L. africana has several orphan proteins with 112 proteins showing < 30% sequence identity with their homologues. Rigorous sequence searches and complementary approaches were employed to annotate 156 uncharacterized protein sequences and 28 species-specific proteins. For 122 proteins we predicted potential functional roles, 43 of which we associated with protein- and nucleic-acid binding roles. Further, we analysed domain content and variations in their combinations within Afrotheria and identified 141 unique functional domain architectures, highlighting proteins with potential for specialized functions. Finally, we discuss the potential relevance of highly represented protein families such as MAGE-B2, olfactory receptor and ribosomal proteins in L. africana and E. edwardii, respectively. Taken together, our study reports the first comparative study of the Afrotherian proteomes and highlights salient molecular features.Afrotheria is a clade of African-origin species with striking dissimilarities in appearance and habitat. In this study, we compared whole proteome sequences of six Afrotherian species to obtain a broad viewpoint of their underlying molecular make-up, to recognize potentially unique proteomic signatures. We find that 62% of the proteomes studied here, predominantly involved in metabolism, are orthologous, while the number of homologous proteins between individual species is as high as 99.5%. Further, we find that among Afrotheria,  It contains the six orders of the elephants (Order Proboscidea), sea cows (Sirenia), hyraxes (Hyracoidea), aardvark (Tubulidentata), elephant shrews (Macroscelidea), golden moles and tenrecs (Afrosoricida). This diverse group of animals show distinct differences in sizes, preferred habitats, diets etc. Although these species bear little resemblance to each other, molecular data offers evidences for their evolutionary relationships2. Afrotheria is a peculiar clade characterized by lack of testicular descent through the loss of functional forms of RXFP3 and INSL3 genes3. Unique features such as abundant copies of tumor suppressor TP53 gene, olfactory receptors and leukemia inhibitory factor are characteristic of elephants6. Likewise, studies within Afrotheria show that the elephant and its closely related species, manatee, are associated with loss of genes involved in bile acid synthesis and low metabolic rates to maintain body temperature9. Others such as the cape golden mole are known for their high sensitivity to seismic vibrations10. The diverse biology of Afrotheria, coupled with the availability of substantial genomic and cognate protein sequence data now enables comparative studies of Afrotherian proteomes. Such studies are useful to understand Afrotherian genome evolution and will provide the platform to probe for potential molecular signatures that can underlie their evolutionary adaptations.The Afrotheria superorder represents a diverse group of mammals that differ distinctively from each other1. Detailed comparative studies involving the distribution of various functional and structural domains within the Afrotheria could help to recognize protein domains that are specifically amplified or occur in distinct combinations. Considering that Afrotheria is a unique clade of extremely divergent species with distinct phenotypes, such a comparative analysis on protein sequences could shed light on characteristic molecular features of Afrotherian species that could influence their evolutionary adaptations.Protein-centric analysis involving Afrotherian species have mainly focused on understanding species phylogenyLoxodonta africana), Florida manatee (Trichechus manatus), aardvark (Orycteropus afer), cape golden mole (Chrysochloris asiatica), cape elephant shrew (Elephantulus edwardii) and lesser hedgehog tenrec (Echinops telfairi). Specifically, we adopted a protein-centric approach to compare their proteomes and analysed the number and nature of orthologous proteins, extent of sequence conservation and probed for species-unique proteins. In addition, we have performed and compared domain assignments of these proteomes to determine if any specific domain expansions are observed within the Afrotherian species studied here. It is well recognized that multi-cellular metazoans and eukaryotes share similar domains but achieve functional variety through domain accretions and differences in domain architectures11. Therefore, we specifically probed for unique domain architectures in the Afrotherian proteomes for signatures that may potentially contribute to phenotypic variability. Our assessments show that certain domain families are highly represented in individual Afrotherian species. In addition, some domain combinations are unique to Afrotherian species and hitherto unseen in other organisms. These findings have broad implications on the evolutionary adaptations of these species that are distinct not only from each other but also from other non-Afrotherian mammals.In this study, we used several comprehensive and complementary approaches to compare the proteomes of six Afrotherian species namely African elephant   with all other Afrotherian species, affirming that it is the most distant member among the species studied here2. When compared with a non-Afrotherian species such as H. sapiens, the average sequence identity is 84% with fewer homologous proteins between them. Additionally, we observe that the average pairwise sequence identity matrix of the Afrotherian proteomes is asymmetric (Table 12.Phylogenetic studies on the evolution of Afrotheria have shown that their origin is monophyleticE. telfairi and C. asiatica), Macroscelidea (E. edwardii), Paenungulata (L. africana and T. manatus), and Tubulidentata (O. afer) might have occurred very rapidly in the late Cretaceous period. Consequently, morphological evidences of their divergence might have been erased through subsequent evolution12. Indeed, when we examine the number of orthogroups and their orthologous proteins  shared with other order members compared to T. manatus , suggesting that in the Paenungulata order L. africana has diverged extensively  within the Afrotheria  with their homologs. These percentages are higher than what has been observed in L. africana (38%), T. manatus (30%) and O. afer (39%). Notably, we find that L. africana has 112 ‘least’ conserved proteins, the highest number among Afrotherian species considered here  category. In 14. To do this, we mapped GO terms to every protein in the proteome based on gene information obtained from the respective human homolog. As a result, we could associate 169 biological processes common to about three-quarters of these proteomes. Since GO terms were available at different hierarchical levels, we traced their parent terms to the primary level for each of the biological processes and then compared the extent of protein conservation. We find that the proportion of ‘moderately’ and ‘least’ conserved proteins associated with biological processes such as signaling, response and biogenesis is higher than other primary biological processes considered here  that are currently annotated as ‘Uncharacterized proteins’ through homology searches in NRDB and found statistically significant matches for 2185 query proteins. For 156 proteins, the homologs were detected only in Afrotherian proteomes at high sequence identity, suggesting that they are Afrotheria-specific. To improve their functional annotations, we employed an integrated approach , we identified the ral-guanine nucleotide dissociation stimulator-like (RGL) protein (XP_023398420.1) as a paralog at 97% sequence identity, covering full length of its sequence. Motif recognition in RGL and XP_023398370.1 predicted that both sequences contain disordered and proline-rich regions in addition to conserved sequence motifs , of which 3187 domains are common to all. The rest of the domains are common to a few or unique to a species . Similar trends are observed in the “Nine Cysteines Domain of family 3”, GPCR, G-protein coupled receptor (E. telfairi-18: H. sapiens-7), MAGE family (Melanoma Antigen Gene) (L. africana-27: H. sapiens-13), Prothymosin (C. asiatica-14: H. sapiens-2) and other domains, suggesting unique functional roles for these domains in Afrotherian species. Additionally, “Nineteen complex-related protein 2”, Pacifastin inhibitor (LCMII), Cancer susceptibility candidate 1, 4Fe-4S single cluster domain, Nucleoporin FG repeat region and few other domains also appear to be exclusive to Afrotheria. Over-representation of a domain might point to its recruitment towards specific functional roles, although this remains to be verified.The availability of sensitive domain recognition methods enables the comprehensive enumeration of protein domains in each proteome and facilitates characterization of trends in their distributions. Such analysis might offer clues on the selection of domains to meet functional requirements specific to an organism. Therefore, we predicted domain make-up of each protein in our dataset to examine if specific domains are over-, under-, or unrepresented in any of the proteomes. Domain assignments were performed using hmmscan against the Pfam database. We find that out of a total of 16,712 Pfam families, 6068 are associated with nearly 92% of the proteomes showing that their coverage is uniform amongst Afrotheria .We compared domain architecture of Afrotherian proteins to probe for novel domain combinations and species-unique trends, if any, in their distribution. For the dataset of 105,534 proteins for which functional domains were assigned, we find that 31% of the query proteins are single domain proteins (with domain assignments covering the full length of the proteins) while 69% are multi-domain proteins. However, in some instances, assignments covered only a portion of the entire length of the protein leaving the rest unassigned. We believe that such regions might harbour domains that have either diverged extensively or are yet to be identified and characterized. Hence, we considered a protein with at least 100 residues of unassigned region as a potential multi-domain protein apart from proteins where more than one domain could be identified. In doing so, we observe that the proportion of multi-domain proteins in Afrotheria increases with 17. Differences in domain architectures among homologous proteins are usually functionally relevant since domain accretion can bring about lineage-specific and complex domain architectures11. Therefore, we assessed protein complexity based on domain composition and the linear order of domains (domain arrangement) from the N–C terminus of a protein. Here, we defined protein complexity as the total number of distinct domains present in a protein and assigned it a complexity index. Our results show that proteins with only one distinct domain are very common (~ 6642) in the dataset, suggesting that a large contribution to the multi-domain nature of the proteins is made by repetitive domains or unassigned regions. This trend holds true for reference proteomes as well. Our results show that a significant contributor to single domain proteins showing a complexity index of 1 in L. africana  and ~ 50% lower than in H. sapiens  and E. telfairi (311) possess higher number of unique architectures as compared to other species (< 260). This trend holds true with the exclusion of cases resulting from tethered domains or domain repeats  at either end of the common domain architecture, where only tethered domain architectures are considered or variation in the number of repeating units of a domain  was predicted to possess two repeating copies of a Tap RNA-binding domain tethered with nuclear transport factor 2 (NTF2) domain. In addition, it has leucine-rich repeat regions and a protein partner interacting motif . Similar domain architecture is reported in Q9H4D5, a human nuclear RNA export factor 3 (NXF3) in which its Tap RNA-binding domain is known to bind snoRNAs and its NTF2 domain is known to interact with proteins to regulate trafficking of snoRNAs between nucleus and cytoplasm18. The extensive similarity of XP_023396935.1 with this protein suggests that it might function as a nuclear RNA export factor  occur as repeats with 362 domains observed commonly in all the Afrotherian proteomes. Of these, 34 domains occur as repeats in novel combinations  showed that the nature and distributions of structural domains are similar between Afrotheria and reference proteomes.Clearly, although the overall functional domain make-up is comparable across the Afrotheria, differences exist in their combinations that could account for proteins responsible for specific adaptations or the evolution of new functional systems. We sought to analyze the extent of structural diversity within the Afrotherian proteomes by associating domains in the ‘Structural Classification Of Proteins’ (SCOP) database to each Afrotherian protein using the SUPERFAMILY database (details in “E. edwardii (230) and least in L. africana (203). Such ‘multi-domain’ class proteins are predominantly associated with GO terms that are involved in processes such as negative regulation of fibrinolysis, blood coagulation, fat cell proliferation and platelet activation. Overall, when we consider the proportion of the multi-domain proteins having this class, we find it highest in O. afer (61%) and least in E. telfairi (50%),  of species-unique structural domain architectures . MAGE proteins are a highly conserved group of proteins in eukaryotes that are primarily known as cancer antigens, due to their abundance in various cancers19. They are grouped into ten subfamilies under two types (I and II) depending on where they are expressed. Type I MAGE  are expressed only in testis and their somatic cell expression is associated with cancers. Type II MAGE proteins (D-L and Nectin) are implicated in the regulation of neuron differentiation and apoptosis19. We observed that MAGE-B (type I) family proteins are highly represented in Afrotheria and reference species  , suggesting that MAGE-B2 family genes might have extensively duplicated. The physiological function of MAGE-B2 family proteins expressed in spermatids during post-meiotic stages of spermatogenesis, is to protect the germline under nutrient stress conditions20. It promotes cell proliferation upon binding with histone acetylase 1 that activates E2F transcription activation. L. africana that is slow to breed likely needs a constant production of high-quality gametes. Therefore, one plausible role for this protein, which is restrictively expressed in testis, might be to assist in cell proliferation and gamete protection.Some domain families are found in large numbers in Afrotherian proteomes suggesting that proteins harbouring such domains might have been selectively recruited for various functions. Comparison of functional and structural domain distributions among Afrotheria revealed that melanoma-associated antigen (MAGE) homology domain is one such domain, found to occur in 30 proteins in ies Fig. A. In par5. Since these are of seven sub-types, we sought to determine their distribution in the Afrotherian proteomes. Here, we find that the olfactory receptor (OR) is remarkably abundant in L. africana as compared to other Afrotheria and the reference proteomes  among the Afrotheria. This protein family is known to be the major sensory system for soil nematodes, which are blind and deaf21, and plays an important role in chemoreception. It mediates physiological responses to diverse stimuli like light, odorants, bitter and sweet tastants, pheromones, lipids etc.22. Its presence in E. edwardii, that is predominantly active in the day and shows several adaptations related to burrowing, suggests that it may be employed to perceive chemical stimuli. Additionally, the absence of fruit fly and insect-specific odorant and chemosensory receptors in Afrotheria suggests that they are probably non-essential for the Afrotherian sensory system. Further, we find that the olfactory receptor is associated with unusual domain architectures in Afrotheria that are thus far not reported. In C. asiatica and E. edwardii, the olfactory receptor domain is associated with a DNA-binding motif called the CUT domain (XP_006877995.1) and a transcription factor Pcc1 domain (XP_006900058.1), which regulates genes involved in cell cycle progression and polarization, respectively  are involved in sensory mechanisms and their gene copies are known to be extensively expanded in some mammalsmes Fig. a. Interen XP_00687995.1 an25. In E. edwardii, we find that the number of copies of these twelve domains is higher than in other Afrotheria and reference species , of which 104 are observed in Afrotherian proteomes. For twelve domains, we find that the distribution varies within Afrotheria Fig. c. These 17. Comparative genomics is an effective tool to study evolutionary history and utilizes the information on similar and dissimilar molecular signatures to identify adaptive mechanisms of species28. Here, we have attempted to understand the differences between Afrotheria, comprising of African-origin animals that share limited resemblance superficially. Indeed, earlier studies have reported differences in aspects such as cancer resistance, olfaction, respiration and metabolic rates etc. in these animals8. The availability of genomic data provides an opportunity to perform a comparative study based on their proteome sequences and constituent domains.Mapping morphological differences between species to variations in their genome is a daunting and challenging task. Most traits are the cumulative result of accumulation of changes in sequence or variations in the way protein domains are arranged or interact with each otherC. asiatica, E. edwardii and E. telfairi have higher proportion of moderately conserved proteomes than when compared with L. africana, T. manatus or O. afer. In terms of functions, proteins that are involved in specific processes such as signaling, metabolic and cellular processes and response to stimulus have diverged extensively between L. africana and E. telfairi.Our studies show that the overall genome size is comparable in the six species studied here, with 62% of the proteomes being orthologous. These proteins predominantly participate in well conserved processes such as metabolism and cellular regulatory processes. Among the six species, ~ 99.5% of some proteomes are homologous, implying close relationships between species. This is also observed when we group the sequences based on their sequence similarity. Indeed, our results show that L. africana and T. manatus, both paenungulates  is high in Afrotheria. Previous studies have also suggested that such proteins are relatively recently evolved and might have been recruited for specialized functions30. The comparative studies performed here have served to enumerate not only a comprehensive list of functional and structural domains in the Afrotherian proteomes but also highlight many domains that occur in high copies in Afrotheria but not in the reference genomes or vice versa. Indeed, our detailed tables are useful to recognize examples such as the novel domain associations of the KRAB domain in Afrotheria and the relatively poorer representation of receptors such as 7tm_3, Takusan domains etc., that are highly represented in other organisms (M. musculus). We provide these detailed lists of domain distributions and anticipate that they will be useful to probe for species-unique domains and further study by others   for Loxodonta africana (GCF_000001905.1), Trichechus manatus (GCF_000243295.1), Orycteropus afer (GCF_000298275.1), Chrysochloris asiatica (GCF_000296735.1), Elephantulus edwardii (GCF_000299155.1) and Echinops telfairi (GCF_000313985.1). For comparative studies, we considered only the longest translated protein sequence per gene, to minimize the uncertainties associated with expression and function of the predicted isoforms. A total of 21,094, 19,108, 19,511, 19,638, 20,443 and 18,858 curated protein sequences from L. africana, T. manatus, O. afer, C. asiatica, E. edwardii and E. telfairi were used for all analyses in this study, unless stated otherwise.Whole proteome sequences were retrieved from the NCBI database  and query coverage threshold (75%). Protein queries with no homologs from Afrotheria were subjected to TBLASTN searches against genomic and mitochondrial DNA sequences of Afrotherian genomes. In such searches, queries that detected hits at E-value better than 10–10 and 75% query length coverage were considered as homologs.Homology searches were performed with every Afrotherian protein sequence as a query in the non-redundant database of protein sequences (NCBI). To find orthologues, both OrthoFinder (with default parameters) and reciprocal BLASTP searches were performed among all six Afrotheria–4) and query coverage threshold (75%)34. Additionally, for queries that did not find homologues, we performed PSI-BLAST searches without filters to account for low-complexity and compositionally biased regions. When these searches also failed, we performed HHblits against the uniclust30 (2017) database35 at an E-value cut-off of 10–3 and query coverage threshold of ≥ 60%.Queries with no orthologs or homologs identified within Afrotheria were employed in searches for homologues in other eukaryotic genomes using PSI-BLAST searches against the RefSeq database (NCBI). Hits were identified at the end of four iterations and selected based on E-value (10We grouped the results into four categories (1) ‘Orthologs’: proteins with bi-directional hits within Afrotheria or as identified by OrthoFinder. (2) ‘Homologs within Afrotheria’: proteins with uni-directional hits . (3) ‘Homologs outside Afrotheria’: proteins with hits only from non-Afrotherian species. (4) ‘Species-unique’: proteins that failed to recognize homologs in any search method.36. Afrotherian orthologs of each protein were aligned using Clustal Omega, ignoring columns with more than 95% gaps and then concatenated with a total of 4757 aligned residue positions37. The tree was constructed using maximum likelihood method (RAxML program) using 1000 bootstrap replications. The best bi-partitioned tree was used to infer phylogenetic relationship38.Protein sequence-based phylogenetic tree was constructed using concatenated sequence alignments of 20 different proteins . This was performed to capture the different evolutionary rates of individual proteins39. Associations of gene ontology terms to proteins with p-value > 0.05 or inferences through electronic annotation (IEA) were ignored. We considered only those biological processes in which at least 75% of proteins in the process are associated with Afrotherian proteomes through GO mapping. Through direct relationships, lower level or intermediate biological process terms were mapped to their parent terms at the first level in biological process hierarchy, to compare across species.For function annotation, gene ontology terms were mapped to all six Afrotherian proteomes using the gene symbols of human homologs. Mapping was done by gProfiler using SCS algorithm for computing p-value from multiple testing correction41. Proteins with no homologs (orphan proteins) in such searches were deemed as species-unique proteins. They were screened for low complexity regions using the NCBI-seg program42. Transmembrane regions and signal