-ray diffraction patterns of HACS, hydro-micro-HACS, and composite presented diffraction peaks at 2θ of 5.6°, 17°, 19.5°, 22°, and 24° . These pThe HACS, hydro-micro-HACS, and composite viscosity were 362, 270, and 234 cP, respectively. An increase in the viscosity of the paste during the heating phase of gelatinization indicated the water absorption level and swelling capacity of the starch , while ap > 0.05). The significant decrease in the weight loss rate of the composite may be due to the zein and starch combination. At maximum HACS, hydro-micro-HACS, and composite decomposition rates, the decomposition temperatures were 316.98, 317.84, and 309.41 °C, respectively. The increased decomposition temperature during the dual treatments (p < 0.05) indicated enhanced thermal stability, while the internal molecule structure became firmer. The decreased decomposition temperature of the composite may be caused by the partial replacement of the starch by zein.The weight loss rate of the HACS, hydro-micro-HACS, and composite was 80.99%, 80.29%, and 74.83% , respectp < 0.05) (p < 0.05) the energy required for gelatinization (∆H). The subsequent increase in the To and Tp and decrease in ∆H were consistent with the modification results obtained in waxy maize starch via hydrothermal treatment and crosslinking [p < 0.05) after hydrothermal treatment [p > 0.05), while the ∆H decreased significantly (p < 0.05). The decreased ∆H could be attributed to the partial replacement of the treated starch by zein. Therefore, the starch concentration was reduced, and less energy was required to destroy the crystalline structure during gelatinization [The hydro-micro-HACS showed a significant increase in the To and peak gelatinization temperature (Tp) compared to HACS ( < 0.05) . Dual trslinking . Hydrothslinking . Molavi reatment . The comnization .The RS levels in the samples were used to characterize the degree of enzymatic hydrolysis. The RS content in HACS, hydro-micro-HACS, and composite was 47.12%, 57.28%, and 62.74%, respectively. The hydrothermal and microwave treatment and compounded zein increased the RS by 21.56% and 33.15%, respectively. Brumovsky and Thompson found that hydro-micro-HACS displayed a resistance level of 43.9% compared to native starch at 18.4% . Zhong eHydrothermal and microwave treatment and zein increase the RS content in HACS by 21.56% and 33.15%, respectively. Hydrothermal and microwave treatment decreases the crystallinity, viscosity, and ∆H of HACS by 16.98%, 25.41%, and 49.18%, respectively. The zein encapsulation of the modified starch granules increases the average particle sizes and b* value of the composite by 118% and 124%. The crystallinity, viscosity, lightness, weight loss rate, and decomposition temperature of the composite decrease by 28.68%, 13.33%, 3.02%, 6.80%, and 2.65%, respectively. The results reveal that hydrothermal and microwave treatment perfected the crystalline structure of the starch granules, while zein encapsulation facilitates enzymatic hydrolysis. Therefore, developing a new method for RS preparation is essential, and further exploration of the application in various food matrices is required.The HACS was supplied by Hengrui Technology Co., Ltd. . The RS test kits were acquired from Ireland Megazyme Company, and zein was purchased from Sinopharm Chemical Reagent Co., Ltd. . The anhydrous ethanol was obtained from Tianli Chemical Reagent Co., Ltd. , while the sodium hydroxide was purchased from Tianjin Kemeo Chemical Reagent Co., Ltd. .Hydrothermal treatment occurred according to a method described by Sui et al. . The moiAfter hydrothermal treatment, the moisture content of the HACS was adjusted to 35% with distilled water and equilibrated at room temperature for 24 h. The sample was microwaved at 40 W/g for 4 min and refrigerated at 4 °C for 10 h, after which it was dried at 40 °C to a moisture content of 10%. The samples were ground and sieved using an 80-mesh sieve for further analysis. w/w) was dissolved in ethanol (70%) and stirred in a water bath at 30 °C until completely dissolved. Then, hydro-micro-HACS (at a 1:5 ratio of zein to starch) was slowly added while rapidly stirring in a 50 °C water bath for 1 h. Next, cold water was added at 80 times the protein content, stirred quickly for 30 min, and allowed to settle. After removing the supernatant solution, the precipitate was dried at 45 °C, ground, and sieved using an 80-mesh sieve for further analysis. Zein  at an accelerating voltage of 3 kV in high-vacuum conditions.The color of the HACS, hydro-micro-HACS, and composite was measured using a Konica Minolta colorimeter CR-400  and the L*(the degree of lightness), a*(the red-green axis), and b* (the yellow-blue axis) values were recorded.The HACS, hydro-micro-HACS, and composite particle size distributions were determined using a laser diffraction particle size analyzer .The X-ray diffraction patterns were determined using an X-ray diffractometer  at a scanning speed of 4°/min, angles ranging from 5° to 45° (2θ), 40 kV, and 30 mA. The MDI Jade 6.0 software  was used to analyze the crystallinity of the samples.w/v), which were placed in separate sealed bottles. The samples were