Recently, there has been great interest in the application of polysaccharides in the preparation of diverse biomaterials which result from their biocompatibility, biodegradability and biological activity. In this work, the investigations on chitosan/poly(aspartic acid)-based hydrogels modified with starch were described. Firstly, a series of hydrogel matrices was prepared and investigated to characterize their swelling properties, structure via FT-IR spectroscopy, elasticity and tensile strength using the Brookfield texture analyzer as well as their impact on simulated physiological liquids. Hydrogels consisting of chitosan and poly(aspartic acid) in a 2:1 volume ratio were elastic (9% elongation), did not degrade after 30-day incubation in simulated physiological liquids, exhibited a relative biocompatibility towards these liquids and similar swelling in each absorbed medium. This hydrogel matrix was modified with starch wherein two of its form were applied—a solution obtained at an elevated temperature and a suspension obtained at room temperature. Hydrogels modified with hot starch solution showed higher sorption that unmodified materials. This was probably due to the higher starch inclusion  when this polysaccharide was given in the form of a hot solution. Hydrogels modified with a cold starch suspension had visible heterogeneous inequalities on their surfaces and this modification led to the obtainment materials with unrepeatable structures which made the analysis of their properties difficult and may have led to misleading conclusions. Polysaccharides as biodegradable, biocompatible and biologically active compounds belong to the group of substances with attracting growing interest in studies on the development of innovative materials for biomedical applications . One of Due to chitosan possessing many functional groups in its chemical structure it is possible to use this polysaccharide for preparation of carriers for various active substances which may form bonds with the mentioned groups . Chitosa3O4 nanoparticles were investigated by Nezami et. al. They proved that the developed polymers were pH-responsive materials able to control the release of guaifenesin [Another polysaccharide widely considered for biomedical applications is starch. There is growing interest from scientists is in its application for the development of innovative dressing materials. For example, hydrogel dressings based on a combination of itaconic acid, starch and Feifenesin . In turnifenesin  and Waghifenesin  while Deifenesin .Considering their properties, a material containing both chitosan and starch could have potential for biomedical use. Hydrogel dressings based on starch, chitosan and poly containing additionally nano zinc oxide showing antibacterial activity were developed by Baghaie et al. . Next, nIn this work, a synthesis methodology and investigations on chitosan/poly(aspartic acid)-based hydrogels modified with starch are presented. Firstly, a series of investigations was performed to select a hydrogel matrix consisting of both above-mentioned reagents in an appropriate volume ratio. For this purpose, the analysis of swelling properties, elasticity and tensile strength, interactions with simulated physiological liquids as well as characterization of the chemical structure via Fourier transform infrared (FT-IR) spectroscopy of obtained hydrogel matrices were conducted to determine the matrix with the most beneficial properties. Subsequently, selected polymer matrix was modified with starch wherein its two forms were used—a solution prepared at high temperature and a suspension obtained at room temperature. Chitosan/poly(aspartic acid)-based hydrogels modified with starch were investigated including the analysis of their surface morphology, chemical structure, mechanical properties (elasticity and tensile strength) and sorption capacity. The main purpose of the research was to evaluate the usefulness of obtained starch-modified hydrogels for biomedical applications.Chitosan , diacrylate poly(ethylene glycol) , 2-hydroxy-2-methylpropiophenone  and L-aspartic acid (≥98%) were purchased in Merck . Starch  was bought in Avantor Performance Materials Poland S.A. . All reagents were applied without further purification.Chitosan/poly(aspartic acid)-based hydrogels were obtained via the photopolymerization process. This method was selected because it allows to obtain materials in a short time and there is no need to use any toxic organic solvents which is significant in terms of potential application of designed materials for biomedical purposes. Here, one of the most important parameters is an adequate proportion of applied reagents (particularly a crosslinker and a photoinitiator). However, this may be verified, e.g., during incubation studies where uncrosslinked reagents may affect pH of incubation liquids.Firstly, 1% chitosan solution in 0.05% acetic acid and 0.5% aqueous solution of aspartic acid were prepared. Next, adequate amounts of these solutions were mixed with crosslinker and photoinitiator and such prepared mixtures were treated with UV radiation for 2 min. A VP-60 EMITA lamp was used as a source of radiation . Compositions of obtained hydrogels are presented in Prepared hydrogels were next dried at room temperature and samples were selected for modification with starch. The studies performed included the evaluation of sorption properties of obtained polymers, determining their behavior in selected liquids, assessment of their mechanical properties and characteristics of their chemical structure. The hydrogel sample with the most favorable properties was subsequently subjected to the modification process.Starch-modified hydrogels were obtained analogously to the unmodified materials. Briefly, 1% chitosan solution was mixed with 0.5% aspartic acid solution :Q—swelling ratio, g/g; m—weight of sample after swelling, g; m0—weight of sample before swelling, g.The research was performed using selected simulated physiological liquids including hemoglobin (1% aqueous solution), SBF , RingerThe results were presented in a form of bar charts showing the swelling ratio calculated for each tested hydrogel sample depending on the type of the liquid absorbed and the sorption time.The study was performed both for unmodified and modified hydrogels. The analysis of swelling sorption of unmodified polymers aimed at determining the impact of the composition of hydrogel matrix on this property. In turn, swelling tests of hydrogels modified with starch were conducted to evaluate whether the polysaccharide introduced into the polymer matrix affected the sorption capability of such modified materials.Evaluation of the behavior of obtained hydrogels in environments simulating selected physiological liquids is important in terms of their potential biomedical application. Such a study included the immersion of hydrogel samples for 30 days in 50 mL of distilled water, SBF, hemoglobin and Ringer liquid (the same liquids in which the swelling ability was determined). The incubation was performed at 36.7 °C wherein such conditions were applied to simulate the temperature of human body. The study involved the pH measurements of liquids with incubated samples made using the pH-meter Elmetron CX-701 . Analysis was performed for chitosan/poly(aspartic acid)-based hydrogels.−1 .Fourier transform infrared (FT-IR) spectroscopy was applied to verify the presence of functional groups in the structure of both unmodified and modified hydrogel polymers. Importantly, samples of chitosan/poly(aspartic acid)-based hydrogels after incubation studies in simulated physiological liquids were also subjected to the FT-IR technique to determine the impact of these environments on the structure of tested polymers. The analysis was conducted at room temperature via the Thermo Scientific Nicolet iS5 equipped additionally with ATR diamond accessory . The FT-IR spectra of analyzed samples were recorded within the range of 4000–500 cmSubsequently, the surface morphology of both unmodified hydrogels and polymers containing starch was characterized. The analysis was performed using the Jeol 5510LV Scanning Electron Microscope . Before the analysis, hydrogel samples were dried at room temperature and sputtered with gold. The study was performed at room temperature.Studies on the mechanical properties of hydrogels are significant because they may provide information on such parameters of the tested polymers as their elasticity or tensile strength. In turn, the knowledge of such properties allows proper evaluation of the suitability of the materials under specific conditions of use. Studies were performed by means of the Brookfield CT3 texture analyzer  according to the ISO 37 type 2 and ISO 527-2 type 5A standards. Firstly, polymers after the synthesis were treated using the ZCP020 Manual Cutting Press to prepare paddle-shaped hydrogel samples  which were subsequently dried at room temperature under pressure to keep their shape and to avoid wrinkling. In order to perform the measurements, paddle-shaped hydrogels were fixed between the jaws of the texture analyzer using clamps. When the measurement started, the jaws parted which resulted in the samples’ stretching. The measurement was carried out until the hydrogel ruptured.The analysis was performed both for unmodified hydrogels and hydrogels containing starch. The investigations aimed at determining the impact of the composition of the hydrogel matrices on their mechanical properties as well as to determine whether the starch introduced into the polymer affected these properties.Results of the investigations on the swelling ability of chitosan/poly(aspartic acid)-based hydrogels in selected simulated physiological liquids are presented below in As it may be noticed considering above-presented results, the volume ratio of the reagents used during the synthesis of hydrogel matrices affected the swelling properties of tested hydrogels. It was observed that the sample obtained using 10 mL of chitosan solution and 20 mL of aspartic acid solution showed the highest swelling ratio in tested liquids. In turn, the lowest swelling ability was reported for samples 30/0 and 20/10, i.e., for hydrogels prepared using 0 mL and 10 mL of aspartic acid solution, respectively. Thus it may be stated that the amount of the aspartic acid in hydrogel matrix affected the sorption of analyzed materials. The larger amount of this reagent