peptides were predicted using TMHMM, TOPCON, Phobius, PrediSi, SignalP and SecretomeP programs46. Disordered regions, three-dimensional structure and gene ontology terms were predicted using IUPred2A and PSIPRED server47.To improve annotation of uncharacterized Afrotherian proteins, we probed for functional details of non-Afrotherian homologs recognized through BLASTP searches. In addition, InterProScan, functional and structural domain assignments were performed for each query sequenceH. sapiens, M. musculus, E. caballus and B. taurus that constitute our reference dataset, using hmmscan40 against PFAM database (version 31.0). Only reliable domain assignments were considered using default E-value and domain hmm coverage filter (60%)25. Common problems in assignment such as domain overlap, splitting and incomplete assignments were treated as follows (1) in case of domain overlap (overlap region > 40% of either domain), only the longer domain was considered. (2) In case of splitting with no intervening domain(s), individual domain parts were stitched together if their domain coverages were > 60%. (3) Where intervening domains were present, each of the assigned domains was considered. Partially assigned domains were considered only for studying domain architectures. Further, to quantify domain composition of individual proteins, we defined the term domain complexity index of a protein as the total number of distinct functional domains present in it.Functional domain assignments were performed on 108,383 Afrotherian protein sequences and 84,376 canonical proteins from 41. Default E-value thresholds were employed to assign domains. In case of domain overlap, longer domain or domain with lower E-value was considered. In both functional and structural domain analyses, to identify multi-domain proteins, we considered 100 or more residues of unassigned region as potentially a domain. In addition, we have considered two scenarios while identifying species-unique domain architectures (i) domain tethering and (ii) domain repeat. In tethering (i), domain architecture of two proteins from different organisms can differ by the presence of another domain at either end of the common architecture (e.g. A-B vs A-B-C). Such cases may arise because of domain recombination or domain fusion. In such instances, to determine unique architecture only domain instances of A-B-C were considered in the counts, since the combination of A-B is also observed in A-B-C. In the case of domain repeat (ii), differences in the domain architectures between two proteins may result from variation in the number of copies of a domain (e.g. A-B-C vs A-B-B-C). Occurrence of the same basic unit (A-B-C) in different copies because of repeating copies of a domain were considered as domain repeats and therefore, not considered further to identify species-unique architecture.Structural domain assignments were made by searches against the HMM models in the SUPERFAMILY database that uses SCOP domain definition48 and superfamily annotations by Vogel and Chothia49. These terms were used to interpret potential functional role of proteins having unusual and novel structural domain architectures.Structural domains that contribute to species-unique domain architectures were associated with GO terms using domain-centric gene ontology methodSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


In an effort to study natural fiber formation, such as, e.g., spider silk, we present a model, which is capable of forming biomimetic fibrillar nanostructure from a hydrogel micellar network. The latter consists of interacting atomic groups which form cores of micelles, and of flexible chains forming the shells of the micelles. Micelles are connected in a compact network by linearly stretched chains. The structural elements of the network can be transformed during deformation from micellar into fibrillary type and their evolution is found to depend significantly on strain rate. Our model suggests a set of conditions suitable for the formation of nanostructured fibrillar network. It demonstrates that a fibrillar structure is only formed upon sufficiently fast stretching while, in contrast, the micellar gel structure is preserved, if the material is pulled slowly. We illustrate this key aspect by a minimalistic model of only four chains as part of the whole network, which provides a detailed view on the mechanism of fibril formation. We conclude that such a simplified structure has similar functionality and is probably responsible for the formation of nano-structured molecular fibrils in natural materials. The arrangement of molecules in natural materials is assumed to be optimal for their efficient functioning, motivating thus the efforts to mimic the natural arrangement also in man-made materials. Natural materials comprise a sequence of atomic groups, assembled in hierarchical structure on the nanometer and supermolecular level. Tensile deformation plays a key role in the formation of biological materials, such as spider silk, where the final structure consists of nano-crystalline inclusions incorporated in the rubbery network. In fact, the drawing of fibers is frequently used in laboratory and in industry  and polyethylene glycol (PEG) molecules. The fibers of polyethylene contain domains of crystalline phase , methylene group (CH2), and oxygen (O). AA-groups, and of flexible shells of micelles comprised of PEG chains. The micelles are organized into a micellar network . The estimated stretching speed of electrospun material in the article of Wang and Hashimoto (−7s−1). Nevertheless, a clear qualitative correlation between stretching rate and the process of orientation was observed in our simulation too. The results for one stretching rate of network model were calculated from 4 independent simulations with different initial atomistic configuration.These stretching rates are high in comparison to those used in real experiments. Molecular dynamics simulation deals with times in the range of hundreds of ns or μs. Stretching rates in classic deformation take approximately  in the network. The strength of a physical network is proportional to the volume fraction of PCLs. Therefore, the damage can be estimated as a decrease of the volume fraction of PCLs. A structural recovery means that the structure after deformation has been restored to the original one . DT is itself a mathematical algorithm, which creates a triangular 3-dimensional mesh from the interacting AA-groups in the cores of micelles. The distribution of connecting lines is related to the fraction of clusters. Detailed information about the modeling method and the parameters of the models are provided in The volume fraction of physical clusters is calculated in this work by triangulation of space between the interacting groups, namely, by 3A disadvantage of the network model is that one cannot specifically control the mutual position of particular chains, the combination of angles between stretched chains as well as their end-to-end distances, even though the network model shows the evolution of the entire network structure.The auxiliary “4-chains model” is a minimalistic one, composed of only four flexible chains which span interacting groups. The four chains model is a structural element which is present also in the network model . This siP2), which describes the orientation of different segments (each segment standing for two consecutive bonds) of the chains. The orientation of a segment is determined with respect to the fiber vector. The latter is sum of the end-to-end vectors of all four chains. P2 describes the correlation of the fiber orientation with the individual segments of the chains, whereby P2 = −0.5, when the segments are oriented perpendicular the to fiber, P2 = 0, for randomly oriented segments, and P2 = 1, when all segments are parallel to the fiber:The bundles and fibrils were identified in the network model and in the four chains model . We charwhere θ is the angle of a segment with the fibril vector.The relaxation was analyzed immediately after the system was deformed. In fact, the relaxation contains several effects which occur during different time scales. That is why we performed three relaxation simulations lasting 1, 100, and 100,000 ps (100 ns) with the same initial configuration and initial random seed.t:Each relaxation is characterized by specific correlation function, taken as the average of dot products of vectors in time 0 and x is a specific property at zero time and at time t. Observables are the inertia (velocity vector), bond/angle relaxation (bond vector), conformation relaxation (bond vector), relaxation of physical clusters  and relaxation of bundles and fibrils (bundle vector). Details are presented in where In our recent study  is presented in Next we explore intermediate stretching rates between “slow” and “quick” in more detail. The first finding is, that there is ~50 ns period after the moderate deformation. During this period the damage persists before the onset of relaxation.Another observation is that the network undergoes only partial relaxation see , wherebyIt has been suggested in the literature, that the changes can be caused by solvent effect. It has been known that a similar effect of the small monomer and solvent molecules can play important role in the structure transition of the deformed networks  groups. The positions of these interacting groups are fixed within the existing clusters. Three consecutive polyethylene glycol monomers (each monomer made of three atomic groups) separated by AA-groups define a SFC. In the present model, all SFCs consist of 11 segments.For the detailed structural analysis of the fiber formation mechanism we consider a structural component in the atomistic configuration, namely a short flexible chain, SFC . Such chAA-groups at both ends of a chain may belong to the same physical cluster so that this SFC is a loop. Alternatively, both chain-ends can be part of different clusters and the SFC is then a connecting chain. The motion of chain-ends is thus restricted inside, or between physical clusters, while the other segments of the chain can move freely in space. So in our model we deal with freely rotating chains with fixed chain ends and weak non-bonding interaction. Their motion in space is then determined by thermal displacements of the chains.Two re, of unrestricted SFC should display a Gaussian peak. The actual end-to-end distance distribution, however, is more complex , or a chain (unstretched or stretched) bridges two different clusters.A histogram of end-to-end distances, plex cf. . Accordire ≤ 0.42 nm, mostly re ≈ 0.35 nm). Both ends of the chain are connected to groups within the same cluster. The groups themselves are nearest neighbors.The first class are loops of type 1 (re ≤ 0.65 nm). Similar to the previous type, loop 2 connects the interacting groups inside a single cluster. The groups, however, are not nearest neighbors.The second class is a loop of type 2 (0.42 ≤ re ≤ 0.85 nm), that span two neighboring physical clusters. Such chains are flexible and the segments can move perpendicular to the chain backbone. They can reach relatively large distances away from the connecting line of the chain ends.The third class is that of unstretched chains (0.65 ≤ re ≥ 0.85 nm), connecting two physical clusters. The chain segments reside on the connecting line between interacting groups.The fourth class is a stretched chain  by using an orientational order parameter, P2(cosθ), Equation (2), where θ stands for the angle between the chain segments and the fiber vector, P2 stays positive, P2 ≥ 0.3, yet less than unity on average, indicating that the segments in fibrils are not