gelatinized in an autoclave at 126 °C for 30 min, while their viscosity levels were measured at 50 °C using a DV-Ⅲ rotary viscometer.The HACS, hydro-micro-HACS, and composite were mixed with distilled water to obtain slurries  and differential scanning calorimeter . The sample  was placed in a platinum pan and heated from 40 °C to 600 °C at a heating rate of 10 °C/min to obtain the weight loss and weight loss rate values. The composite  was mixed with 7.5 μL in an aluminum pan, which was hermetically sealed and equilibrated at room temperature for 24 h. The sample and reference were scanned from 20 °C to 160 °C at a heating rate of 10 °C/min.w denotes the RS content (%), E is the absorbance difference between the sample and the blank reagent, F signifies the conversion from the absorbance value to the microgram—an absorbance value of 100 μg d-glucose was determined during the GOPOD reaction, F = 100 , and W is the dry starch weight (g).The HACS, hydro-micro-HACS, and composite enzymolysis were determined using a Megazyme RS detection kit . The samThe RS levels were used to characterize the HACS, hydro-micro-HACS, and composite enzymolysis. The RS content in the composite was calibrated using the starch percentage in the composite and was calculated asp < 0.05 was considered statistically significant based on the analysis of variance (ANOVA) test.All measurements were performed in triplicate. The data were analyzed using SPSS Version 16.0. Duncan’s pairwise comparison was used to assess the differences between the mean values, and


Nonspecific lipid transfer proteins (nsLTPs) have been categorized as panallergens and display widespread occurrence across plant-kingdom. Present study, investigated B-cell epitopes for LTPs from chickpea, mung-bean, cowpea, pigeon-pea, and soybean via in silico methods. In-silico predicted regions were evaluated for epitope-conservancy and property-based peptide similarity search by different allergen databases. Additionally, the in-silico predicted regions were compared with the experimentally validated epitopes of peach-LTP. Sequence-homology studies showed that chickpea and mung-bean LTPs shared significant homology, i.e., >70% and >60%, respectively, with other LTP allergens from lentil, garden-pea, peanut, etc. Phylogenetic-analysis also showed chickpea and mung-bean LTPs to be closely related to allergenic LTPs from lentil and peanut, respectively. Epitope-conservation analysis showed that two of the predicted B-cell epitopic regions in chickpea and mung-bean LTPs were also conserved in other allergenic LTPs from peach, peanut, garden-pea, lentil, and green-bean, and might serve as conserved B-cell epitopes of the LTP protein family. Property-distance index values for chickpea and mung-bean LTPs also showed that most of the epitopes shared similarity with the reported allergens like-lentil, peanut, apple, plum, tomato, etc. Present findings, may be explored for identification of probable allergenicity of novel LTPs, on the basis of the reported conserved B-cell epitopes, responsible for potential cross-reactivity. Food allergy is defined as a hypersensitivity reaction induced upon exposure to a particular food allergen (protein) that occurs reproducibly on subsequent exposures. Several characteristics account for allergenicity of a food protein like ability to elicit an immunoglobulin E (IgE) mediated immune response, thermal stability, pepsin resistance, glycosylation, enzymatic activity, etc. ,2. HowevIgE-antibodies generated during a type-1 hypersensitivity reaction, in response to an allergen, specifically interacts with the antigen at discrete regions referred as antigenic-determinants or B-cell epitopes ,9. SpatiPlant LTPs display a ubiquitous presence throughout the plant kingdom and have been divided into two subclasses on the basis of structural organization, i.e., lipid transfer protein 1(LTP1) and lipid transfer protein 2 (LTP2) with a molecular weight of 9 kDa, and 7kDa, respectively . Both thDue to geographical diversity the dietary consumption of individuals varies among different regions in India. Previous studies, have reported highest prevalence of legume allergy followed by allergy to milk, egg, fish, and eggplant among the Indian population ,22. AlmoIn the present study, allergenic potential of five LTPs from different legumes, namely, chickpea, mung-bean, cowpea, pigeon-pea, and soybean were assessed by sequence homology and phylogenetic analysis, using online web-servers and allergen databases. Further, B-cell epitopes were identified for the LTPs using eight web-servers. In silico predicted epitopes were assessed for ‘epitope-conservancy’ and ‘property-based peptide similarity index  by different tools. B-cell epitopes predicted for the LTPs employed in the study were also compared with the experimentally validated epitopes from peach LTP . Due to www.ncbi.nlm.nih.gov; accessed on 25 January 2021). Further, for comparison with the reported allergenic LTPs, the amino acid sequences for LTPs from peach (AGW21358), garden pea (AJG44053), lentil (AAX35806), green-bean (ADC80502), and peanut (ABX56711) were also retrieved from NCBI.Amino acid sequences for five legume LTPs viz. chickpea (CAA05771), mung-bean (CCF23017), cowpea (CAA56113), pigeon-pea (XP_020207090), and soybean (XP_003549896) were retrieved from National Center for Biotechnology Information (NCBI)  and Structural Database of Allergenic Proteins , respectively, for prediction of probable allergenicity. Briefly, two search modes were employed in the present study, i.e., ‘full length FASTA alignment’ and ‘80 amino acid sliding window’, respectively. Both the search approaches have previously been described by several groups in details [Involves, comparison of the query protein with the reported allergenic proteins and depending on the percent identity between the two, the potential of a novel protein to act as an allergen is determined ,27. In thttps://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 25 January 2021). Subsequently, based on the MSA results, the evolutionary relatedness among different legume LTPs was evaluated by constructing a phylogenetic tree (Cladogram or Phylogram), using the neighbor-joining (NJ) algorithm without distance corrections .Amino acid sequences retrieved for different LTPs employed in the study were subjected to multiple sequence alignment using the Clustal Omega multiple sequence alignment program .Secondary structures for all the legume LTPs were predicted by the GOR IV method . Briefly, each of the query protein, i.e., LTPs from chickpea, pigeon-pea, mung-bean, soybean, and cowpea were individually compared with a set of five allergenic LTPs  using default settings. Evolutionary scoring was depicted with the help of three distinct signs, i.e., ‘*’ for fully conserved residues, while ‘:’ and ‘.’ for partially conserved residues. Non-conserved residues were devoid of any symbol. The descending order of the evolutionary score was ‘*’ > ‘:’ > ‘.’.Evolutionary conservation of each amino acid residue for all the five legume LTPs was assessed using the Clustal Omega MSA program (Linear epitopes predicted for different legume LTPs were assessed for ‘peptide similarity search (PD Index)’ with the reported allergen sequences in SDAP database. PD index delineates similarity between the query peptide (linear predicted epitope) and other allergen sequences in SDAP database with similar overall physiochemical properties. Peptides with identical sequences display a PD index of 0, while peptides with conservative amino acid substitutions show a lower PD index, usually less than 4. On the contrary, random unrelated peptides display a PD index of more than 10. Therefore, in the present study, the threshold for the PD index was set at < 4, in order to limit less similar matches.https://www.ebi.ac.uk/Tools/msa/clustalo/ accessed on e 25 January 2021). Briefly, MSA was performed for all the fifteen LTPs with default settings. Depending on the MSA profile obtained, percent conservation score was calculated for each residue corresponding to the experimentally validated epitopes of Pur p 3. The score was calculated by the formula given below, where ‘X’ corresponds to each residue of the validated epitopes of peach LTP.Amino acid sequences for fifteen allergenic LTPs from different food crops namely, wheat (P24296.2), cabbage (AAA32995.1), celery (E6Y8S8.1), maize (P19656.1), tomato (NP_001316314.1), grape (P80273.2), hazelnut (Q9ATH2.1), walnut (ACI47547.1), sunflower (CAA63340.1), apricot (P81651.2), plum (P82534.1), cherry (Q9M5X8.1), almond (Q43017.1), pear (Q9M5X6.1), and apple (Q9M5X7.1), were retrieved from NCBI. Evolutionary conservation of three experimentally validated B-cell epitopes of Pur p 3, among fifteen allergenic LTPs was assessed using the Clustal Omega MSA program  and reported allergenic LTPs from peach, garden pea, lentil, green bean, and peanut. LTPs constitute a well characterized category of food allergens, and some of the examples include Pru p 3 (from peach), Tri a 14 (from wheat), Len c 3 (from lentil), Pis s 3 (from garden pea), Sola l 3 (from tomato), Act c 10 (from kiwi), Mal d 3 (from apple), Pha v 3 (from green bean), Ara h 9 (from peanut), etc . Since, Phylogenetic tree obtained via Clustal Omega showed  chickpea).Previous studies have reported, LTPs as panallergens from diverse sources, viz., fruits, vegetables, cereals, pollens, etc. ,43. TillSupplementary Table S1Three B-cell epitopes were predicted for chickpea LTP by majority of the web servers employed in the study . First pThree B-cell epitopes were predicted for mung-bean LTP and all the three were overlapping linear and conformational epitopes  . Around Presence of identical epitopes/or some regions of the identical epitopes, among two related or distinct proteins contributes to potential cross reactivity . AlthougFive B-cell epitopes were predicted for cowpea LTP by majority of the webservers. As like chickpea LTP, the first predicted stretch (1st 29–35) was entirely a linear B-cell epitope, while rest of the predicted regions were overlapping linear and conformational epitopes , respectively . Among tTotal four epitopes were predicted for pigeon-pea LTP, of these the first predicted region (1st 25–39) was specifically a linear B-cell epitope, while other predicted residues constituted overlapping linear and conformational B-cell