in the polymer matrix, the better swelling properties of hydrogels. Considering the swelling ability of all tested hydrogels, it may be concluded that a larger amount of aspartic acid compared to the amount of chitosan in the analyzed polymer matrices caused an increase in their sorption capacity. This was probably caused by the chemical structure of aspartic acid which contains many hydrophilic groups resulting in larger sorption of liquids. Thus the larger the amount of this acid in the hydrogel matrix, the better sorption properties of such obtained materials. For example, Suhail et al. reported that an increase in the content of aspartic acid in swelled polymers leads to an increase in a hydrophilicity of hydrogel network and thus to its higher swelling ability [Analyzing the results obtained in all tested liquids, it was observed that the lowest swelling properties were reported for Ringer liquid. It may be caused by the presence of various  ions in this solution. They contributed to the increase in the crosslinking degree of swelled hydrogel which, in turn, led to the decrease in its swelling ability. Additional crosslinks may form between the polymer chains which may result in the decrease in the volume of free spaces in the polymer structure which is available for absorbed liquid. On the other hand, the highest sorption capacity was observed in hemoglobin. Lack of additional ions in this liquid meant that any additional crosslinks were not formed, and in turn, the liquid freely penetrated the hydrogel structure resulting in an increase in its sorption capability.Considering graphs showing the results of swelling investigations of obtained hydrogels it may be noted that the most beneficial properties in terms of the future application of developed materials for biomedical purposes showed sample 20/10 (obtained using 20 mL of chitosan solution and 10 mL of aspartic acid solution). This hydrogel matrix exhibited similar swelling properties in each tested liquid. Such a behavior is beneficial for use in medicine and pharmacy because the material obtained using the mentioned amounts of the reagents will behave in a similar manner in each environment occurring in the human body. Importantly, this sample did not show averaged values of swelling ratio among all tested hydrogels therefore such a prepared matrix exhibited a potential for a wide range of applications in medicine and pharmacy. When applying such a material, its desired property is to absorb as much liquid as possible. During the application of the tested materials in cosmetics, e.g., in a form of moisturizing eye pads, a large sorption capacity of such applied hydrogels is not beneficial because it might cause the skin to dry out and the swelled material might become too heavy and thus uncomfortable.Changes in pH of the solutions during the 30-day incubation of hydrogels are shown below in Analyzing above-presented results, it may be observed that in the case of all immersed samples pH values of liquids measured during the incubation were only slightly different. The buffering properties of the analyzed hydrogels were most visible in the SBF solution thus such an environment seemed to be the most adequate for further studies on the developed matrices in terms of their use for biomedical purposes.Performed incubation investigations allowed to indicate that the most promising results were observed for sample 20/10 (obtained using 20 mL of chitosan solution and 10 mL of aspartic acid solution). This matrix showed a relative biocompatibility towards each tested simulated physiological liquid. In pH values measured during the incubation of this sample maintained at the same level, only slight deviations were observed. pH changes observed during the whole period of the incubation of sample 20/10 seemed to be the lowest which, in turn, indicated good buffering properties of this polymer and its good stability in tested simulated physiological liquids.The presence of functional groups in the hydrogels’ structure was verified using FT-IR spectroscopy. Obtained FT-IR spectra of chitosan/poly(aspartic acid)-based polymer matrices are presented in Furthermore, hydrogel samples after incubation studies were also subjected to the FT-IR analysis to determine the impact of such an immersion on their structure. The resulting FT-IR spectra of samples both before and after incubation in tested liquids are shown in −1 corresponding to C–H group were observed. They probably derived from the photoinitiator applied, which has an aromatic ring in its chemical structure. Next, in the case of all samples bands within the range 3300–3250 cm−1 were also noticed which corresponded to OH groups of hydrogel matrices consisting of chitosan and aspartic acid. Moreover, bands within the range 1750–1730 cm−1 corresponding to carbonyl group as well as bands at 1100–1000 cm−1, probably derived from chitosan, were also reported.Based on the above-presented FT-IR spectra it was possible to identify the functional groups occurring in tested hydrogels as well as to check the impact of their 30-day incubation in simulated physiological liquids on the chemical structure of these polymers and their potential degradation. On obtained FT-IR spectra of all analyzed samples wide bands at 3000–2970 cmIt was proved that on the FT-IR spectra of both samples before and after incubation in simulated physiological liquids bands corresponding to the same functional groups were indicated. The bands observed differed only in the intensity. In the case of FT-IR spectrum of sample 0/30 after incubation in hemoglobin a significant decrease in the intensity of the majority of bands (apart from the band corresponding to the distilled water which overlapped probably with the band deriving from the sample before the modification) was observed. It may be caused by the partial degradation of incubated materials.Based on the spectroscopic analysis it was concluded that sample 20/10 showed the most beneficial properties compared to the other tested ones. This polymer matrix did not degrade during 30-day incubation in various simulated physiological liquids and did not show any structural changes as a result of such an incubation. Furthermore, it was noticed that the differences between the intensities of bands visible on the FT-IR spectra of this sample before and after incubation were the slightest compared to the differences observed on the FT-IR spectra of the other tested samples.Subsequent analysis involved determining selected mechanical parameters of obtained chitosan/poly(aspartic acid)-based hydrogel matrices. Results of performed investigations are presented below in Based on the results of performed mechanical investigations, the dependence between the elasticity of tested hydrogels and their chemical compositions may be clearly observed. The lowest tensile strength was reported for samples which consisted only of one reagent, i.e., chitosan (sample 30/0) or aspartic acid (sample 0/30). The hydrogel sample containing the same amounts of these reagents also did not show satisfactory properties—it exhibited the biggest tensile strength wherein its elasticity was the lowest compared to the other tested samples.Sample 20/10 was selected for further investigations because this hydrogel showed the most beneficial mechanical properties among all tested samples. This material was characterized by the highest elasticity, did not deform during the analysis and exhibited appropriate tensile strength. In terms of the application of the developed materials for biomedical purposes such properties are highly desirable. Mechanical investigations are important in view of the potential biomedical uses—e.g., the elasticity of such materials is significant in the case of their application as wound dressings applicable on wounds placed in sites with high mobility.Swelling properties of chitosan/poly(aspartic acid)-based hydrogels modified with starch were also investigated to evaluate the impact of this polysaccharide on hydrogels’ sorption ability. Results of performed analyses are shown below in Verifying the results obtained as a result of studies on swelling properties of chitosan/poly(aspartic acid)-based hydrogels modified with starch it was observed that hydrogel polymers containing this polysaccharide showed better swelling properties after 72 h of the study compared to the unmodified samples. This result from the fact that the introduction of starch into the hydrogel matrix enhanced its hydrophilicity  which was also proved by Parvathy et al.  and IsmaFor example, the swelling ratio of unmodified hydrogel (sample 20/10) after 72 h sorption in distilled water was 3.32 g/g while this parameter in the same swelling conditions for hydrogel sample modified with 5.0 mL of “cold starch” was 4.29 g/g and 4.59 g/g for hydrogel containing 5.0 mL of “hot starch”. The same was observed for 72 h swelling in Ringer liquid, i.e., 3.33 g/g for unmodified hydrogel, 4.36 g/g for sample with 5.0 mL of “cold starch” and 4.03 g/g for sample with 5.0 mL “hot starch”, respectively.Only samples modified with “cold starch” (2.5 mL) did not exhibit higher swelling ratios compared to the samples without this additive wherein in the case of the swelling performed in hemoglobin even worse sorption capacity was observed (3.80 g/g for unmodified sample and 3.39 g/g for sample with 2.5 mL of “cold starch”). This was probably caused by too little starch suspension used for the modification of hydrogels. The temperature of the starch suspension introduced into the hydrogel matrix may also be the cause of the swelling properties of such modified materials not increasing. Due to the fact that the starch suspension introduced into the reaction mixture was at room temperature, the molecules of starch did not dissolve in the solvent (distilled water) and the mixture added was only a suspension of starch in distilled water. Thus the starch inclusion was higher when this polysaccharide was introduced in the form of a hot solution than as a cold suspension. The introduction of a high temperature starch solution into the reaction mixture subjected to the photopolymerization process resulted in the increase in the values of swelling ratios calculated for all tested liquids (regardless of the volume of the introduced solution). Therefore, it may be concluded that such modified materials in the majority cases are appropriate for application in