perfectly oriented along the fiber vector. Apparently, for the chains, which make an angle 40° and larger (with no formation of fibrils), the value P2 becomes close to zero, which reflects a random orientation of the segment. Interestingly, even the chains transformed into random loops show some increased orientation in the end phase of simulation when the end-to-end distance becomes very small (≈2 nm), presumably as a consequence of a secondary orientation of loops.We have tried to quantify the process of fibril formation . Several SFCs could be aligned under certain conditions into a stable fibril. It seems, however, that the route from several SFCs to fibril is not direct. There occur transient objects, which we refer to as bundles. Bundles are composed of 3 or more aligned SFCs, and they appear to serve as precursors to permanent fibrils. In contrast to fibrils, however, bundles can exist temporarily. In this section, we focus on the behavior of bundles. The SFCs, which form a bundle, can be either of stretched type or unstretched. As a rule, bundles connect two clusters of One can analyze several aspects of bundle properties, for example their number and weight, change of orientation in space, rotation, and twisting. The basic analysis includes the number of the bundles per simulation box as well as the number of chains in the bundle. Average values and standard deviations were calculated in four independent samples of the network. We identified 7 ± 1 bundles during 10 × “quick” deformation, 9 ± 2 bundles during “quick” deformation of the box, and 10±2 in the slow deformation, so that the difference between slow and quick deformation appears statistically insignificant. The number of bundles looks small, yet, 22% of all atomic groups of the organic network participate in bundles. The bundles are mostly composed of 3 SFCs whereas bundles composed of four or five chains, occur rarely .AA-groups play a role.Bundles of SFCs do rarely exist in the beginning of the simulation, and most of them appear during the deformation. The majority of bundles seems to appear in the final phase of the simulation, suggesting a predominantly dynamic nature of the process. The formation of bundles requires apparently certain time, whereby the segmental displacements of The behavior of bundles during simulation was observed. The average size of bundles is constant during the simulation, however, individual chains can fluctuate from one to another bundle. Regarding the geometry of bundles, we first examine the aspects, which are related to the formation of fibrils: rotation and twisting. These aspects were selected since they have different course in the slow and quick mode.In what follows we describe the rotation of the bundle about a rotation axis pointing along the bundle. One end of the bundle is at the center of mass of the chain ends, associated with the bundle, and the same applies to the other end of the bundle. Bundle rotation is then observed when the chain end-groups move with respect to bundle axis see , wherebyWe find significant difference in the average twisting intensity between slow and quick deformation. The twisting is enhanced during the quick deformation, where we observe also increased formation of stable fibrils . TherebyNo fibrils or bundles persist after the “slow” deformation, whereas almost 100% of fibrils persist after the “quick” deformation. The consequences of moderate deformation for the relaxation behavior are generally indicated in We investigated the effect of twisting also in our four-chain model, starting with unstretched chains as an initial configuration. Upon twisting, the potential energy of the four chains was found to decrease whereas when this model was simulated without twisting, the energy remained constant. It was found that the chains can twist only slightly. The most stable atomistic configuration has a twisting angle 2.5°.The model is simplified and therefore there are doubts about the validity of results mainly in relation to behavior of real hyrogels. From the structural aspects, our model resembles a micellar network with aggregates comprising cores, formed by hydrophobic Acrylic Acid (AA)-clusters, intermediate layer from poly-lactide-glycolide and shells, formed by hydrophilic polyethylene-glycol (PEG) chains. Model micelles are composed of two layers: core from AA, and hydrophilic PEG shell.The proposed model provides insight into the relationship between gel structure and deformation response. The model response shows effects, which are observed in real hydrogel materials during deformation: hysteresis during forward- reverse deformation, yield stress, solvent redistribution during deformation . The autocorrelation function identifies whether the interacting Bundles or fibrils  change their axis orientation in space. The time autocorrelation function of bundle orientation was calculated from a 100 ns period with interval of 0.1 ns.The relaxation was analyzed by means of time-autocorrelation functions which were fitted by exponential decay. A characteristic parameter of the exponential function is the half-time of relaxation. Our network reveals several relaxation processes with significantly different relaxation times:For comparison with the derived relaxation times, we recall that the quick deformation takes 10 ns, and the slow one lasts 100 ns, as the most relaxation processes are significantly faster than the deformation. Only the relaxation of physical clusters and bundles appears comparable to the interval of deformation.The relaxation times were calculated for several stretching rates. The fastest stretching rate is 10 × faster than the “quick” deformation considered in the previous section. The range between fastest and slow deformation was divided into seven intervals on a logarithmic scale. A table of all relaxation times is presented in the A summary of the typical relaxation times after the deformation is presented in The next process pertains to the fluctuation of bonds and angles. The relaxation process is observed within the range of hundreds of fs. It describes the reorientation of covalent bonds. The relaxation is driven by thermal motion of molecules as it was observed also in the reference sample without deformation.The conformation relaxation by rotational motion of chain segments occurs within tens of picoseconds. This motion is predominantly driven also by thermal motion, however, the degree of deformation plays a role too.The process of segmental displacements is related to the reconstruction of micellar structure and takes place within the range of nanoseconds. Macroscopically, the micellar structure does not change, but the individual physically interacting atomic group can switch from one to another physical cluster (core of micelle). That relaxation process is unambiguously a response to deformation. We did not observe it in the reference simulation box without deformation.During the longest interval of tens of nanoseconds one observes a reorientation of bundle axes. The reorientation implies rotation of the bundle. In the same time, we observe also a second wave of segmental hops as a means of reconstruction of the physical clusters. We can assume that in that interval, the bundles are stabilized and this affects the physical clusters.We observed a relaxation of bundles at the intermediate stretching rate (0.5× quick) along with temporarily persistent fibers. On the other hand, at the stretching rate 0.2× “quick” the relaxation of bundle orientation cannot be analyzed, because bundles are not stable. As well the recovery of physical network starts immediately after deformation.Bundle axis orientation and the accompanying segmental hops are specific to the fast deformation. They are not observed after the slow simulations or without deformation.The correlation between cluster reconstruction and bundle orientation is presented in Thus, during the relaxation one can observe the differences between quick and slow deformation. During the slow deformation predominantly the physical network undergoes relaxation whereas after the quick deformation one observes a relaxation of the bundles and fibrils.Wi) as a dimensionless quantity, which indicates the typical ratio of elastic and viscous forces in our structure. Wi is calculated from the deformation rate  for selected deformations and relaxation processes. The processes can be subdivided into 3 groups: viscous (v) (Wi < < 1), viscoelastic (ve) (0.5 < Wi < 2), and elastic (e) (Wi >> 1).Wi. The other simulations, when we observe the formation of fibers, show either the viscoelastic reorientation of molecular bundles or viscoelastic reconstruction of physical clusters or micelles.The Weissenberg number clarifies the correlation between the relaxation times and formation of fibers. The simulations, when we observe complete recovery of the structure, contain only the viscous processes according to It has been mentioned in the Introduction that the fibrillar structure of self-assembled natural materials reveals an alternating sequence of stiff and soft blocks of proteins   is also observed in the deformed sample as demonstrated in In this work we present molecular dynamics simulations of a model, which shows the ability to form ordered fibrillar structures on molecular level. A significant result of our study is the finding that the relevant mechanism is sensitive to strain amount and rate. The fibrillar structure is formed only by both quick and large deformation.We identified some factors, which are important for the formation of nanostructured fibrilar material. During deformation, one must achieve synchronized unfolding of macromolecules during deformation. The synchronized unfolding is activated by quick deformation. Another aspect is the ability of the chains to form bundles of aligned macromolecules. Such bundles can be stabilized by twisting and thus transformed into fibrils. Whether the macromolecules can form fibrils depends on the molecular structure. The molecular structure has been investigated here by a simplified four-chains model, which elucidates the conditions necessary for a material of certain composition to form fibrils.The structural transformations are reflected in the relaxation process of the sample. The relaxation after fibril formation is manifested in the reorientation of fibrils and bundles. In the case of slow deformation, where fibrils are not formed, the process of reorientation is not observed.The present model makes it possible to consider new materials, which are able to form fibrillar structure. The primary structure according to our findings can be produced in a laboratory. When one combines an appropriate primary structure and satisfies the conditions of preparation/extrusion, a nanostructured fibrilar material similar to spider silk can be achieved also in a laboratory.All datasets generated for this study are available within the article and from the corresponding author on request.JZ carried out the MD calculations and analyzed the data. JZ, AM, and JJ interpreted the results and developed the theoretical scheme. JZ and AM wrote the paper.JJ was employed by the company SCITEG a.s. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PICK1 is a multi-domain scaffolding protein that is uniquely comprised of both a PDZ domain and a BAR domain. While previous experiments have shown that the PDZ domain and the linker positively regulate the BAR domain and the C-terminus negatively regulates the BAR domain, the details of internal regulation mechanisms are unknown. Molecular dynamics (MD) simulations have been proven to be a useful tool in revealing the intramolecular interactions at atomic-level resolution. PICK1 performs its biological functions in a dimeric form which is extremely computationally demanding to simulate with an all-atom force field. Here, we use coarse-grained MD simulations to expose the key residues and driving forces in the internal regulations of PICK1. While the PDZ and BAR domains do not form a stable complex, our simulations show the PDZ domain preferentially interacting with the concave surface of the BAR domain over other BAR domain regions. Furthermore, our simulations show that the short helix in the linker region can form interactions with the PDZ domain. Our results reveal that the surface of the βB-βC loop, βC strand, and αA-βD loop of the PDZ domain can form a group of hydrophobic interactions surrounding the linker helix. These interactions are driven by hydrophobic forces. In contrast, our simulations reveal a very dynamic C-terminus that most often resides on the convex surface of the BAR domain rather than the previously suspected concave surface. These interactions are driven by a combination of electrostatic and hydrophobic interactions. Protein Interacting with C Kinase-1 (PICK1) is a multi-domain mammalian membrane protein  BAR domain with PDZ domain and linker and (2) BAR domain with C-termini. These truncations are modeled after experimental work .” Users must click the “advanced” button (after selecting “MD” option) located at the top-right of the web page to use this force field. Both systems started from the PDB structures described above with periodic boundary conditions set at 10,000.0 Angtroms. No secondary structure restraints were applied. Distance restraints were manually added to the input files generated by the UNRES server to maintain the structure of the BAR and PDZ domains but not the linker or the C-terminus. It should be noted that the UNRES server does not include keywords to add distance restraints. Since PICK1 is much larger than the proteins used to parametrize the force field, a higher temperature (350K) was used for all canonical MD simulations of the two systems simulated. It should be noted that the 350K used here does not directly correspond to 350K in a biological system. Rather, a temperature of 350K is used to estimate a temperature between 300K and 350K in a biological system based on the evaluation of the previous work  were generated using the UNRES server at 0 155104 .” Users Root mean square deviation (RMSD) and radius of gyration (Rg) analysis were performed to quantify the flexibility of the dynamic system. Frequency refers to the proportion of frames with the given distance. For each of the two systems, contact maps were used to reveal the major interactions between any pair of residues. Contact was defined as any two Cα atoms at least five residues apart with a distance separation of 8Å or less. In System 1, the PICK1 complex is in the proposed inactivated state as the protein was neither in complex with ligand nor in proximity with the lipid membrane. As expected for inactivated PICK1, the PDZ domains formed contact with a wide range of residues located on the concave face and side surface of the BAR domains, as seen in Detailed residue-residue interaction analysis revealed that the short helical portion of the linker region forms significant interactions with the PDZ domain. Key residues of PDZ-linker interaction were elucidated by identifying the most prevalent contacts, in this case, forming a contact in 13% of the frames. Ten key interaction pairs were identified between the PDZ domain and the linker, as shown in These ten key pairs are hydrophobic interactions between the βB-βC loop, βC strand, and αA-βD loop of the PDZ domain and the short helix fragment of the linker region, as shown in The interaction pattern between the PDZ and the BAR domains is quite different than the interactions between the PDZ domain and the linker. The top ten contact residue pairs are shown in To identify the preferred regions on the dimeric BAR domain which interact with the PDZ domains, cluster analysis revealed the five most probable positions of the PDZ domains in space. While cluster analysis reveals the most probable positions of the PDZ domain in respect to the BAR domain, RMSD and radius of gyration analysis reveal that the system has widely dynamic movements. In efforts to capture this range of motion and make a direct comparison to data reported by previous experiments  and radius of gyration (Rg) analysis was performed to quantify the flexibility of System 2 (the BAR domain with the C-termini), as shown in The previous literature  of the BAR domain but rather include neighboring residues. Surprisingly, the short helical fragment in the linker can form extensive interactions with the PDZ domain, potentially outcompeting the BAR domain. The biological function of the helical fragment may be more than just help to align to the BAR domain on the lipid membrane.The interaction pairs demonstrate the significance of the βB-βC loop  of the PDZ domain in initiating PDZ-BAR and PDZ-linker contact. Previous structural prediction via small-angle X-ray scattering (SAXS) analysis was unable to determine the orientation of the PDZ domain in PDZ-BAR interactions, but made the prediction that the βB-βC loop of the PDZ domain would orient toward the concave surface of the BAR domain (Madasu et al., Previous experimental results (Jin et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AS performed data analysis, carried out simulations, and wrote the manuscript. YH designed research strategy, performed data analysis, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The research and development of modern metallic materials goes hand in hand with increasing their lifetime via optimized deformation processing. The presented work deals with preparation of an Al/Cu clad composite with implemented reinforcing Cu wires by the method of twist channel angular pressing (TCAP). Single and double pass extrusion of the clad composite was simulated numerically and carried out experimentally. This work is unique as no such study has been presented so far. Detailed monitoring of the deformation behavior during both the passes was enabled by superimposed grids and sensors. Both the sets of results revealed that already the single pass imparted significant effective strain ), especially to the Al matrix, and resulted in notable deformation strengthening of both the Al and Cu composite components, which was confirmed by the increased punch load and decreased plastic flow velocity (second pass compared to first pass). Processing via the second pass also resulted in homogenization of the imposed strain and residual stress across the composite cross-section. However, the investigated parameters featured slight variations in dependence on the monitored location across the cross-section. As regards to intensive plastic deformation processing, the main focus has been on conventional Fe-based materials, although non-ferrous metals are given attention, too, primarily due to their wide applicability in various industrial branches. However, based on the increasing demands of the industry, the everlasting research of the forming processes increases their applicability also for innovative and newly developed materials. Nevertheless, the aims of the processing are similar regardless of the used material: the materials subjected to intensive plastic deformation are prepared to meet high demands, including the one on having ultra-fine-grained structures (UFG).m). In other words, the high deformation strengthening rate results in accumulation of dislocations inside the grains, which, in combination with high strain rate sensitivity coefficient m, significantly suppresses the development of material failure during deformation processing and thus increases plasticity of the material during forming. Nevertheless, ways how to achieve combinations of high strength and high plasticity exist; such material behavior is characterized as the strength and ductility paradox [Among the preferred methods used to prepare UFG structures within (non)ferrous metals and alloys are severe plastic deformation (SPD) methods, such as equal channel angular pressing (ECAP) ,2, twist paradox . The met paradox ,10. This paradox .One of the alternative approaches for achieving favorable combinations of mechanic, electric, or magnetic properties is also preparation of composite materials, among the individual types of which are clad composites ,13. VariThe methods based on deformation processing, which have so far been applied to prepare clad composites, are e.g., rolling and related methods such as accumulative roll bonding  and asymThe presented report comprises a detailed study characterizing the deformation behavior of an Al-Cu clad composite during the TCAP process, which enables to impose higher values of effective strain during each pass when compared to conventional ECAP . TheThe aim of the work was to provide a detailed characterization of the effects of TCAP (twist channel angular pressing) processing on the Al/Cu clad composite .Bc. The deformation route was based on the Bc deformation route known from conventional ECAP, i.e., the composite billet was subjected to +90° rotation before the second pass [Since the work is unique, as this method has not been used before to process any composite material, the study does not only involve the experimental work, but also numerical analysis. The composite billet consisted of Al sheath and reinforcing Cu wires , and wasond pass ,36. Howe2 as the lubricant, at the room temperature of approx. 25 °C and extrusion rate of 5 mm s−1. The friction was in the simulation determined by the Coulomb friction with the values of µ = 0.02. The values of the mentioned boundary conditions were selected based on the results previously acquired within the study mutual comparison of predicted and experimentally acquired values in which was successfully performed [ω = 90° (twist rotation angle), φ = 90° (channel bending angle), β = 40° (twist slope angle), ψ = 20° (angle of the arc of curvature in which the two channels intersect)  .The second part of the study deals with practical realization of the multiple TCAP process. The selected materials were commercially pure Cu (99.97%) with the chemical composition of 0.0074 Ni, 0.0058Sn, 0.0030Zn, 0.0031Fe, 0.0023Si, bal. Cu (in wt.%), and commercially pure Al (99.97%) with the chemical composition of 0.125Fe, 0.10Si, 0.020Cu, 0.020Zn, 0.015Mg, 0.015Mn, 0.015Ti, bal. Al (in wt.%). Before completing the composite, both the component metals were pre-annealed in a furnace at 500 °C for 30 min. The dimensions of the samples prepared for extrusion were matching the parameters given in the simulation, i.e., square 12 mm × 12 mm cross-section and 130 mm length.The structure analyses of the extruded clad composite samples were performed via the electron backscatter diffraction (EBSD) method (scanning electron microscopy (SEM)). Preparations of the samples for the analyses were carried out by grinding on SiC papers and final electrolytic polishing. SEM investigations were done using a Tescan Lyra 3 FIB/SEM microscope equipped with a NordlysNano EBSD camera . The EBSD scans were acquired on samples tilted by 70° with the steps of 50 nm and the accelerating voltage of 20 kV. To enable comparison with the predicted results, the presence of residual stress in the structures of the extruded billets was evaluated by analyzing the internal grains misorientations in the rainbow color scheme in the scale from 0° , to 15° . Overall structure scan of the cross-section of the final extruded billet was performed on the polished sample using the OLYMPUS DSX1000 digital microscope .Last but not least, the mechanical properties of the composites were investigated via microhardness measurement performed on transversal cross-sectional cuts using a Zwick/Roell testing machine . The applied load was 200 g, and the loading time per indent was 10 s.