epitopes , respectively . Most ofOut of five epitopes predicted for soybean LTP , the last predicted stretch was a linear epitope, while other four predicted regions were overlapping linear and conformational epitopes . SimilarAs like, chickpea and mung-bean LTPs, triads were attempted for cowpea, pigeon-pea, and soybean LTPs, respectively . HoweverMoreover, it is important to mention that three experimentally validated epitopes of peach LTP, viz., epitope 1st , epitope 2nd , and epitope 3rd  , corroboAmong all the query proteins studied, maximum number of evolutionary conserved amino acid residues was observed for chickpea LTP, followed by mung-bean, pigeon-pea, cowpea, and soybean, respectively. Around 31 residues were fully conserved in chickpea LTP with reference to the allergenic LTPs, and 27 residues showed conservation with a score of >0.5 in the Gonnet point accepted mutation (PAM) 250 matrix. This implied that these 27 residues were mostly conserved, and if any variation existed, then they were replaced with amino acids having strongly similar properties to the original residues . FurtherOn overall analysis of the chickpea LTP sequence, four regions were identified as identical across LTPs from different allergenic sources, these were ‘T-T-P-D-R-Q-A’, ‘L-P-G-K-C’, ‘I-P-Y-K-I-S’, and ‘T-S-T-N’. Depending on the wide spread presence of these regions among the predicted epitopes of LTPs, these regions may contribute to potential cross reactivity and may also account for conserved B-cell epitopes of LTPs. It was observed that in ‘T-T-P-D-R-Q-A’ except for ‘P’ and ‘A’ all the residues were partially or fully conserved, suggesting that this predicted B-cell epitope may also be referred as an conserved B-cell epitope of LTPs and may be explored for evaluating potential allergenicity of novel LTPs. Next, all the residues in ‘L-P-G-K-C’ and ‘T-S-T-N’ were fully conserved, except for ‘K’ in former and ‘T’ in latter, both of which displayed partial conservation. Lastly, in ‘I-P-Y-K-I-S’ all the residues were fully conserved, except I and K, former displayed partial conservation, while latter was not conserved. Therefore, these regions may be categorized as conserved B-cell epitopes of legume LTPs or as conserved residues in the LTP allergen family as a whole .In mung-bean LTP, 30 residues were fully conserved, and 24 of the residues were conserved with a score of >0.5 in the Gonnet PAM 250 matrix . Three rTotal number of fully conserved residues in cowpea, pigeon-pea and soybean were 11, 14, and 10, respectively. While the count for the partially conserved residues were 15, 14, and 19, in the same order as above. Since, no specific regions present in the predicted epitopes of these three legume LTPs, were shared by the allergenic LTPs, therefore, the conservation of the epitopic regions was not analyzed for these three legume LTPs .As previously described in Similarly, for mung-bean LTPs, the predicted linear epitopes were subjected to a peptide similarity search in SDAP database and only those allergenic sequences were selected which displayed a PD index of less than four. The allergenic sequences which displayed similarity with mung bean predicted linear epitopes , were frTill date several studies, and significant amount of clinical data has already validated the antigenic dominance of peach LTPs among other LTP allergens . TherefoFurthermore, previous studies have reported the allergenic potential of chickpea and mung bean LTPs via sequence homology and multiple sequence alignment studies ,51. HoweLTP hypersensitivity in patients is diverse and complex; therefore, a generalization cannot be deduced on the basis of few studies with limited number of LTP positive patient samples. For instance, ELISA inhibition of kiwi LTP with Pur p 3 positive serum does not render complete abolishment of the IgE binding of the former, implying that kiwi LTP display some unique antigenic determinants not recognized by Pur p 3 specific IgE’s . SimilarWith the increase in the diversity of the food proteins being consumed, i.e., exploration of new food sources as alternative protein supplements for the human body, the burden on the food safety testing protocols is constantly increasing. Therefore, under such circumstances an ideal scenario would be to assimilate as much information as possible for all known and related food proteins. So that the information obtained may be utilized for evaluating potential allergenicity of new/novel food proteins. Consequently, in silico studies were explored as a preliminary testing approach, in the present study, for investigating the potential allergenicity associated with five legumes LTPs. Along-with sequence homology studies several other in silico tools/methods were explored for evaluating structural similarities among the allergenic LTPs and the query proteins. Depending on the results obtained in the present study , it was inferred that chickpea and mung-bean LTPs displayed significant homology, phylogenetic relatedness, high epitope-conservancy, and antigenicity with the reported allergenic LTPs, and might act as potential allergens.