medicine, for example, in a form of wound dressings of which task is to inhibit heavy bleeding, because starch-modified hydrogels are able to absorb larger volumes of liquids compared to unmodified polymers.Results of FT-IR spectroscopy of hydrogels containing starch are shown in Based on the FT-IR spectra of starch-modified hydrogel materials before and after modification it was observed that the material did not significantly change its chemical structure. On the spectra of modified polymers any new bands which could derive from the additive (starch) were not observed. On the other hand, a dependance between the intensity of bands from hydrogels with starch solution (“cold starch”) and starch suspension (“hot starch”) was observed. Bands deriving from samples modified with 2.5 mL of starch solution (“hot starch”) showed higher intensity than bands observed on the FT-IR spectra of samples containing starch solution prepared at room temperature (“cold starch”). The same conclusion may be drawn analyzing the bands deriving from samples modified with 5 mL of starch solutions. This may be caused by the fact that in the case of modification of hydrogels using “hot starch” a higher starch inclusion in obtained hydrogel matrices may be observed. This, in turn, is related to the larger number of hydrophilic groups specific for this polysaccharide and finally to the higher intensity of bands deriving from them. Moreover, functional groups present in the structure of starch duplicated with functional groups of the reagents forming the hydrogel matrix. This, in turn, resulted in the increase in the intensity of bands on the FT-IR spectra of samples modified with the high temperature starch solution. An example of such a functional group may be a carboxylic group which occurs both in the structure of starch and chitosan which is a main component of hydrogel matrix.For every sample, SEM imaging was performed for four different areas (with two magnifications: ×250 and ×500). Here, in On above-presented images a morphological structure of the surface of obtained hydrogel polymers may be observed. Each analyzed sample ) was tested using two different magnifications, ×250 and ×500, and in four different areas. Unmodified hydrogel sample (a) showed a very homogeneous surface. Its surface is smooth and not corrugated. On the other hand, the surfaces of starch-modified materials are completely different. Hydrogel modified with cold starch suspension (b) showed visible inequalities on its surface which are heterogeneous. This, in turn, may confirm the addition of starch in a form of a solution—not a suspension—which caused such heterogeneous surface formation. It may also make the analysis of the sorption properties of such modified samples and their mechanical properties difficult and lead to the misleading conclusions . However, in the case of the modification of hydrogels with hot starch solution (c) the surface of obtained polymers was homogeneously corrugated, and importantly, it was possible to prepare a repeatable structure corrugation.Thus it may be concluded that the materials modified with cold starch suspension did not show repeatable characteristics of the morphological structure, therefore these polymers are not attractive in terms of future studies and potential biomedical applications.Results of studies on the mechanical properties of chitosan/poly(aspartic acid)-based hydrogels containing starch including determining the elongation and the tensile strength under the tension applied are presented below in Performed mechanical studies allowed to state that the materials modified with 2.5 mL of hot starch solution exhibited a high elasticity showing at the same time slightly worse tensile strength than unmodified hydrogels. The introduction of 2.5 mL of cold starch suspension into the hydrogel matrix resulted in the preparation of materials with comparable tensile strength to the unmodified material. Nonetheless, such modified hydrogels also showed lower elasticity  than a hydrogel without this polysaccharide. This is strictly related to the above-presented SEM images in which the highly heterogeneous structure of tested materials was observed. Starch present in the hydrogel matrix in the form of a cold suspension probably did not form any bonds with the polymer matrix which could improve its mechanical properties, i.e., elasticity.Thus it may be concluded that the introduction of the hot starch solution into the hydrogel matrix resulted in the improvement of its mechanical properties—the improvement of such properties by this polysaccharide was also noticed by Almeida et al. . NonetheIn the case of the hydrogel sample modified with 5 mL of the cold starch suspension, the analysis of its mechanical properties was not possible due to technical reasons—such a modified material, due to its rigidity and fragility, cannot be placed between the jaws of a texture analyzer.The volume ratio of chitosan and aspartic acid used for the synthesis of hydrogels affected their properties. It was proved that the larger the amount of aspartic acid compared to the amount of chitosan, the higher swelling ability of the hydrogels.Hydrogels based on chitosan and poly(aspartic acid) in a 2:1 volume ratio showed the most favorable properties, i.e., were elastic (9% elongation which was two times larger compared to the other samples), did not degrade after 30-day incubation in various simulated physiological liquids and did not show any structural changes as a result of such an incubation, and exhibited a relative biocompatibility towards tested liquids and similar swelling in each absorbed medium.Hydrogels modified with starch added as a hot solution showed higher sorption capacity and greater elasticity than polymers without this polysaccharide.Unmodified hydrogel sample showed smooth and very homogeneous surface. On the other hand, hydrogel modified with cold starch suspension had visible inequalities on its surface which were heterogeneous. Such heterogeneous surface formation of samples modified with cold starch suspension may make the analysis of their swelling and mechanical properties difficult and lead to misleading conclusions . The surface of hydrogels modified with hot starch solution was homogeneously corrugated and it was possible to prepare a repeatable surface corrugation.The differences in the properties of materials modified with starch solutions prepared at different temperatures were caused by the fact that at hot temperature starch dissolved in water, and this additive was added in the form of a homogeneous solution. The application at room temperature resulted in the preparation of a starch suspension in which the starch particles were dispersed in the solvent and they were probably heterogeneously distributed in the polymer matrices. This, in turn, affected the morphology, elasticity and tensile strength of the hydrogels modified with such a suspension of this polysaccharide.


African swine fever (ASF) is a viral disease of pigs caused by ASF virus (ASFV). High mortality and the lack of available treatments have severely impacted the swine industry resulting in huge global economic losses. In response to the dire necessity for vaccines, this study aims to identify highly conserved cytotoxic T-cell epitopes in ASFV structural proteins pp220, pp62, p72, p30, and CD2v through immunoinformatics approach.50 binding scores >500 nM were filtered out. Epitopes which are classified to be potentially toxic and cross-reactive with the swine proteome sequences were all excluded from the study. The epitopes were docked with the swine leukocyte antigen-1*0401 (SLA-1*0401) wherein the binding affinity, the binding energy, and the root-mean-square deviation (RMSD) per residue of epitope-SLA complexes formed were determined and compared with the influenza epitope as positive control.The amino acid sequences of the structural proteins were retrieved from the National Center for Biotechnology Information protein database. The sequences were evaluated in CD-HIT Suite wherein resulting representative sequences were aligned in Clustal Omega. Highly conserved sequences were identified in the Protein Variability Server which were used as reference sequences for the cytotoxic T-cell epitope mapping. Epitopes were predicted using the tools in Immune Epitope Database. Peptides which bind to the swine major histocompatibility complex with ICA total of 112 highly conserved fragments with Shannon variability index ≤0.1 were identified. These include 66, 12, 26, 6, and 2 highly conserved fragments from ASFV proteins pp220, pp62, p72, p30, and CD2v, respectively. From these reference sequences, 35 nonameric peptides were selected for the list of candidate cytotoxic T-cell epitopes. These include 26 epitopes for pp220, 7 for pp62, 6 for p72, and one each for p30 and CD2v. Bioinformatics analysis classified the peptides as non-toxic. Further evaluations of epitopes showed that these are less likely to cross-react with the domestic swine proteome sequences. This study identified candidate epitopes from pp220 , and pp62  which can bind to at least two widely distributed SLAs in pig populations. The immunogenicity of candidate peptides RSNPGSFYW, DFDPLVTFY, AIPSVSIPF, and VVFHAGSLY was validated by the acceptable binding affinities, binding energies, and RMSD of the peptide-SLA complexes formed. Results were also comparable with the crystal structure of an SLA-epitope complex in the database.in silico evaluations showed that the identified highly conserved cytotoxic T-cell epitopes may be used as part of future vaccine formulations against ASFV infection in domesticated pigs. Nonetheless, these findings require in vitro and in vivo validation before application.This is the first study to identify highly conserved cytotoxic T-cell epitopes in the structural proteins of ASFV. Overall, the results of  Ornithodoros erraticus) and wild pigs through feeding, or less frequently through direct transmission between wild pigs. In domesticated pigs, ASFV can be readily transmitted resulting to lethal hemorrhagic fever. The widespread of ASF has been a great challenge for swine breeding. Although it does not pose a risk to human health, ASF has resulted to huge economic losses around the world [Asfivirus within the family Asfarviridae, is an icosahedral enveloped virus with double-stranded DNA as its genetic material. The viral particle consists of an inner