−1.The deformation behavior of both the component metals was predicted with the use of an elastic-plastic model defined via the Newton–Raphson convergent algorithm. The mesh of the clad composite billet consisted of 215,871 nodes. The billet was meshed with tetrahedral elements, whereas both the extruder and die were considered as rigid parts. Since severe shear deformations were expected to occur during the simulation, automatic re-meshing was activated. The stress-strain curves, depicted in ε is the equivalent von Mises strain, T is the temperature, A, m1, m2, m3, m4, m5, m6, m7, m8, and m9 are regression coefficients. The values of the individual coefficients for Cu are, respectively, 411.19 MPa, −0.00121, 0.21554, 0.01472, −0.00935, and m5 ÷ m8 are 0. The values of the individual coefficients for Al are, respectively, 151.323 MPa, −0.00253, 0.21142, 0.03177, −0.00654, m5÷m8 are 0.The experimentally acquired stress-strain data were entered into the material flow stress database of the computational software. The Haensel–Spittel equation (Equation (1)) was then used to characterize material behavior during deformation processing:−5 (K−1), 250 (W/(mK)), 1230 (J/kgK) 0.03 and 2800 (kg/m3), and for Cu as 111 (GPa), 0.3, 1.7 × 10−5 (K−1), 394 (W/(m K)), 100,000 (W/m2 K), 398 (J/kg K), 0.7, and 8960 (kg/m3).The boundary conditions defined in the simulation were the temperature of 25 °C, and the values of parameters describing the temperature behavior of aluminum and copper, i.e., Young’s modulus, Poisson’s ratio, thermal expansion, thermal conductivity, heat transfer coefficient, specific heat, emissivity, and density. The parameters were defined for Al as the constants of 72 (GPa), 0.3, 2.4 × 10In order to provide more specific characterization of the material plastic flow of both the composite components, a monitoring grid was superimposed through the billet being extruded, perpendicularly to the longitudinal axis of the composite b. The deAs documented by the predicted results, the component metals exhibited differences in their behaviors during plastic deformation. The imposed strain is an important factor, the distribution of which non-negligibly influences also the distribution of temperature. However, for the Al/Cu clad composite, the temperature development was affected not only by the particular component metal, but also by the localization of the individual monitoring sensor in the deformation zones .The maximum value of temperature was detected in the main deformation zone (MDZ) for both the passes  of the die affected primarily the corner areas of the extruded composite a surface, but also the axial region of the extruded composite a.The effects of the TDZ were evident also on the Al/Cu interfaces, the superimposed grids at which exhibited evident serrations originating from rotations of the clad composite. Moreover, the original monitoring plane exhibited deformation in the extrusion direction. In other words, the deformations of the individual cells of the superimposed grid were not only caused by the tangential plastic flow, but also by its axial component which became dominant after passing through the MDZ a. The prThe cells of the superimposed grid, heavily deformed during the first pass, were subsequently deformed even more during the second pass. The effect of the previous deformation strengthening contributed to relatively uniform deformation of the entire superimposed grid; this was observed also for the Cu wires, the plastic flow of which was supported by the substantial plastic deformation introduced to the Al sheath. The second pass also resulted in full bonding of both the components, as can be seen in the optical microscopy scan of a cross-sectional cut through the extruded billet in Bc, which contributed to substantial reduction of the gradient of the imposed strain across the Cu wires cross-sections. This was also confirmed by the plastic behavior of the superimposed grid, the typical skewness of the cells of which reduced after passing through the MDZ (when compared to the first pass), as well as by the overall velocities of the plastic flow in the individual monitored sensors depicted in The TDZ introduced rotations also during the second pass. However, during this pass, the rotations affected more intensively the axial region of the composite. Overall rotation of the cross-section of the billet, by 180°, was introduced by the selected deformation route The graphical dependencies in The effects of both the TCAP passes on the composite components were different from the viewpoint of residual stress. The composite exhibited prevailing tensile stress during the first pass though the TDZ. This phenomenon can be attributed to two main factors, the first one of which was the rotational movement of the Al sheath, the intensity of which decreased towards the axial region of the billet. The second factor was the dominant plastic flow occurring in the axial region of the composite see a. As theThe stress state within the composite changed after passing through the channel bending, i.e., the MDZ. However, the stress character changed gradually from dominant tensile stress to dominant compressive stress during passing through the channel zone between the TDZ and MDZ due to the increasing resistance against the plastic flow caused by gradual filling of the MDZ  with the composite material. Behind the MDZ, the stress character changed again as axial differences in the plastic flows in different composite regions started to be notable. As evident from The development of residual stress was different during the second pass. The Cu wires featured local presence of tensile stress before entering the MDZ also during the second pass. On the other hand, the Al sheath exhibited predominant occurrence of compressive stress from the very beginning of extrusion. With continuing filling of the MDZ, the tensile stress within the Cu wires gradually transformed to compressive stress b; the grExperimental verification of the predicted data was performed via analyses of internal grains misorientations within both the components of the billet extruded via two consequent TCAP passes. The results of the analyses for the peripheral Cu wire of the billets extruded via single and double TCAP pass are depicted in As can be seen, both the figures feature more or less homogeneous distribution of the areas featuring high misorientations , pointing to the presence of residual stress. However, the occurrence of residual stress was more notable in the structure of the Cu wire from the composite extruded via two TCAP passes, which is in accordance to the predicted results a,b. In aThe maximum loading force during the first pass reached to 40 kN . WhereasThe development of the punch load during the second TCAP pass featured even steeper increasing character from the very beginning of extrusion. Such behavior was imparted by two main influencing factors, both of which affected the development of the punch load during the entire pass. The first factor was the accumulation of deformation strengthening within both the component metals during the previous pass. In addition, the strain imposed during the first TCAP pass resulted in the changes of shapes of the ends of the inserted Cu wires (not shown here), which was the second influencing factor. In the moment in which the billet entered the twist deformation zone, the previous deformation strengthening accumulated in the Al sheath, but primarily in the Cu wires, resulted in the increase in the punch load by up to 50% (when compared to the first pass).Bc exhibited the highest (maximum observed) HV values.The already mentioned deformation strengthening, contributing also to the notable increase in the punch load during the second pass, was experimentally observed via microhardness measurements carried out along the diagonals across the cross-sectional cuts through both the composite billets b. The inThe study featuring processing of the Al/Cu clad composite via the TCAP method revealed variations when compared to processing of single-component billets. Firstly, the deformation behavior of the investigated composite was influenced by the combination of the characteristic properties of both the component metals. For example, the comparison of the presented results with the results acquired during studies dealing with processing of single Al , or Cu , shows dThe deformation behavior, especially the effective imposed strain and plastic flow, of the composite was affected not only by the individual component metals, but also by the deformation zones within the die and the selected deformation route. Although the effects of the individual deformation zones on the values of the effective imposed strain are not equal, their mutual effect, which makes TCAP an advantageous method for processing of multicomponent materials, is non-negligible. Passing of the composite through the TDZ did not introduce as high strain as passing through the MDZ. The MDZ primarily influenced the axial region of the extruded billet, whereas in the TDZ, the highest effective strain values were detected in the (sub)surface regions of the Al sheath. The inter-regions and the axial region of the billet were more or less affected by the imposed strain in the TDZ, too, but the values of the imposed strain were significantly lower within the Cu wires. However, as the result of the variations in the velocities of the axial plastic flow of both the materials in the TDZ, the overall gradients in the observed parameters between the (sub)surface and axial billet regions were more or less reduced. In other words, among the main contributions of the TDZ is that it diminishes the differences between the axial and (sub)surface regions of the extruded composite. Therefore, the TDZ primarily contributes to homogenization of the imposed strain across the cross-section of the clad composite .During TCAP, the locations of the individual Cu wires with respect to the die channel vary, which is not possible for conventional ECAP . By thisThe time dependence of the punch load featured notable oscillations for both the passes, however, the oscillations were more intense during the second pass see a.Such serrated development is typically related to plastic flow instability ,35, whicThe presented study documented the results of numerical and experimental analysis of successful room-temperature preparation of an Al/Cu clad composite by single and double pass of twist channel angular pressing (TCAP). Both the TCAP passes introduced significant shear strain to the component metals, as the maximum effective imposed strain reached up to the value of 5 for the Al sheath of the billet extruded by two passes. The imposed strain contributed to deformation strengthening resulting not only in the increase in punch load by almost 50% and increase in microhardness up to 130 HV for the Cu wires, but also in temperature increase during processing. Nevertheless, the processing temperature was still only ~37 °C during the second pass, i.e., the processing conditions were safe from the viewpoint of introduction of possible structure changes. The results also showed that the processing via multiple passes increased the bonding quality of both the metals, as both the distributions of the imposed strain and residual stress homogenized after the second pass, which was ensured by the favorable combination of twist and bending deformation zones within the single unique die.