The enzymes glucose oxidase (GOx), acetylcholine esterase (AchE) and urease that drive biocatalytic transformations to alter pH, are integrated into pH-responsive DNA-based hydrogels. A two-enzyme-loaded hydrogel composed of GOx/urease or AchE/urease and a three-enzyme-loaded hydrogel composed of GOx/AchE/urease are presented. The biocatalytic transformations within the hydrogels lead to the dictated reconfiguration of nucleic acid bridges and the switchable control over the stiffness of the respective hydrogels. The switchable stiffness features are used to develop biocatalytically guided shape-memory and self-healing matrices. In addition, loading of GOx/insulin in a pH-responsive DNA-based hydrogel yields a glucose-triggered matrix for the controlled release of insulin, acting as an artificial pancreas. The release of insulin is controlled by the concentrations of glucose, hence, the biocatalytic insulin-loaded hydrogel provides an interesting sense-and-treat carrier for controlling diabetes. Biocatalytic control over the stiffness of pH-responsive hydrogels is applied to develop shape-memory, self-healing and controlled release matrices. These include pH,7 heat,8,9 light,10–13 chemical agents14,15 and magnetic fields.16 Different applications of stimulus-responsive hydrogels were suggested, including their use as controlled drug-delivery carriers,17–19 sensing,20 tissue repair,21,22 bone-healing materials,23,24 and actuating and robotic devices.25–29 Within the broad class of stimulus-responsive hydrogels, stimulus-responsive nucleic acid-bridged hydrogels, i.e. hydrogels that consist of polyacrylamide or carboxymethyl cellulose crosslinked by nucleic acids, find growing interest.30,31 Besides the crosslinking of polymer chains by means of duplex nucleic acid bridges,32 hydrogels cooperatively stabilized by duplex nucleic acids and stimulus-responsive nucleic acid crosslinking units exhibit signal-triggered stiffness functions.33,34 For example, cytosine-rich strands undergo reversible pH-triggered reconfiguration between random strands and i-motif structures.35 Guanosine-rich nucleic acids undergo, in the presence of K+-ions/crown ethers, reversible transitions between G-quadruplex and random strand configurations,36 nucleic acid strands functionalized with photoisomerizable azobenzene units demonstrate light-induced formation and dissociation of duplex nucleic acids upon photoisomerization of the azobenzene units between trans and cis states,27,37,38 and triplex T–A·T or C–G·C+ nucleic acids undergo pH-stimulated reconfiguration of the nanostructures.39,40 Indeed, hydrogels cooperatively stabilized by duplex nucleic acid bridges and signal-triggered, reconfigurable cross-linkers were reported, demonstrating control over the stiffness of the hydrogels by means of pH,39,41,42 K+-ions/crown ethers43,44 or light.45–47 Also, aptamer-modified hydrogels were reported as functional matrices for the release of protein drugs.48 Recently, light-induced control over the stiffness of hydrogels through incorporation of plasmonic Au nanoparticles/Au nanorods into duplex nucleic acid-bridged hydrogels was reported.49 These Au nanoparticle-functionalized hydrogels enable thermoplasmonic heating, resulting in the thermal separation of the nucleic acid bridges and control over the stiffness of the hydrogel matrices. In addition, stimulus-responsive hydrogels were suggested to enable various applications, including the development of shape-memory,39,41,50,51 self-healing matrices,15,46,47 assembly of triggered and switchable drug release materials52 and carriers,53 the design of hydrogels exhibiting triggered mechanical bending properties,54 the control over ion-transport through nanopores,55 and the development of switchable electrocatalytic hydrogel materials.43,44Stimulus-responsive hydrogels undergoing gel-to-liquid, gel-to-solid or gel-transitions between variable stiffness states attract continuous interest due to their diverse potential applications.56 or sensing,57 and enzyme-loaded hydrogels were used as functional matrices for the biocatalysed, triggered degradation of the matrices and release of loads.58 Nonetheless, the enzyme-loaded DNA-based hydrogels, where the biocatalysts are retained and caged in the hydrogels within the process of the biocatalytic control over the stiffness of the hydrogels , are, to the best of our knowledge, unprecedented. Such enzyme-triggered, non-degradable hydrogels are anticipated to introduce new stimulus-responsive materials. Furthermore, the reversible enzyme-driven release of loads from the hydrogels may have important biomedical applications reflected by switchable drug release and the elimination of immunogenic responses. The unique switchable reconfiguration of nucleic acids59 suggests that the coupling of enzymes with stimulus-responsive hydrogel materials leads to functional materials that feature shape-memory, self-healing and controlled drug-release properties.Here we report the incorporation of enzymes into DNA-based hydrogels to yield biocatalytic control over the stiffness of the matrices, especially, we demonstrate the integration of glucose oxidase (GOx), acetylcholine esterase (AchE) and urease in polyacrylamide hydrogels cooperatively stabilized by