core coated by a thick protein core shell enclosing its genetic material. The subsequent layers are the lipid envelope surrounding the protein core shell, and the viral capsid which forms the outermost cover of an intracellular virion. Virions budding out of the host cell carry a portion of the host plasma membrane forming an extra layer of envelope [CP2475L which product is cleaved and post-translationally processed to p150, p37, p14, and p34. Structural protein pp62, a relatively shorter polypeptide, is encoded by gene CP530R. Similar to pp220, this polyprotein is proteolytically cleaved into mature p35 and p15 virion proteins. The post-translational products of pp220 and pp62 play roles in viral particle assembly and form the major component of the viral core shell [B646L (VP72) which serves as a major protein component in viral capsids and functions in the formation of ASFV capsids [CP204L gene and is most abundantly expressed during the early phase of infection. It has crucial functions in viral entry and is known to be one of the most antigenic ASFV proteins [EP402R encodes the structural glycoprotein CD2v which consists of transmembrane region, extracellular domain , and cytosolic domain . CD2v is associated to immune response modulation and lymphocyte function impairment which enhance the virulence of ASFV in domestic swine [African swine fever (ASF) is a viral disease of pigs with mortality approaching 100% -4. ASF ihe world . ASFV, tenvelope . The lenenvelope . ASFV enenvelope . Both ppre shell ,10. Findre shell . Antigen capsids . Anotherproteins ,14. Studic swine . In addiic swine .Due to the significant roles of pp220, pp62, p72, p30, and CD2v in ASFV replication and assembly, disporting key properties including immunogenicity, abundant expression, and virulence mechanism, these structural proteins can be suitable immunotherapeutic targets in designing vaccines and treatments against ASFV. At present, there are no anti-viral agents and vaccines available against ASFV . The sigin vitro and in vivo evaluations are conducted.In the pursuit of this compelling demand, the researchers utilized immunioinformatics approach to provide potential immunotherapeutic agents against ASFV. Immunoinformatics tools and databases can aid in hastening and reducing the cost required to identify immunogenic proteins and peptides for vaccine designs ,19. IdenThus, this study aims to identify cytotoxic T-cell epitopes from the conserved amino acid sequences of pp220, pp62, p72, p30, and CD2v in ASFV. The use of highly conserved sequences in vaccine development is one of the most important factors that must be considered to resolve immune epitope evasion in the rapidly mutating viral antigens.Ethical approval is not necessary for this type of study.The study was conducted from April to June 2021. The study was conducted at the Polytechnic University of the Philippines.http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit) with 1.00 as sequence-identity cutoff. Unique representative sequences for each protein were aligned in Clustal Omega  to generate multiple sequence alignment. To identify highly conserved sequences for each protein, Shannon variability threshold H ≤0.1 was used in the protein variability server (PVS) tool (http://imed.med.ucm.es/PVS/). The first sequence in the alignment was used as a reference and the varying residues were masked. Fragments with ≥9 residues were kept for cytotoxic T-cell epitope identification.ASFV protein sequences of pp220, pp62, p72, p30, and CD2v were retrieved from the National Center for Biotechnology Information protein database on April 17, 2021. Search results were filtered using 2475-2476, 530-534, 645-646, 203-204, and 360-404 for the sequence length of pp220, pp62, p72, p30, and CD2v, respectively. The lists of representative sequences for each antigen were obtained using CD-HIT Suite (50 <500 nmol/dm3 are classified as good binders [https://webs.iiitd.edu.in/raghava/toxinpred/multi_submit.php) to identify potentially toxic peptides that can cause damage to cells through the default SVM Method [Cytotoxic T-cell epitopes which can bind to widely distributed swine leukocyte antigens (SLA) SLA-1*0101, SLA-1*0401, and SLA-1*0801 in swine population  were map binders ; thus, t binders . AlthougM Method . Peptide50 binding affinity for SLA-1*0401 was docked with the cleaned PDB structure of SLA-1*0401 in GalaxyPepDock server . The resulting top score structures of SLA-epitope complex were further refined in the GalaxyRefineComplex server . Refined SLA-epitope structures were viewed using the iCn3D tool (https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html). The dissociation constant (Kd) and the binding energy (∆Gbind) of each SLA-epitope complex were calculated in PRODIGY (https://bianca.science.uu.nl/prodigy/) at 300 K. This webserver tool uses non-interface surface properties and intermolecular contacts for predictive models [d and ∆Gbind values for SLA-1*0401-bound epitopes were compared with that of the influenza epitope bound to SLA-1*0401. To determine the stability of SLA-epitope complex formation, molecular dynamic simulation was performed in MDWeb server (http://mmb.irbbarcelona.org/MDWeb/) to obtain the root-mean-square deviation (RMSD) plot per residue. Molecular dynamics parameters include C-alpha Brownian dynamics in 100 ps time, 0.01 ps time change, 3.8 Ǻ distance between alpha carbon atoms, 10 step output frequency, and 167.36 kJ/mol Ǻ2 force constant. This process employs the GROMACS MD setup with solvation using force-field Amber-99sb [The crystal structure of SLA-1*0401 bound to influenza epitope (PDB ID: 3QQ3) was retrieved from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB). The PDB file was cleaned to obtain the isolated structure of SLA-1*0401 for subsequent docking procedures. For each structural antigen, epitope with the highest ICe models . Estimatber-99sb .Herein, the reference sequences identified for the ASFV structural proteins pp220, pp62, p72, p30, and CD2v have Shannon variability index ≤0.1. Sequences of pp220, pp62, p72, p30, and CD2v yielded 66, 12, 26, 6, and 2 highly conserved fragments, respectively. 50. A total of 35 highly conserved epitopes from the five structural proteins of ASFV were identified. These include 26 peptides from pp220, 7 from pp62, 6 from p72, and one each from p30 and CD2v. Analysis revealed that seven of these epitopes (four in pp220 and three in pp62) can bind to at least two widely distributed SLAs. In this study, all candidate cytotoxic T-cell epitopes have MHC IC50 ≤500 nmol/dm3 with positive TAP and proteasome scores.All resulting cytotoxic T-cell epitopes mapped using the reference sequences were filtered by excluding potentially toxic and cross-reactive peptides. 50) to SLA-1*0401 was employed for the docking procedures. The candidate epitope YQYNTPIYY in CD2v binds to SLA-1*0801; thus, it was not included in the docking procedures. Peptides docked to SLA-1*0401 include pp220 RSNPGSFYW (P1) with MHC IC50 392.5, pp62 DFDPLVTFY (P2) with MHC IC50 324.8, p72 AIPSVSIPF (P3) with MHC IC50 384.1, and p30 VVFHAGSLY (P4) with MHC IC50 132.3. The influenza epitope NSDTVGWSW bound to SLA-1*0401 was used as a control to serve as a supporting reference for positive binding. The PDB structure of SLA-1*0401 was utilized for the validation procedures in this study because the crystal structures of the other two SLAs (0101 and 0801) are not available in the RCSB protein data bank. Thus, for every antigen, the epitope with the lowest binding affinity (with the highest ICd) and the binding energy (DGbind) of each SLA-epitope complex formed were calculated. bind values with very small Kd. Notice that the DGbind and Kd values of identified cytotoxic T-cell epitopes in this study are comparable to the influenza epitope-SLA complex. To evaluate if the binding of epitopes to SLA-1*0401 is favorable, the dissociation constant which are less than 1.0 and the negative binding energy (DGbind) of the candidate epitopes P1, P2, P3, and P4 with SLA-1*0401 (50) cytotoxic T-cell epitopes  from pp220, pp62, p72, and p30 in the docking procedures and molecular dynamics evaluations with SLA-1*0401. In addition, the value of Kd, DGbind, and RMSD plot of influenza A H1N1 virus cytotoxic T-cell epitope bound to the crystal structure of SLA-1*0401 was also evaluated as a control, to further highlight the positive binding of identified candidate cytotoxic T-cell epitopes to SLA. The recent emergence of ASFV in new countries and continents has significantly impacted worldwide pork production due to its high mortality . At pres binders . Anotherreactive . CandidaA-1*0401 , indicatin vitro and in vivo assays will be conducted in the future before application of the candidate epitopes inferred in this study.This study has provided preliminary data for the potential immunotherapeutic targets against ASFV. A computational approach was conducted in the analysis of highly conserved sequences and epitopes mapped from the structural proteins of ASFV. In addition, only cytotoxic T-cell epitopes of ASFV pp220, pp62, p72, p30, and CD2v were covered in this study. B-cell and helper T-cell epitopes can also be analyzed from the identified highly conserved sequences in succeeding studies. As with the step-by-step processes in any vaccine development, it is anticipated that In silico analysis showed that the candidate epitopes can be safely used in vaccine formulations as they are classified non-toxic and are less likely to cross-react with the domestic swine proteome. The peptides can potentially cover wider domestic swine populations and can bind to their corresponding SLA alleles with stability which indicates that the epitopes are potentially immunogenic. Finally, highly conserved cytotoxic T-cell epitopes identified in this study may avoid immune evasion to offer longer and more effective protection against ASFV infection. The use of these candidate epitopes as part of future peptide or recombinant vaccines is anticipated to be validated both in vitro and in vivo in subsequent studies.This is the first work to identify highly conserved cytotoxic T-cell epitopes in the structural proteins pp220, pp62, p72, p30, and CD2v which have important roles in the virulence of ASFV. LRMH: Conceived the study. LRMH and EPB: Drafted the manuscript and analyzed the data. Both authors revised the manuscript and approved the final manuscript.