Strychnos madagascariensis Poir is an underutilized fruit that is considered a valuable food during droughts and famine. The aim of this research was to characterize the nutritional composition and the flour functional properties, for the use as a potential food source. Seed flour was analysed using a standard enzymatic assay for sugars, acid/neutral detergent analysis for fibre, ether extraction for fat and HPLC for strychnine. Results showed that the seeds contained 41% reducing sugars and 53% fibre. The mineral composition, determined using microwave-assisted acid digestion and inductively coupled plasma optical emission spectrometry (ICP–OES), showed that the seeds contained high quantities of iron (15.78 mg/100 g) and manganese (9.86 mg/100 g). The flour water absorption index (1.37 g/g) was substantially higher than that of wheat, brown rice and tapioca flours and the oil absorption index showed similarities to the reference flours (1.09 g/g). The flour peak  and final viscosities  were significantly (p < 0.001) higher than the reference flours. This study was the first to quantify the strychnine content (0.08%) in the seeds. Results suggest that the seeds have good potential for food product development; however, further processing is essential to ensure safety for consumption. Many developing countries are faced with the problem of food insecurity, which is threatening the quality of life of inhabitants. This issue is also on the rise as the world population is rapidly increasing, placing a higher demand on the currently available food staples and imported food goods . Thus, nCoupled with the threat of climate change and a drastic reduction in rainfall (particularly in southern Africa), agricultural food production has been greatly affected, thus worsening the food security status and increasing the dependence on imported staple foods. It has been proposed that the use of underutilized indigenous food crops may improve food production as certain plants are well adapted to the environment and require substantially low amounts of water, low agronomical inputs, as well as other characteristics that allow them to adapt to current climate changes. Moreover, deviation from the current agricultural mono-cropping systems and the introduction of a larger variety of underutilized crops may contribute greatly in the adoption of agro-biodiversity, which benefits the environment .Strychnos (Monkey orange) as a new food crop due to specific species within this genus. These species are known for their production of edible fruits and high drought tolerance, making them suitable for arid regions [S. madagascariensis Poir (commonly known as black monkey orange) belongs to the family Loganiaceae. It is a native southern African tree, which is distributed in Limpopo, North-West, Mpumalanga, Eswatini, KwaZulu-Natal and Botswana. The tree produces high fruit yields, containing numerous large translucent seeds. According to Shaffer [S. madagascariensis is consumed (ranging between bitter to very sweet in taste) as it is believed that the seeds may contain the toxic alkaloid, strychnine. However, there has been contradictory claims surrounding the toxicity of the seeds as Govender [A rising interest has developed in South Africa, around the genus  regions . Particu Shaffer , only thGovender  reportedGovender . Thus, tS. madagascariensis were obtained from the Agricultural Research Council (ARC) in Nelspruit, South Africa . The seeds were removed from the fruit pulp and freeze-dried for five days. The seeds were then milled and further ground using a coffee grinder . The ground material was then passed through a 250 µm sieve and stored in airtight zip-lock plastic bags. Reference flours were purchased at local supermarkets: uncooked, gluten-enriched wheat flour , long-grain gluten free brown rice flour , dehulled split chickpea flour , coconut flour  and tapioca flour .Ripe (orange) fruits of The moisture content of the flour samples was determined using the MLB 50-3N moisture meter . The total fibre content of the seeds was calculated using the acid detergent fibre (ADF) fraction and the neutral detergent fibre (NDF) fraction. These components were determined using the methods set out by Goering and Van Soest , in whicThe mineral composition  was determined according to Santos , with slStrychnine was quantified using high performance liquid chromatography (HPLC), UV detection (254 nm), 25 cm × 4.2 mm ID column  and the methods outlined in the NIOSH  Manual of Analytical Methods (NMAM) . Strychn®-5 ms column (30 m × 0.25 mm ID × 0.25 μm) . 1 µL was injected in splitless mode with helium as the carrier gas. The initial oven temperature was set at 70 °C, held for 0.5 min, ramped at 10 °C/min to 150 °C, held for 2 min, ramped at 10 °C/min to 330 °C and held for 3 min. Subsequent data obtained were processed by evaluating each of the three replicates and selecting only the compounds found within each replicate. Once this was done, the common compounds found within S. madagascariensis seed flour were compared to those of the reference flours to construct a table of comparison.Sample preparation for metabolite profiling was done according to the methods set out by Adebo  with slig for 10 min at 25 °C. The supernatant was then discarded, and the pellet was weighed. The WAI and OAI were calculated using the formula:The water absorption index (WAI) and the oil absorption index (OAI) of the flour samples were determined using the methods set out by Julianti . ApproxiS. madagascariensis seed flour and the reference flours were determined using the methods of Yu [g for 15 min. The supernatant was decanted into its respective pre-weighed glass petri dishes and evaporated at 105 °C, for 24 h. They were allowed to cool to room temperature and the resulting pellets weighed. Solubility and swelling power were calculated using Equations (3) and (4), respectively:The swelling power (SP) and the solubility (S) of ds of Yu  with min−1 using a resolution of 4 cm−1.FTIR spectroscopy has gained popularity in the food industry as it is a rapid and non-destructive technique for the analysis of food compositions. This technique is simple and requires little to no sample preparation as samples may be analysed in their original state . The IR w/w, flour in dH2O). The suspensions were then equilibrated for 1 min at 50 °C and stirred for 30 s at 960 rpm. The stirring speed was then reduced to 160 rpm and the temperature was increased to 91 °C at a rate of 11.08 °C/min. Once the samples had reached the desired temperature, they were held at that temperature for a further 2.5 min. The temperature was then decreased to 50 °C and held for 1 min, after which the critical points  were determined.The pasting properties of the test and reference flours were determined using a rapid visco analyser (RVA)  following the methods of Kongolo . SuspensThe gel texture properties of the test and reference flours were analysed using a TA.XT Plus Texture Analyzer  and a 2 mm cylinder probe. The pastes produced in The colour properties of the test and reference flours were determined using the tristimulus Commission Internationale de l’Eclairage’s (CIELAB) scale, using a CR-10 chromameter . The CIELAB values L * (white = 100 and black = 0), a * (positive = red and negative = green) and b * (positive = yellow and negative = blue) were recorded. The chroma, yellowness and flour colour index (FCI) were calculated using Equations (6)–(8), respectively:Except for FTIR, all other analyses were done in triplicate. Genstat for Windows 19th Edition  was used for statistical analyses. A one-way analysis of variance (ANOVA) and Tukey’s post hoc test was performed to differentiate between the means at the 95% probability level.S. madagascariensis seeds is far greater than that of dried broad beans (22.9%), a popular fibre-rich food [S. madagascariensis seeds for the possible alleviation of mineral deficiencies. However, further investigation is required to quantify the amount of prebiotic dietary fibre present in the seeds. S. madagascariensis seeds contain moderate amounts of energy , which is substantially lower than that of commercial flours , wheat (Ca = 14 mg/100 g and Fe = 1.2 mg/100 g) and brown rice flour (Ca = 11 mg/100 g and Fe = 2 mg/100 g). The K content was found to be substantially higher than that reported for peeled bananas (241 mg/100 g) wheat flour (105 mg/100 g) and brown rice flour (289 mg/100 g), while the Mn content greatly exceeded that found within wheat (0.63 mg/100 g) and brown rice flour (4 mg/100 g). The seeds also contained Mg (79.25 mg), P (94.20 mg), Na (16.45 mg), Zn (1.36 mg) and Cu (0.72 mg). The rich mineral composition thus suggests that the use of the seed of S. madagascariensis may have the potential in alleviating some mineral deficiencies associated with developing/underdeveloped countries. Moreover, Fe deficiency is considered to be the most predominant micronutrient deficiency across the world [When comparing the micronutrient composition  of the she world . In southe world .Strychnos are considered to be toxic due to the presence of strychnine. The strychnine content of S. madagascariensis seeds (0.08%) was found to be substantially lower than that of S. nux-vomica (~1.1%) [Strychnos nux-vomica is the main member of the genus that has made it popular—it is cultivated in many parts of the world and is a medicinally important plant, specifically in Indian and Chinese remedies. Although the seeds are the main target, the leaves, bark and fruit are utilized for herbal remedies as well after they have been processed (boiled or cooked) in order to reduce the quantity of strychnine. These techniques might not be effective in reliably reducing the strychnine content [S. nux-vomica are used for an array of therapeutic and clinical applications, which include increasing appetite, reducing fevers, and being used as an aphrodisiac and a tonic [S. nux-vomica significantly improved the medicinal efficiency and greatly reduced the toxicity, in comparison to the unprocessed seed powder. Another study conducted by Choi et al. [S. nux-vomica seeds, and reported an effective reduction in the strychnine content, among other alkaloids. These studies, although developed for the production of medicine and herbal remedies, reveal that strychnine may be substantially reduced. Further research is required to develop methods suitable for food product development, to efficiently eliminate the strychnine content within S. madagascariensis seeds and ensure safety for consumption.Many species within the genus  (~1.1%) . However content ,30, nor  a tonic . Further a tonic  revealedi et al.  evaluateS. madagascariensis seed flour. The seed flour shared some compounds with the reference flours; however, the majority of the compounds identified were unique to S. madagascariensis. The compounds shared between the seed powder and reference flours include pyrazole, vinyl acrylate, ethylene acetal and methoxy-phenyl-oxime. To ensure