two pH-responsive crosslinking motifs . The bioA was prepared by the copolymerization of acrylamide and the acrydite nucleic acid monomers (1) and (2) (see the ESI1) + (2)) on the polymer chains was evaluated by UV spectroscopy to be 1 : 58 (DNA : acrylamide), Fig. S1.1) includes a self-complementary sequence, while tether (2) is designed to generate a T–A·T triplex in the presence of the strand (3). The polymer PA, in the presence of the strand (3), glucose oxidase (GOx) and urease, yields a GOx/urease-loaded hydrogel cooperatively crosslinked by (1)/(1) duplexes and (2)/(3)/(2) T–A·T triplexes (path I). An additional, related, biocatalytic hydrogel included the loading of acetylcholine esterase (AchE) and urease in the (1)/(1) and (2)/(3)/(2) cooperatively crosslinked hydrogel (path II). The loading of the enzymes GOx, urease and AchE in the hydrogels corresponds to 23, 28 and 27 units, respectively /(1) units. The lower degree of crosslinking yielded hydrogels with lower stiffness. Treatment of the lower-stiffness hydrogel with glucose (path I) resulted in the aerobic oxidation of glucose to gluconic acid and H2O2. The biocatalytically formed gluconic acid acidified the hydrogel, regenerating the stiffer hydrogel crosslinked by the (1)/(1) duplex and the (2)/(3)/(2) triplex bridges. Similarly, subjecting the lower-stiffness hydrogel to acetylcholine (path II) led to the formation of choline and acetic acid; the process acidified the hydrogel and restored the hydrogel of higher stiffness cooperatively stabilized by the (1)/(1) and (2)/(3)/(2) bridging motifs. The enzyme-driven and switchable stiffness properties of the hydrogels were confirmed by rheometric measurements. 1)/(1) and T–A·T cross-linkers exhibits G′ and G′′ values corresponding to 90 Pa and 9 Pa (curve a′ and a′′). The treatment of the hydrogel with urea led to the urease-catalyzed hydrolysis of urea and to the pH-stimulated dissociation of the T–A·T triplexes, resulting in a hydrogel with lower stiffness, G′ = 55 Pa and G′′ = 5 Pa (curve b′ and b′′). The reversible treatment of the hydrogel with urea and glucose allowed switchable control over the stiffness of the hydrogel (1)/(1) and (2)/(3)/(2) revealed a higher stiffness, G′ = 110 Pa and G′′ = 12 Pa, curve a′ and a′′ as shown in G′ = 50 Pa and G′′ = 4 Pa (curve b′ and b′′). Similarly, the cyclic treatment of the hydrogel with urea and acetylcholine switched the stiffness of the hydrogel between lower and higher values  and the strand (4), and polymer PC functionalized with the nucleic acid strands (1) and (5) were prepared  was hybridized with the strand (4) to yield polymer  consisting of duplexes extended by single-strand toehold domains. Similarly, the strand (5) associated with PC was hybridized with strand (7) to yield a polymer  consisting of duplexes, where the hybridized tether (7) includes a single-strand domain. The single-strand domains associated with the tethers (6) and (7) are complementary to each other. Mixing the two polymers  and , in the presence of the enzymes, yielded the hydrogels cooperatively crosslinked by the (1)/(1) bridges and by the duplexes generated between the single-strand domains of (6) and (7) associated with (4)/(6) and (5)/(7) duplexes linked to  and , respectively. Strand (7) in  participating in the bridging of the chains is, however, engineered to include a cytosine-rich sequence capable to form the i-motif structure at acidic pH. This provides the principle for biocatalytically guided control over the stiffness of the hydrogels, as outlined in 2O2 (path I), while subjecting the AchE/urease matrix to acetylcholine resulted in the hydrolysis of acetylcholine to choline and acetic acid (path II). The product, gluconic acid or acetic acid, acidifies the hydrogels to state II, due to the partial reconfiguration of sequence (7) into the i-motif structure and the dissociation of the (6)//(7) duplexes. Treatment of the lower-stiffness hydrogels with urea resulted in the urease-catalyzed hydrolysis of urea to ammonia that neutralizes the hydrogels. This resulted in the dissociation of the i-motif structures and the regeneration of the high-stiffness hydrogels cooperatively crosslinked by the (1)/(1) duplexes and the (4)/(6)//(7)/(5) bridges. Thus, by the cyclic biocatalytic activation of the GOx/urease or AchE/urease-loaded hydrogels cooperatively crosslinked by the (1)/(1) and (4)/(6)//(7)/(5) bridges and the partial pH-stimulated dissociation of one of the bridges via the formation of i-motif units, the hydrogels were reversibly switched between higher- and lower-stiffness states. The quantitative stiffness properties of the hydrogels were evaluated by rheometry, as shown in 1)/(1) and (4)/(6)//(7)/(5) cross-linkers revealed G′ = 124 Pa and G′′ = 10 Pa (curve a′ and a′′), and the glucose-induced separation of (4)/(6)//(7)/(5) by the formation of i-motif units yielded a softer hydrogel, G′ = 46 Pa, G′′ = 6 Pa, curve b′ and b′′ as shown in G′ = 100 Pa and G′′ = 5 Pa (curve a′ and a′′), as compared to the i-motif dissociated hydrogel that showed lower stiffness, G′ = 49 Pa, G′′ = 4 Pa, curve b′ and b′′ in Besides the biocatalytically stimulated pH-controlled stiffness of the