The impact of HLA matching on graft survival has been well characterized in renal transplantation, with a higher degree of matching associated with superior graft survival. Additionally, living donor grafts are known to confer superior survival compared to those from deceased donors. The purpose of this study is to report our multi-decade institutional experience and outcomes for patients who received HLA-identical living donor grafts, which represent the most favorable scenario in kidney transplantation. We conducted a retrospective analysis of these graft recipients performed at a Duke University Medical Center between the years of 1965 and 2002. The recipients demonstrated excellent graft and patient survival outcomes, superior to a contemporary cohort, with median patient and graft survival of 24.2 and 30.9 years, respectively, among Duke recipients vs. 16.1 and 16.0 years in a cohort derived from national data. This study offers a broad perspective on the importance of HLA matching and graft type, and demonstrates a historical best-case-scenario in renal transplantation. The importance of histocompatibility has been recognized since the inception of transplantation surgery as a field, even prior to the elucidation of human leukocyte antigen (HLA) in 1958 by Jean Dausset . In 1954The Duke kidney transplant program began in 1965 with an intentional focus on selecting HLA-identical donor-recipient pairs using a combination of skin grafting, mixed lymphocyte reactions, and microcytotoxicity assays , with exSince then, the field of kidney transplantation has evolved considerably. Changes in donor selection, organ preservation, and, most importantly, immunosuppression have transformed kidney transplantation from an experimental therapy to a routinely performed procedure for patients with end-stage renal disease. Although the first successful clinical kidney transplant was performed in living identical siblings , in the HLA matching was once considered an essential factor in donor selection based on the association of mismatch with deceased donor allograft survival ,13,14. CHaving performed kidney transplantation since 1965, the Duke University Medical Center is home to a unique cohort of HLA-matched LDKT recipients with ultra-long term follow up. Given the historical and ongoing relevance of HLA matching in transplantation, the purpose of this study is to characterize our multi-decade institutional experience and outcomes for patients who received what is considered the most optimal graft: HLA-identical kidneys from a living donor.This study is a retrospective analysis of HLA-identical living donor kidney transplants performed at a single institution between the years of 1965 and 2002. Of the 2262 renal transplants performed at this institution during this time period, 118 allografts were utilized from HLA-identical donors. Of these 118 patients transplanted, 23 patients were excluded due to lack of patient survival data, 18 were excluded for lack of graft survival data, 3 were excluded due to lack of immunosuppression data, and 2 were excluded due to having a known deceased donor after further review. We were therefore able to obtain complete records and include 72 of those individuals. Of special note, HLA typing in this cohort was robust, with 35/72 (49%) having sufficient familial genotypic information to ensure that donor and recipient were HLA genetically identical. For donors about whom this degree of genetic information was not available, they were considered serologically identical using the serological techniques available at the time.Additionally, we constructed two cohorts using the UNOS Standard Transplant Analysis and Research (STAR) file to examine approximate patient and graft survival for both a historical cohort (relative to our experience) and a modern cohort. For the historical cohort, we included all patients transplanted prior to 2003  with an age greater than 14 , who had no prior transplants , who received only a kidney  from a living donor . For the modern cohort, we examined all patients transplanted after 2003, using the same criteria that was used for the historical cohort, which yielded a total of 73,623 transplant recipients. As a sensitivity analysis, we determined the death-censored graft survival in a subset of our UNOS cohort, including only patients who had 0/6 HLA mismatches at HLA-A, -B, and -DR.We examined patient- and death-censored graft survival in this cohort from the time of transplant until 31 March 2019.Patient characteristics were summarized using appropriate measures of central tendency. Patient- and death-censored graft survival were plotted using Kaplan–Meier curves. Median survivals were determined with 95% confidence intervals.Seventy-two (72) patients from our cohort met the inclusion criteria. Of this total, 31 (44%) were female. The cohort was young , mostly white (74%), and the most common cause of renal disease was chronic glomerulonephritis. Many patients were on hemodialysis (42%) at the time of transplant or had been on dialysis for a short duration . Most patients were initially managed on azathioprine and nearly all received steroids . Of noteThough detailed donor information was not available, the majority (69/72) were living related donors with only 3 living unrelated donors. The cohort was transplanted between 1965 and 2002; the distribution of transplants over time is shown in p < 0.001 for both). As a sensitivity analysis, we compared death-censored graft survival among patients with 0/6 HLA mismatches in the UNOS cohorts relative to the Duke cohort. The median death-censored graft survival in the historical UNOS cohort was 16.0 years (95% CI 14.8–16.5) and 16.9 years (95% 16.1–∞) in the modern UNOS cohort compared to over 30 years in the Duke cohort.Overall, 10-year patient and graft survival were 86% and 77%, respectively . The medThough data were limited and there were no consistent biopsy protocols over the full time period, 23/72 (32%) of our cohort experienced acute rejection within the first year of transplant. This number decreases slightly to 17/72 (24%), if including only biopsy-proven rejection. Overall, 50% of patients died with a functioning graft.Historically, living related donors comprised the initial supply of kidney allografts; however, over time, due to ethical considerations and scale of demand, unrelated deceased donors have become the predominant source . As our It is well-established that grafts utilized from living-related donors demonstrate a longer median survival than those recovered from deceased donors  . Furtherp = 0.003), donor BMI (odds ratio (OR), 1.22 *1.04–1.46); p = 0.01), and minimization of immunosuppression ; p < 0.001). Of interest, patient and graft survival rates were not statistically different according to rejection at 1, 5, and 10 years post-transplant [One interesting aspect of this analysis is the high incidence of early acute rejection events in the Duke cohort. At least 24% (17/72) of patients experienced biopsy-proven acute rejection within the first year. In spite of this, patients had excellent survival and graft outcomes. Recently, a multi-center retrospective analysis showed an incidence of 12.9% of biopsy-proven acute rejection in living donor HLA-identical kidney transplant recipients, with an average occurrence within 24 months after transplantation. In their study, factors associated with rejection were recipient age ; ansplant . Multiplansplant ,28. Howeansplant ,30. ThesOne aspect of transplantation this study highlights is the potentially salutatory effects of kidney exchanges. Kidney exchange mechanisms allow for the broader sharing of grafts from living donors in order to improve overall “compatibility”—including HLA matching—between donor and recipient . Indeed,There are several limitations in this study that deserve mentioning. First, we recognize our cohort is a small, select group of ideal recipients from a center with a large transplant experience. However, this does not negate the finding that these outcomes exceeded those in both historic and contemporary cohorts. We also did not control for demographic differences between the UNOS-derived cohorts and those from our institution, which limits comparisons. Additionally, we were only able to obtain complete follow-up information on a fraction (~60%) of the total cohort. However, the included and excluded patients were relatively similar, differing only in age at transplant (those included were older), azathioprine usage (less in the included), and mycophenolate usage (more in the included group). The HLA typing in this cohort is distinct from that used by UNOS during the majority for the study and therefore limits formal comparisons. However, it also presents and opportunity to examine exceedingly well-matched individuals outside of the three HLA loci generally reported to UNOS. Additionally, any analysis of acute rejection events in this cohort is limited both by the vast changes in technology and terminology that occurred over the interval in which this cohort was observed. The first Banff Conference on transplant pathology—the modern institution for standardizing pathologic analysis of transplant specimens—was not held until 1991. However, given the fact that there was likely underdiagnosis of rejection events, the lack of association between rejection and poor outcomes, which comports with other contemporary data, is of interest.In conclusion, this report represents a unique analysis of patients who underwent HLA-identical renal transplantation at a single center with ultra-long follow up. Although the relationship between graft survival and rejection rates with an increasing degree of HLA match is well established, this study offers a broad historical perspective on the importance of HLA matching. Additionally, the observation of very favorable graft outcomes in spite of relatively high levels of acute rejection may indicate a moderation of the deleterious effects of acute rejection in genetically similar individuals.