that the seed flour of S. madagascariensis is safe for consumption and food product development, further analysis is essential to quantify the compounds listed in S. madagascariensis seed flour were observed through evaluating their WAI, OAI, solubility and swelling power. The WAI of flours has direct implications on its use in food products; typically, flours with high WAI are sought after for food products requiring high viscosities such as baked products, soups and gravies [S. madagascariensis seed flour and the reference flours are indicated in S. madagascariensis seed flour was found to be significantly different (p < 0.001) to the reference flours used. However, it was observed to be substantially high , showing the second highest WAI just below that of coconut flour, thus indicating the seed flours potential as a thickening agent. The WAI is of particular importance during the processing of the flour, as it has direct effects on its pasting properties and represents the ability of a flour to associate with ‘under-water’ conditions  where such interactions are limited. The OAI of the seed flour showed similarities to brown rice flour, chickpea flour and tapioca flour. However, the OAI was substantially lower than that of wheat and coconut flour. Owing to the lack of starch in the seed flour, it is assumed that the WAI and OAI are attributed to the high fibre and sugar content of the flour, thus indicating the presence of cellulose and sucrose as these components are known for their water-holding capacities [The absorption properties of  gravies , while t gravies . The WAIpacities .S. madagascariensis seed flour compared to those of reference flours. Water solubility is an indication of the soluble solid content of the flour, thus indicating the degree of starch degradation in relation to the level of maturity of the produce [p < 0.001) high percentage of solubility across all the temperatures in comparison to the reference flours. Additionally, the seed flour showed a gradual increase in solubility with an increase in temperature, with a peak solubility at 100 °C. The SP of the flours is an indication of its water absorption properties [S. madagascariensis showed significant difference (p < 0.05) within this temperature range (60–100 °C). This is possibly due to the lack of starch granules within the seed flour, which hinder the increase in swelling. A similar pattern was observed for coconut flour, where no substantial increase in swelling occurred. The remaining reference flours  all showed a gradual increase in SP with an increase in temperature. However, the overall swelling capacity of S. madagascariensis seed flour is similar to the maximum SP of the reference samples; suggesting that the seed flour possesses similar water absorption characteristics. produce . The seeoperties . The seeS. madagascariensis seed were analysed using FTIR and were compared to those of the reference flours, as depicted in the FTIR spectra below  within the same regions as the reference flours. These regions include N–H (3350–3310 cm−1), O–H (3000 cm−1), C = C (1650–1600 cm−1), N–O (1550–1500 cm−1), C–O (1150 cm−1) stretching and O–H (1440–1395 cm−1) and C = C (995–985 cm−1) bending groups . The major peaks were identified through the use of IR spectroscopy correlation tables [−1 and 3300 cm−1 are indicative of the moisture content as the water molecules possess a strong absorption within this range, due to the O–H stretching and H bending vibrations [S. madagascariensis flour showed differences to most of the reference flours (excluding coconut flour) within the C–O stretching regions, of 1085–1050 cm−1 (arrow 1) and 1150–1085 cm−1 (arrow 2). These regions represent the primary alcohol group and the aliphatic ether group [S. madagascariensis showed similarities to those of the commercial flours, suggesting that it may be used in similar food products.Flour properties of ra below A,B. The n tables . Regionsbrations . S. madaer group . The FTIS. madagascariensis seed flour to those of the reference flours used. The dashes in the gel texture column indicate data that could not be used, due to instrumental errors that occurred during the analysis. Flour pasting is defined as the process post-gelatinization, which includes the swelling and disruption of starch grains, and the release of molecular components. The presence of large amounts of fibrous material may hinder swelling and affect the viscosity of the sample during heating [S. madagascariensis seed flour showed the second highest peak viscosity (3149 RVU) and the highest through the final and setback viscosities, as well as a relatively low breakdown viscosity. This was surprising as the flour had 0% starch  than that of the reference samples, contradicting expectations as it possesses a high peak and final viscosities. This indicates that the seed flour gel regressed much more easily than that of the reference flours. This could be due to the lack of starch, which is essential in maintaining the gels’ shape.Gel texture is a significant factor in food product development, as it indicates the ability of the gel to retain its structure and moisture content post processing . The gelS. madagascariensis was done according to the CIELAB scale and compared to that of the reference samples (p < 0.05) to the reference flours used. The seed flour showed a higher degree of redness (a * = +0.9) and yellowness (b * = +12.7) compared to the reference samples, except for chickpea flour which had a yellowness value of +19.5, which may be due to the presence of the fruit pulp that was not completely removed from all of the seeds. Moreover, when comparing the colour of the pastes obtained from RVA , which exceeds those of some commonly consumed products. The fibre content is of importance as this fraction may contribute greatly to the functioning of the gastrointestinal tract, while the high iron content may prove to be beneficial in combating iron deficiencies in South Africa. Although the seeds of S. madagascariensis hold valuable nutritional potential, it is crucial to develop food processing methods to reduce the strychnine content prior to their use in the food industry. Further research should be directed towards quantifying other phytochemicals, which might present anti-nutritional properties. The functional properties of S. madagascariensis seed flour may be beneficial in food product development as it possesses a high water absorption capacity, as indicated by its water absorption index and swelling power. Moreover, its high peak and final viscosities suggest that it may be used as a thickening agent in sauces or soups. The colour properties may be a hindrance, as the flour and the paste are substantially darker than the popular commercial flours. However, the consumer acceptability of varying flour colours is currently increasing. The commercialization of this underutilized fruit may also benefit the surrounding rural communities, as it provides entrepreneurial opportunities for the development of new food sources and the cultivation of this species. However, it is essential to rule out any toxicity hazard of the seeds’ prior use in the food industry.The seeds of


The mechanisms responsible for the trafficking of carboxylate ions across cell membranes are becoming clearer. Related research article Sauer DB, Trebesch N, Marden JJ, Cocco N, Song J, Koide A, Koide S, Tajkhorshid E, Wang DN. 2020. Structural basis for the reaction cycle of DASS dicarboxylate transporters. eLife9:e61350. doi: 10.7554/eLife.61350Small carboxylate ions such as citrate and succinate are intermediates in the citric acid cycle, which is a crucial metabolic pathway in aerobic organisms. However, small carboxylate ions also have many other roles: for example, they function as signaling molecules in processes ranging from DNA transcription and replication  to heat Cells rely on transmembrane proteins belonging to the DASS family  to move small carboxylate ions into and out of cells. There are two clades in the DASS family: cotransporters that import carboxylate ions from the bloodstream into cells , and antVibrio cholerae – had been determined  sliding up and down the scaffold domain between an inward-facing state and an outward-facing state . HoweverLactobacillus acidophilus. Remarkably, they were able to obtain structures for the previously elusive outward-facing state for both. Moreover, they determined the structures when a carboxylate ion was bound to the transport domain and also for the substrate-free case. Relative to the inward-facing state, the transport domain in the outward-facing state is rotated by an angle of 37° and is about 13 Å further away from the inside of the cell  to a final state in which the succinate ions were inside the cell and the antiporter was in an inward-facing state: the researchers used an approximate structure for the final state as the actual structure for the inward-facing state in LaINDY has not been determined yet. Jointly, the experiments and simulations lend strong support to the elevator mechanism.One may ask: how does the cotransporter or antiporter make sure that the carboxylate ion has been loaded into the elevator before the button is pressed? The experimental structures suggest that a 'passport check' is enforced via electrostatic effects. In the absence of the carboxylate ion, the binding site on the transport domain has a positive net charge, which makes it difficult for this domain to pass through the membrane, because charged particles prefer polar environments (such as aqueous solutions) to the nonpolar environments found in the membrane. Loading the negative carboxylate ion onto the binding site neutralizes the postive charge, allowing the transport domain to cruise through. This mechanism limits a form of unproductive transport called slippage  in both cotransporters and antiporters.The work of Sauer et al. also highlights interesting differences between the cotransporter and the antiporter. For the VcINDY cotransporter the positive charge on the transport domain is provided by sodium ions, and the binding of the carboxylate ion to the transport domain leads to large changes in conformation. It also appears that the relatively weak initial binding of sodium ions in the cotransporter is strengthened by the arrival of the carboxylate ion. In contrast, the positive charges in the LaINDY antiporter are provided by amino acid residues rather than sodium ions, and the conformational changes caused by binding appeared to be small and local.It would be interesting to explore the effects of introducing mutations to the residues at the sites of the sodium ions in VcINDY cotransporters or the equivalent sites in LaINDY antiporters. Would it be possible to engineer a sodium-independent cotransporter? And could a cotransporter be converted into an antiporter, and vice versa? Answers to these questions will lead to a deeper understanding of the structure-function relationships for proteins belonging to the DASS family.