hydrogels by means of T–A·T triplex responsive bridges, the design of biocatalytic matrices that control their stiffness by pH-responsive i-motif structures was also demonstrated, as shown in 1)/(1) and the enzyme-responsive bridges, to the respective substrate transforms the hydrogel into a lower-stiffness, quasi-liquid state that is stabilized only by the (1)/(1) duplex bridges. The residual duplex bridges provide, however, a memory reflected by the dictated entanglement of the polymer chains. Upon the counter biocatalyzed regeneration of the cooperatively bridged hydrogels, the duplex “memory” units provide instructive information to regenerate the shaped, higher-stiffness hydrogels. This is exemplified in 1)/(1) duplexes and (2)/(3)/(2) triplexes, as biocatalytic shape memory matrices. The stiff biocatalyst-loaded hydrogels were prepared in molds and were extruded as a triangle. Subjecting the hydrogels to urea separated the triplex bridges, resulting in lower-stiffness, shapeless matrices crosslinked only by the duplexes (1)/(1). Treatment of the quasi-liquid, shapeless matrices with glucose or acetylcholine resulted in the neutralization of the hydrogels, and the memory-guided reshaping of triangle-shaped hydrogels stabilized by the two cooperative crosslinking units. In analogy, 1)/(1) and (4)/(6)//(7)/(5) cross-linkers. The reversible transitions of the triangle-shaped and the shapeless, quasi-liquid hydrogels were realized upon the treatment of the hydrogels with glucose/urea or acetylcholine/urea.The biocatalyzed control over the stiffness of the different hydrogels was then used to develop hydrogel matrices exhibiting shape-memory properties. That is, subjecting a shaped, higher-stiffness hydrogel, crosslinked by the duplexes (2)/(3)/(2) T–A·T triplexes and the (4)/(6)//(7)/(5) bridges (D and PE). This hydrogel reveals switchable stiffness properties in the presence of three substrates, i.e., glucose, acetylcholine and urea. In the presence of glucose or acetylcholine (path I), the acidification of the higher-stiffness hydrogel (state I) leads to the separation of the (6)//(7) duplex units into the i-motif structures and the formation of the lower-stiffness hydrogel (state II). The reverse addition of urea neutralizes the hydrogel, leading to the dissociation of the i-motif units and to the regeneration of the (4)/(6)//(7)/(5) bridges (state I). Besides, treatment of the higher-stiffness hydrogel (state I) with urea leads to the urease-catalyzed hydrolysis of the substrate and to a basic pH environment. This leads to the separation of the (2)/(3)/(2) T–A·T triplexes, and the resulting (4)/(6)//(7)/(5) bridges provide the sole crosslinking units, yielding a hydrogel of lower stiffness, state III (path II). The reverse treatment of the hydrogel in state III with glucose or acetylcholine neutralizes the basic condition of the hydrogel, recovering the hydrogel to state I that exhibits higher stiffness. G′ = 177 Pa and G′′ = 18 Pa  and state III  show lower stiffness. 2)/(3)/(2) triplex bridges and (4)/(6)//(7)/(5) crosslinking units. Subjecting the triangle-shaped hydrogel (state I) to urea leads to lower-stiffness, quasi-liquid state III, where the (4)/(6)//(7)/(5) bridges act as internal memory. The neutralization of the matrix upon the addition of glucose results in the memory-guided regeneration of the shaped, stiff hydrogel in state I. The subsequent acidification of the shaped hydrogel by acetylcholine leads to shapeless, low-stiffness state II that includes only the (2)/(3)/(2) triplex bridges as memory. The subsequent treatment of the hydrogel with urea neutralizes the matrix, resulting in the (2)/(3)/(2)-guided regeneration of the stiff triangle-shaped hydrogel. Finally, the treatment of the stiff neutral hydrogel with glucose re-acidifies the matrix, leading to the shapeless matrix crosslinked only by the (2)/(3)/(2) memory bridges.In addition, all three enzymes GOx, AchE and urease were integrated into one hydrogel cooperatively crosslinked by the /(6)//(7)/(5) bridges, was cut into two pieces that were treated with glucose to yield low-stiffness hydrogel pieces. Their physical connection did not lead to any self-healing, after a time interval of 1 h, and shaking the system led to the separation of the two pieces. The physical connection of the pieces, followed by the treatment with urea, led to neutralization of the hydrogel, the separation of the i-motif units, and to the cooperative stabilization of the interlinked boundary to a self-healed, intact hydrogel crosslinked by the T–A·T and (4)/(6)//(7)/(5) bridges (ESI Video 12)/(3)/(2) and the (4)/(6)//(7)/(5) bridges was cut into two pieces that were treated with urea. This stimulated the separation of the T–A·T bridges, thus forming the low-stiffness hydrogel crosslinked only by the (4)/(6)//(7)/(5) bridges. The physical connection of the two pieces did not lead to self-healing. Treatment of the low-stiffness, physically connected matrices with acetylcholine or glucose neutralized the hydrogels, leading to self-healing through the formation of the stiff matrices crosslinked cooperatively by the (2)/(3)/(2) and (4)/(6)//(7)/(5) bridges, as shown in 4)/(6)//(7)/(5) bridges /(1) and (4)/(6)//(7)/(5) bridges was applied to demonstrate the switchable ON–OFF release of insulin. A higher-stiffness hydrogel was prepared in the presence of GOx/urease and coumarin-labeled insulin, as show in F and PG). The addition of glucose resulted in its aerobic GOx-catalyzed oxidation. The formed gluconic acid acidified the hydrogel matrix and led to the separation of (4)/(6)//(7)/(5) bridges through the reconfiguration of (7) into the i-motif structure, resulting in the release of insulin. Subjecting the hydrogel to urea resulted in the urease-catalyzed hydrolysis of urea and the neutralization of the hydrogel. This recovered the higher-stiffness hydrogel cooperatively stabilized by (1)/(1) and (4)/(6)//(7)/(5) bridges and blocked the release of insulin. The release of insulin could be reversibly switched between the ON/OFF states by the cyclic treatment of the hydrogel with glucose and urea, The biocatalytic control of the stiffness of the hydrogels was further applied to develop stimulus-responsive drug-loaded hydrogel matrices. Specifically, the hydrogel cooperatively crosslinked by , a negligible release of insulin was observed, implying that at these concentrations of glucose, the pH changes in the matrix are too low to change the stiffness of the hydrogel and induce the release of insulin. At a higher glucose concentration, 200 mg dL−1 or 400 mg dL−1, effective release of insulin was observed. Of particular interest, is the insulin release profile at an elevated glucose concentration of 200 mg dL−1 that is relevant for diabetes control. The release of insulin proceeded for ca. 30 min, reaching a saturated state due to a decrease in glucose concentration consumed in the processes. Indeed, monitoring the glucose concentrations with a glucometer revealed a rapid decrease of glucose in the hydrogel (−1) switched-on the release of insulin /(1) and (4)/(6)//(7)/(5) bridges, treated with DNase at different time-intervals. We did not observe a decrease in the GOx-activity after a time-interval of 3 days, implying that no GOx release and no hydrogel degradation of the hydrogel occurred within this time-interval. Presumably, the hydrogel matrix protects the DNA-bridging units from being digested by DNase within this time-interval. It should be noted that the enzymes incorporated in the hydrogel matrices retained >90% of their activity as compared to the native enzymes.The GOx-catalyzed release of insulin from the hydrogel matrix suggests that the enzyme-responsive hydrogel could introduce a concept for tailoring an “Artificial Pancreas”, where the hydrogel acts as a carrier for the autonomous glucose-guided release of insulin. Accordingly, we argued that the GOx-stimulated pH changes in the hydrogel could be controlled by the concentrations of glucose. Thus, we integrated GOx and coumarin-labeled insulin in a higher-stiffness hydrogel matrix, as shown in hydrogel . Subject insulin , implyin2O2 or by the AchE-catalyzed hydrolysis of acetylcholine to acetic acid and choline. By the selective pH-stimulated separation of the respective nucleic acid crosslinking units, the control over the stiffness of the hydrogels was demonstrated. In fact, other enzymes, e.g., phosphatase or carbonic anhydrase, could be also used to control the pH conditions in hydrogels. The enzyme-guided switchable control over the stiffness of hydrogels provides novel methods to develop biocatalytically driven shape-memory and self-healing matrices. In addition, the biocatalytically switched stiffness properties of the hydrogels were used for the substrate-triggered controlled drug release. Specifically, the glucose-triggered control over the stiffness of the GOx/insulin-loaded hydrogel and the glucose dose-controlled release of insulin provide means for the autonomous regulation of glucose levels by the released insulin. One could envision the deposition of the GOx/catalase/insulin-loaded hydrogel, described in this paper, on a capillary microneedle array as a functional device acting as an artificial pancreas. The hydrogel/microneedle array may act as a patch on the skin for the autonomous sensing of glucose in subcutaneous fluids followed by the autonomous release of insulin and its sequestered delivery to the body.63The present study has integrated enzymes in DNA-based hydrogels as a means to control their stiffness. Two or three different enzymes were loaded in nucleic acid bridged polyacrylamide hydrogels. The biocatalysts inducing the acidification of the hydrogels were glucose oxidase (GOx) and acetylcholine esterase (AchE) and the enzyme that introduced basic conditions into the hydrogels was urease. The hydrogels were cooperatively crosslinked by two kinds of pH-responsive bridges, where T–A·T bridging units were dissociated under basic conditions generated by the urease-catalyzed decomposition of urea, and the duplex nucleic acids were separated by their reconfiguration into i-motif structures under acidic conditions, generated by the biocatalytic aerobic GOx-catalyzed oxidation of glucose to gluconic acid and HThere are no conflicts to declare.SC-011-D0SC01319F-s001SC-011-D0SC01319F-s002SC-011-D0SC01319F-s003SC-011-D0SC01319F-s004