Structures with variable stiffness have received increasing attention in the fields of robotics, aerospace, structural, and biomedical engineering. This is because they not only adapt to applied loads, but can also combine mutually exclusive properties. Here inspired by insect wings, the concept of “triple stiffness” is introduced and applied to engineering systems that exhibit three distinct deformability regimes. By implementing “flexible joints,” “mechanical stoppers,” and “buckling zones,” structures are engineered to be not only load‐bearing and durable, but also impact‐resistant. To practice the performance of the design concept in real‐life applications, the developed structures are integrated into 3D printed airplane wing models that withstood collisions without failure. The concept developed here opens new avenues for the development of structural elements that are load‐bearing, durable, and impact‐resistant at the same time. Inspired by insect wings, the concept of ‘triple stiffness’ is introduced and implemented into structures that reach a compromise between load‐bearing, durability, and impact‐resistance. The structures are further incorporated into the wings of 3D printed airplane models that can withstand collisions and quickly recover. This study inspires the design of novel adaptive structures in robotics, aerospace, biomedical, and structural engineering. They constantly respond to the applied loads, by adjusting their stiffness and flexibility, through different design strategies, which often include structural gradients, flexible joints, and mechanical interlocking.Flexibility and stiffness are two key characteristics of a structural element. However, they are often mutually exclusive; changing one of them causes a reciprocal change in the other one. A solution to combine the flexibility and stiffness in a mechanical system lies in an adaptive trade‐off between them. Interestingly, such a combination can often be found in biological structures.2, 3 This has usually ended up with mechanical systems that display two deformability regimes , depending on the magnitude of applied loads. The variable, yet reversible, stiffness levels have provided such “double‐stiffness” structures with a major advantage over the conventional single stiffness structures: they can adapt to applied loads. That is why the “double‐stiffness” structures have found plenty of applications in biomedical, aerospace, robotics, structural engineering, etc.In recent years, researchers have applied either of the above‐mentioned strategies to engineering structures.11 Thi Second, the linear response of “double‐stiffness” structures makes them more predictable and therefore, facilitates their modeling and controllability for robotic applications. Third, the properties of “double‐stiffness” structures in each deformability regime can be readily tuned by adjusting their geometric features. This enables them to reach a wide range of variable stiffness, which is especially important for shape morphing systems.“Double‐stiffness” structures present multiple advantages over commercially available components that gradually change stiffness in response to loads . A piece‐wise linear stiffness change, as seen in many “double‐stiffness” structures, makes them particularly attractive for practical applications. This is due to three main reasons. First, there is no time delay in the process of stiffness change of “double‐stiffness” structures, which enables the system to be highly dynamic.13 Sec In another scenario, a flexible structure, turns into a stiff one to restrict deformability. Khaheshi et al., for example, developed a durable structure that is initially flexible, but becomes stiff through an interlocking effect to prevent detrimental large deformations. Each of the two approaches, however, represents a drawback. The impact‐resistant structures developed based on the former approach are often less durable than those implementing the latter approach, when subjected to the same displacement. In contrast, the durable structures developed based on the latter approach could fail under mechanical impacts or excessive stresses. How can an engineering structure gain the advantages of the two described approaches, that is, load‐bearing, durability, and impact‐resistance?The approaches to achieve a reversible change in the stiffness, however, could be highly different. One scenario is that an initially stiff, that is, load‐bearing, structure turns into a flexible one to prevent failure under excessive loading. This transition could be triggered, for instance, by an external force upon a mechanical impact.16 In FigureThis question prompted the current study. To find the answer, we used bioinspired strategies Figure1A, to e22.1We used computer aided design (3D computer graphics software Blender v. 2.8) and 3D printing (Prusa i3 MK2.5S) to design and fabricate our structure. The developed 3D model and its 3D printed prototype are illustrated in Figure −1, Colorfabb) for 3D printing of the model .Our main model, called as the “reference model” was composed of a membrane and three reinforcing elements, which crossed each other and created a joint‐like structure at the center of the model Figure . There wThree other models were developed by the removal of one of the design strategies from the “reference model”: i) “no flexible joint model,” by the removal of the flexible joints between the reinforcing elements; ii) “no mechanical stopper model,” by the removal of the spikes and iii) “no buckling model” by the removal of the buckling zones  . The test continued until a stabilized response was reached. For all tested specimens this happened below 100 loading cycles . The force in the last loading cycle was demonstrated as a percentage of the force in the first loading cycle, and used as a measure of the durability  of the specimens. In each set of experiments, at least three specimens of each type were tested.In order to characterize the mechanical behavior of our 3D printed structures under loading, we performed three sets of experiments: i) static, ii) dynamic and iii) fatigue tests to measure their load‐bearing capacity, impact‐resistance, and durability, respectively Video . A displ2.3Figure−1 . When the loading exceeds a set threshold (here ≈12 N), at which the maximum load‐bearing is reached, the cross elements suddenly buckle and the model flexes to prevent failure  flexible joints, (ii) mechanical stoppers, and (iii) buckling zones. Our results confirmed that the implementation of flexible joints enhanced the durability of the 3D printed models, in comparison with the “no flexible joint model.” This is because the flexible joints reduce the stress concentrations at the junctions of reinforcing elements. This finding is supported by a previous report, which showed that the stress within flexible joints of insect wings is up to 80 times smaller than that developed in fused joints under the same loading condition.30 Thu Joint‐like structures play a key role in this regard. Due to their “weaker” structure or material, the joints are often more flexible than other wing components. However, joint‐associated spikes, which resemble our mechanical stoppers, provide the joints with the stiffness necessary to withstand aerodynamic forces. The same principle is valid for most engineering structural elements. This is the main reason why engineering structures are not made of the strongest materials; strong materials cannot deform readily and, therefore, tend to be brittle. A similar design strategy as that used in insect wings, that is, joint‐like structures at the junctions of reinforcing elements, could help to improve the resilience of brittle composites and their resistance against fracture.Insect wings, particularly dragonfly wings, inspired our design strategy, that is, combination of the flexible joints and mechanical stoppers. As mentioned earlier, insect wings represent remarkable examples of a balance between load‐bearing and durability. This balance is achieved through a compromise between stiffness and flexibility; Wings need to be stiff enough to withstand aerodynamic loads and flexible enough to deform without failure.32 JoiTo enhance the resistance of our developed structures against mechanical impacts, we implemented a buckling mechanism into our design. This was done by the inclusion of a discontinuity and two structural gradients, which provided the “reference model” with the instability needed under mechanical impacts. While the discontinuity and the structural gradient implemented in the cross elements facilitated the occurrence of the buckling, a thickness gradient, in the form of two marginal strips at the edges of the membrane Figure , helped 36We implemented our “triple stiffness” structure, that is, the “reference model,” into the wings of airplane models and subjected them to mechanical impacts (Video  in which we showed that combining the flexible joints and mechanical stoppers enhanced the flight stability of 3D printed “double‐stiffness” kites under wind gusts. This is mostly because, by slight changes in the configuration, the kites could automatically adapt to the changing flight conditions. We expect a similar effect to occur here. Although, taking into account the inherent difference between the free flight of airplanes and tethered flight of kites, this conclusion should be interpreted with caution.Here we did not analyze the dynamics of the 3D printed airplanes from the aspect of stability, since our main aim was to examine their resistance to mechanical impacts. However, we would like to build on our own previous work,11 in  Some of these challenges are associated with the manufacturing processes, control strategies, long‐term durability, and efficiency of the mechanical parts when applied at different scales. The excellent mechanical performance of our “triple stiffness” structure along with its low‐cost merits and the ability to tune properties within a single material system are characteristics that make it a suitable candidate for scalable development. In addition to these, the deformations exhibited by our structure are fully passive and, hence, there is no requirement for a complex control strategy. The use of manufacturing techniques other than 3D printing by fused deposition modeling, as that used here, and the application of advanced structural composites, such as carbon fiber reinforced polymers, can further improve the performance of our bioinspired design. This is because of the quality issues associated with low‐cost 3D printed parts, which often contain small defects and coarse grains that negatively influence their specific stiffness. Hence, in summary, we expect that our design can be mass produced and implemented to high‐performance adaptive systems.Scalable manufacturing of adaptive structures with variable stiffness levels and their integration with strictly rigid conventional systems still present considerable technical challenges.38 Som The use of variable stiffness structures could also benefit the performance of conventional fixed‐wings aircrafts at different flight conditions. The concept developed here could inspire the design of novel adaptive structures in robotics, aerospace, biomedical and structural engineering.In the end, we conclude that our design strategy is an efficient, yet convenient, solution for the development of engineering structures that combine load‐bearing, durability, and impact‐resistance. These are the three key features of any structural element in order to properly function and survive “extreme” conditions. As a promising outlook, the implementation of the “triple stiffness” structures in wings of flapping robots or drones could facilitate their widespread use in civilian applications, such as safety inspection, environmental preservations, etc.40 TheVideo The authors declare no conflict of interest.Conceptualization: A.K., S.N.G., H.R.; Supervision: S.N.G., H.R.; Investigation: A.K., H.R.; Data Curation: A.K., H.R.; Formal Analysis: A.K.; Funding Acquisition: A.K., S.N.G., H.R.; Methodology: A.K., H.R.; Project Administration: H.R.; Resources: S.N.G.; Validation: A.K.; Visualization: A.K., H.R.; Writing—Original Draft Preparation: A.K.; Writing–Review & Editing: A.K., S.N.G., H.R.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.Supplemental Video 4Click here for additional data file.


A functional wheat bran (FWB) was obtained from wheat grains that were rich in wheat aleurone. The effects of the microwave (MW) power  and treatment time  on the moisture and free fatty acid (FFA) content, lipase activity, and antioxidant activity of the FWB were investigated. The purpose of this study is to stabilize the FWB against lipid oxidation and rancidity and as much as possible to retain its antioxidant activities. MW treatment significantly decreased the FFA content, moisture content, and lipase activity of the FWB. Moreover, MW treatment significantly increased the total phenolic content (TPC) and antioxidant activity of the FWB without drastically altering its color. MW treatment at 7.5 kW and 120 s was found to be optimal for stabilizing the FWB and increasing its antioxidant activity. The stabilized FWB was proven to be far more stable than the control FWB during storage. Thus, MW treatment is an effective stabilization method for the storage and utilization of FWB. Additional research is needed for the exact mechanism of the decrease of FFA content and increase of antioxidant activity of FWB induced by MW treatment. Microwave treatment significantly decreased the FFA content and lipase activity and significantly increased the total phenolic content (TPC) and antioxidant activity of the FWB. The stabilized FWB was far more stable than the control FWB during storage. Microwave treatment is an effective stabilization method for FWB storage and utilization. The FWB was obtained using traditional roller milling after debranning the outermost layers (pericarp layers) of wheat kernels. The storage stability of the FWB, along with the free fatty acid (FFA) content, total phenolic content (TPC), and antioxidant activity after MW treatment, was analyzed.22.1Wheat grains of a hard white winter cultivar (Jinan 17) were obtained from Jiaoqiao seed station. Soybean oil that had been extracted via a leaching method was purchased from Yihai Grain and Oil Industry Co., Ltd. All other chemicals were of analytical grade and purchased from Chemical Reagent Co., Ltd.2.2Wheat grains were cleaned and debranned to remove the outermost layers (approximately 4% of the kernel weight) using a wheat debranner (TPZ‐20). Then, the debranned wheat grains were milled using a laboratory wheat miller (JMFB 70 × 30) to obtain white flour, fine and coarse brans. According to the micronutrient content and antioxidant capacity (unpublished data), the coarse bran was regarded as the FWB. Subsequently, the freshly produced FWB was treated immediately under various MW conditions.2.3A microwave dryer  with a cooling system was used for the FWB treatment. The belt speed and MW power were controlled by a control panel, and the treatment time was controlled by adjusting the belt speed.Each FWB sample was placed on the belt, spread evenly to a thickness of 0.3 cm, and treated at a selected MW power  for a specified treatment time . Immediately after treatment and cooling, the samples were ground and passed through an 80 mesh aperture sieve (180 μm) using a hammer mill . All samples were stored at a temperature of −18°C in a freezer. Untreated FWB was used as a control.2.4The moisture contents of the treated FWB and control samples were determined via method 44‐15A  between the control and treated samples were calculated using the equation ΔE = (ΔL2 + Δa2 + Δb2)1/2 . The CIE color values were recorded as L* (lightness), a* (redness), and b* (yellowness). The total color differences 2.10An accelerated storage test was conducted in a constant‐temperature oven at 45°C for 4 weeks. The FWB that had been treated under optimal treatment conditions and the control were packed in aluminum foil bags (50 g each). The moisture and FFA contents were assayed at 1‐week intervals.2.11p <.05 by using Duncan's test.All experiments were conducted in triplicate. The results are reported as the mean value ± the standard deviation and subjected to one‐way analysis of variance, followed by least significant difference calculations, to assess any differences between the group means using the SPSS 20.0 software package. Significance was defined at 33.1As shown in Figure 3.2The influences of various MW treatment conditions on the color of FWB were presented in Table ΔE) during the MW treatment was only 4.05. Moreover, significant decreases in browning were observed at all MW powers for 90 s and 120 s during the MW treatment, which were characterized by higher L* values and lower a*, b* and ΔE values than those at 30 s and 60 s. The MW treatment might be an effective approach to reduce undesirable impacts of nonenzymatic browning without drastically altering the color of the raw FWB , the lower the FFA contents after treatment for the same period of time. The effect of MW treatment at 10.0 kW on the FFA content of the FWB was similar to that at 7.5 kW. The lowest FFA content was 44.3 mg KOH/100 g d.b. (approximately 49% that of the raw FWB) after MW treatment for 120 s. Zhao et al.,  reported3.5p >.05). Significant increases in the TPC were observed after MW treatment for 90 s and 120 s (p <.05), regardless of the MW power. The increase in TPC of the treated FWB ranged between 9% and 37%. The maximum value (1.66 mg GAE/g d.b.) was obtained from the FWB sample treated at 7.5 kW for 120 s. The results demonstrated that the TPC of FWB increased as a result of MW stabilization. Similar observations were reported by Saji et al., . The highest value of the antioxidant activity was 14.76 mg TE/100 g d.b. for the FWB sample that was treated at 7.5 kW for 120 s, which was approximately 4.3 times higher than that of the control. This could be explained by the strong correlation between the TPC and the antioxidant capacity (Verma et al., The effect of MW treatment on the antioxidant activity of the FWB was presented in Figure 3.6Based on the above results, 7.5 kW for 120 s was identified as the optimal MW treatment conditions to prepare the sample for the accelerated storage test. The moisture and FFA contents are shown in Figure FFA content continuously increased as the storage time increased Figure . Lipase 4The effects of microwave treatment on the stability and antioxidant capacity of functional wheat bran were investigated. MW treatment significantly decreased the FFA content, moisture content, and lipase activity of the FWB and improved the TPC and antioxidant activity of the FWB without drastically altering the color. The MW power contributed more than the treatment time, and 7.5 kW for 120 s was identified as the optimal MW treatment condition. The results of this study show that MW treatment can increase the storage stability and antioxidant capacity of the FWB and could provide a scientific foundation for application of MW in industrial production of FWB.The authors declare no conflict of interest.Ethics approval was not required for this research.


St-g-PAM loaded with ex situ prepared silver nanoparticles was also synthesized by using an accelerated electron beam. The graft copolymers were characterized by chemical analysis, rheology, and differential scanning calorimetry (DSC). The results showed that the level of grafting , intrinsic viscosity and thermal behavior (thermodynamic parameters) were influenced by the irradiation dose, dose rate and presence of silver nanoparticles. The flocculation performances of the synthesized copolymers were also tested on water from the meat industry in experiments at the laboratory level. In the coagulation–flocculation process, the copolymer aqueous solutions showed good efficiency to improve different water quality indicators.Modification of natural polymers for applications in the treatment of waste and surface waters is a continuous concern of researchers and technologists in close relation to the advantages they provide as related to classical polymeric flocculants. In this work, copolymers of starch-graft-polyacrylamide (St- Renewable raw materials are increasingly used nowadays due to both the rapid development of society in recent decades and the growing interest in using natural, biodegradable and affordable resources. As an example, the modification of natural polymers to be used in various applications, such as flocculants for wastewater treatment ,2,3, matOrganic flocculants, typically polymeric in nature, are widely used in the modern processes of potable water and wastewater treatment ,9,10,11.Grafting is an effective way of regulating the properties of biopolymers, which can be “tailor-made” according to the needs and produce highly efficient graft copolymers usable as flocculants ,18. CrosSynthetic polymers based on vinyl monomers, such as acrylic acid and acrylamide, with flocculating features are effective and relatively cheap, with wide applications due to their varied qualities, but being poorly biodegradable and harmful to the environment . They arElectron beam (e-beam) irradiation is an environmentally friendly practice for the synthesis of advanced materials with unique properties. E-beam-induced grafting is a rapid technique that can be performed without an initiator or other chemical catalysts, resulting in low byproduct levels and hazards . MoreoveThe present work deals with the physicochemical and functional characterization of starch-based copolymers and starch-based copolymer-loaded silver nanoparticles synthesized by using electron beam irradiation. Therefore, the purpose of this study was to point out relevant aspects related to new flocculating materials that can be a starting point for a new generation of products with unique functional properties, low toxicity and biodegradable, designed to be effective in environmental protection. At the same time, we aimed to give a deeper insight into a practical and ecological method of radiation-based synthesis for flocculants functionalized with metallic nanoparticles that meet the quality claims, which is able to replace the classical methods involving pollution risks, complex procedures and high costs.Unmodified regular corn starch containing approximately 73% amylopectin and 27% amylose  was purchased from Sigma-Aldrich , and acrylamide  was purchased from Alfa Aesar . Other chemicals were of analytical grade and purchased from SC Chimreactiv SRL . Graft copolymers were synthesized in two steps: (1) preparation of solutions containing starch and monomer; (2) irradiation of solutions by using electron beam.In the first step, starch samples were prepared by gelatinizing powder starch in distilled water. Acrylamide and sodium chloride (7.5%) was added to starch sample with further stirring, resulting in a starch: acrylamide (St:AMD weight ratio = 1:10) homogenous samples. Sodium chloride is one of the critical parameters in electron beam-induced polymerization, and the additional use of NaCl in the range of 5% to 10% can increase the monomer conversion (over 80%) and level of intrinsic viscosity of copolymers, reducing also residual monomer ,36. MoreTherefore, the homogenous samples resulting from the first stage were then subjected to e-beam irradiation. The sample processing was performed in static mode by using an accelerated e-beam provided by a linear accelerator ALID-7 of energy of 5.5 MeV . The electron accelerator installation is of traveling-wave type, using microwaves in the S-band at 2.99 GHz that propagate in a disk-loaded tube of about 2 m long; the microwaves are produced by an EEV-M5125 type magnetron that provides 2 MW of power in pulses of 4 μs . The ALIThe physicochemical properties of synthesized copolymers were evaluated through chemical analysis and rheology, while the functionality as flocculant agents was tested on wastewater from the meat industry.cC) and the unreacted monomer (mR) were evaluated by determination of the vinyl monomer double bonds before and after copolymerization. Thus, the copolymer solution was prepared by dissolving graft copolymer (2 g) in distilled water (200 mL) stored overnight at room temperature (25 ± 1 °C), and further, the obtained solution was magnetically stirred (300 rpm) in order to obtain a homogenous solution. Distilled water (100 mL) and 4 mol/L sodium chloride solution (100 mL) were added to this solution. Volume of 25 mL of copolymer solution was then transferred to a 250 mL Erlenmeyer flask and 10 mL of bromide–bromate solution (containing 35 g of potassium bromide and 2.783 g of potassium bromate per liter) and 2.5 mL of hydrochloric acid (18.5%) were added; then the flask was well stoppered. The solution was left to stand in the dark for 20 min and shaken frequently. After this time, 2.5 mL of 20% potassium iodide was rapidly added to flask content and the excess of the iodine liberated was titrated with 0.1 mol/L sodium thiosulfate (Na2S2O3) solution using 1 mL starch indicator solution (1%). A blank determination was made as well.The monomer conversion coefficient (mR) and the conversion coefficient (cC) were calculated from the following equations:mR is the content of residual monomer (g of unreacted monomer/100 g copolymer), 0.0568 is a constant expressed in g/mL, bV is the volume of Na2S2O3 used for titration of the blank sample (mL), gV is the volume of Na2S2O3 used for titration of graft copolymer sample (mL), gW is the weight of the graft copolymer (g),cC is the coefficient of the monomer conversion (g of reacted monomer/100 g copolymer), iM is the monomer content in sample before irradiation (g of monomer/100 g sample), mR is the content of residual monomer (g of unreacted monomer/100 g copolymer).The residual monomer  using 1 mol/L sodium chloride solution as a solvent. The prepared solutions were measured with a Hoppler type BH-2 viscometer at 25 °C, and the intrinsic viscosity was then determined by extrapolating the linear curve of the reduced viscosities that were calculated as a function of the selected concentrations.The intrinsic viscosity  and lower Hk. These findings indicate that longer polyacrylamide (pAMD) chains are probably formed in the presence of AgNPs as a result of the intermolecular forces induced by metallic nanoparticles, thus obstructing the formation of short or branched chains of pAMD.The copolymer-loaded silver nanoparticles  and decomposition of the synthesized copolymers in comparison with the native starch and polyacrylamide. The DSC curves of the investigated graft copolymers are displayed in Peak I) located at around IT = 340 K can be attributed to the loss of absorbed moisture in the sample, while the second peak (Peak II) located around IIT = 560 K corresponds to the crystalline melting of starch and thermal decomposition.Two endothermic events could be observed in the DSC curve of native starch (St). The first endothermic peak (Peak I) at around IT = 385 K, which corresponds to the loss of absorbed moisture in the sample and the crystalline melting, and a glass transition at around gT = 506 K. It was reported that the degradation of pAMD occurs in the temperature range from 450 to 573 K as a result of the liberation of ammonia (volatile product) and formation of imide groups in the cyclization phenomenon [Polyacrylamide (pAMD) also showed two major events in the DSC curve: an endothermic peak , the DSC curves (p ≤ 0.05) in comparison with pAMD and St. Moreover, molecules of St-g-AMD 2 and St-g-AMD 3 showed that Peak I consisted of two overlapped peaks (IT~387 K and I *T~393 K). On the other hand, the second peak, typical for crystalline melting of polysaccharides, could not be identified for any samples of this batch. The glass transition could also not be detected for the copolymer synthesized by using the smallest dose irradiation (St-g-AMD 1), whereas the other copolymers showed the increase (p ≤ 0.05) of gT value in comparison with pAMD  with the irradiation dose cC (r~0.999) and the residual monomer mR (r~−0.997), respectively, and a good correlation (r~0.831) with the intrinsic characteristic copolymer, namely the viscosity (η). At the same time, the process enthalpy IΔH was very well correlated (r~0.965) with the irradiation dose cC (r~0.599) and mR (r~−0.620).The graft copolymers presented thermal events characteristic to the St and pAMD shifted to higher temperatures in the DSC curves. For copolymers prepared by using a constant C curves a showed ith pAMD . WorzakoBatch 2) compared to the native starch and pAMD. The copolymers St-g-AMD 4 and St-g-AMD 5 obtained by irradiation at 0.5 and 0.6 kGy/min, respectively, displayed two endothermic peaks in a similar manner  in comparison with the gT of homopolymer. At the same time, a reduction (p ≤ 0.05) of the gT values was observed and it was very well correlated (r = 0.921) with the increase in the dose rate IT had very good correlations with the dose rate r = 0.998), as well as with the grafting parameters, namely cC (r = 0.981) and mR (r~−0.985), respectively, and only poor correlation (r~0.677) with the copolymer intrinsic viscosity (η). Moreover, the process enthalpy IΔH was very well correlated with the dose rate r = 0.992), cC (r = 0.985) and mR (r = −0.988) and poorly correlated with the intrinsic viscosity (η) (r~0.693).Changes in the temperatures of the thermal events were also observed for the copolymers synthesized by using a constant irradiation dose of 1 kGy and varying the dose rate  showed significantly (p ≤ 0.05) lower values of peak temperatures while the gT had a higher value (p ≤ 0.05) in comparison with the unloaded copolymer  and higher glass transition temperatures than the polyacrylamide (pAMD). In other words, we can consider that, in terms of the glass transition temperature, the graft copolymers showed better thermal stability than the homopolymer (pAMD). Other reports also revealed that natural polysaccharides grafted with vinyl monomer by means of ionizing radiation had good thermal stability in the field of practical applicability ,70,71. MThe functionality of the graft copolymers as flocculant agents was examined on surface water collected from the meat processing industry.TSS), fatty matters (FM), chemical oxygen demand (COD), and biochemical oxygen demand (5BOD). The characteristics of raw water and the permissible levels of water quality indicators according to the Romanian national guideline are shown in The quality parameters measured were pH, total suspended solids (Flocculation performances of the inorganic coagulants and their combination with a flocculant (graft copolymer) are presented in TSS yield of 83.5% was reached by using St-g-AMD 5(AgNPs) copolymer in the coagulation–flocculation process. The yield of the fatty matters reached 17% when only inorganic coagulants were used and increased to approximately 45% by adding the synthesized copolymers. The copolymers St-g-AMD 1 and St-g-AMD 5(AgNPs) showed the best values of FM yield. The yields for the other investigated indicators (COD and 5BOD) were improved only slightly by adding copolymers to the inorganic coagulants in the water treatment.All synthesized copolymers showed good performances in the coagulation–flocculation process. The pH value of raw water decreased significantly after treatment with inorganic coagulants and then slowly from 7.1 to the value of 6.9 by adding flocculant to the inorganic coagulants in the water treatment. The copolymer presence increased the yield of the total suspended solids from 55% for inorganic coagulants to approximately 80%. A TSS, FM, COD and 5BOD) of water collected from a meat processing plant. Among the graft copolymers, the copolymer-loaded silver nanoparticles St-g-AMD 5(AgNPs) showed superior results by significantly reducing the total organic load  of the tested water.The elimination of suspended solids and as much organic material as possible is an essential trait in the process of wastewater flocculation . TherefocC exceeded 90%, while the residual monomer mR had values below 3%. However, these grafting parameters were much better correlated with dose rate The monomer conversion coefficient η) > 7 dL/g) and Huggins constant Hk below unity.All graft copolymers had high intrinsic viscosity values  and polyacrylamide (pAMD) shifted to higher temperatures in the DSC curves. The identified changes confirmed the grafting process and formation of new chemical bonds between the St backbone and pAMD. At the same time, all graft copolymers showed improved thermal stability in terms of glass transition temperatures higher than the polyacrylamide.The potential of the synthesized copolymers to improve the quality of the surface water from the meat industry was proven in coagulation–flocculation experiments at the laboratory level. Thus, the copolymers were able to reduce the water quality indicators, such as total suspended solids, fatty matters in suspension, chemical and biochemical oxygen demands.“Green” starch-graft copolymers with eco-friendly approaches were successfully synthesized via graft copolymerization in aqueous solutions of acrylamide (AMD) onto a starch substrate (St) induced by an electron beam irradiation. Therefore, for the copolymers prepared in the experimental conditions applied herein, the main findings can be summarized as follows:Therefore, the characteristics of the starch-based flocculants synthesized via graft copolymerization induced by an electron beam irradiation depend on the irradiation parameters, namely the irradiation dose