Families of related proteins and their different functions may be described systematically using common classifications and ontologies such as Pfam and GO (Gene Ontology), for example. However, many proteins consist of multiple domains, and each domain, or some combination of domains, can be responsible for a particular molecular function. Therefore, identifying which domains should be associated with a specific function is a non-trivial task.We describe a general approach for the computational discovery of associations between different sets of annotations by formalising the problem as a bipartite graph enrichment problem in the setting of a tripartite graph. We call this approach “CODAC” . As one application of this approach, we describe “GODomainMiner” for associating GO terms with protein domains. We used GODomainMiner to predict GO-domain associations between each of the 3 GO ontology namespaces  and the Pfam, CATH, and SCOP domain classifications. Overall, GODomainMiner yields average enrichments of 15-, 41- and 25-fold GO-domain associations compared to the existing GO annotations in these 3 domain classifications, respectively.These associations could potentially be used to annotate many of the protein chains in the Protein Databank and protein sequences in UniProt whose domain composition is known but which currently lack GO annotation.The online version of this article (10.1186/s12859-018-2380-2) contains supplementary material, which is available to authorized users. Proteins are macromolecules which carry out many biological functions in living organisms. At the molecular level, protein functions are often performed by highly conserved structural regions identified from sequence or structure alignments, which may be classified into families of domains. Because many protein domains fold into characteristic three-dimensional 3D) structures, there is often a close relationship between protein structure and protein function . CurrentD structuMore generally, there are many millions of protein sequences that currently lack GO annotations. On the other hand, only a relatively small number of distinct protein domain families exist, which are re-used and combined in different ways in different proteins. Indeed, compared to the vast number of different sequences that exist, current domain classifications contain of the order of only 15,000 distinct protein domain families. Therefore, it is natural to suppose that if known protein structure and sequence annotations could be assigned GO terms at the domain level, many of these annotations could be transferred to a potentially very large number of unannotated proteins. However, we emphasize here that our aim is to discover functional annotations for protein domains themselves rather than entire protein sequences, in order to improve domain description and classification by combining structural and functional features. Nonetheless, even the task of associating GO terms with protein domains is a non-trivial problem because, except for single-domain proteins where the mapping is obvious, many different kinds of relationships can occur  numbers to Pfam domains . Becausek-partite graph is a graph whose vertices can be partitioned into k disjoint subsets, such that in each subset no two vertices are connected. If k=2, the graph is called a bipartite graph (or bigraph), and if k=3 it is called a tripartite graph. The CODAC approach is designed to solve problems of bipartite graph enrichment within a tripartite graph framework. The main intuition is to calculate new weighted edges between two sets of items which already contain reliable but sparse associations, and which are indirectly connected through common associations with a third set of items.In graph theory, a X, Y and Z are 3 sets of items and Eis the set of all edges connecting X, Y and Z in the input configuration. Let us consider 3 bipartite subgraphs of X and items of Y in order to generate E∗ denotes an enriched set of edges. New edges may be discovered by exploiting the existing edge distributions in xi of X and yj of Y share the same  set of neighbours {zk} in Z, then it may be supposed that an edge might exist between xi and yj. Figure x2 and y2 because these items are associated with the same subset of items { z1, z3, z4} from Z. Candidate edges found in this way are then scored and filtered, as described in more detail below.Let entclass1pt{minimaX), a set of Pfam domains (Y), and a set of UniProtKB/SwissProt sequences (Z). E1 is the set of edges derived from the MF GO annotation of UniProtKB/SwissProt sequences, E2 is the set of edges derived from the domain contents of UniProtKB/SwissProt sequences, and It is now possible to instantiate our model with a set of MF GO terms  and M2 (dimension |Y|×|Z|), derived from U1 and U2. Each element of the matrix x of X and an item y of Y, according to the number of common associations with the items in Z.Given a tripartite graph PredictAssociations determines a similarity threshold T for filtering the raw scores in C to produce C∗. The matrix C∗ can be interpreted as the weighted biadjacency matrix of the enriched bigraph X and Y. Pseudocode for the core CODAC algorithm is presented in Algorithm 1.The main procedure called X and Z, and between Y and Z. During shuffling, the node degrees of each xi, yj and zk is kept constant, and the shuffled edges are constrained not to overlap the original edges. The shuffled graphs are denoted by C#, may be calculated. This matrix is then used to select |N|=|P| negative examples at random. Taken together, the P positive and N negative examples constitute our “Gold Standard” dataset.In order to determine an edge similarity threshold, we need to define a “gold standard” set of positive and negative examples of associations. Here, we take all of the TP), false positive (FP), true negative (TN), and false negative (FN) predictions for each threshold. We then calculate the recall, R=TP/(TP+FN), precision, P=TP/(TP+FP), and the F-measure, F1=2RP/(P+R). The similarity threshold T that gives the best F-measure with the Training subset is verified using the Test subset and retained to calculate a filtered cosine similarity matrix, C∗, according to Ci,j>T or if the  edge already exists in E3, otherwise, We randomly split the Gold Standard dataset into two groups with equal distributions of positive and negative examples to give a “Training” and a “Test” subset. We then rank the scores of all members of the Training subset, and label them “positive” or “negative” according to a score threshold that is varied from 0.0 to 1.0 in steps of 0.001. This allows us to determine the numbers of true positive . Whenever there is no data for a given pair  in an input graph, the corresponding score There may often be more than one configuration for a graph mentclasspt{minimaequences . To handwd, each weight is varied from 1 to 10 in steps of 0.1, and for each combination of weights a ROC performance curve is calculated using the complete ranked list of consensus scores and our Gold Standard set of positive examples. The combination of weights that gives the largest area under the curve (AUC) is selected and used to calculate the best consensus similarity matrix CS. Then, the PredictAssociations procedure determines the best threshold to filter the consensus similarity matrix CS and to deduce the resulting enriched bipartite graph Receiver-operator-characteristic (ROC) analysis provides an objective way to measure the ability of an information retrieval system to retrieve positive documents as first ranked, i.e. with the best scores . One advx from set X represents a term in an ontology and has a neighbour z in set Z, it is quite possible that all of the ancestors of x present in X should also have z as neighbour. If requested by the user, whenever an edge  is found where z is annotated with an ontology term x, then CODAC will add additional edges between item z and all parents of x present in X. This is illustrated in Fig. Ontologies are often described as taxonomic hierarchies of classes, as is the case for the GO gene ontology . Thus, iZ into clusters having 100% identity. Each cluster is represented by a unique cluster identifier (CID). As shown in Algorithm 3, all source edges  and  from E1 and E2 in which identical zi and zj belong to the same CID, are merged into unique  and  edges, producing A possible source of bias in any data mining approach is the existence of redundant items in the input. This is especially the case for protein entries in UniProt where it is quite possible to have entries with different identifiers but identical amino-acid sequences. In order to deal with this possibility, CODAC groups all items in p-value”, for finding an edge simply by chance. For example, it is reasonable to suppose that an edge  might be predicted at random if x and y are each highly connected to many items in Z. In order to estimate the probability of finding edges by chance, one could generate multiple random graphs by shuffling the edges of a given input graph, as described above for constructing the Gold Standard Negative examples. However, this is quite impractical given the very large numbers of items in X, Y, and Z and the complexity of the filtering procedure that would have to be repeated for each shuffled version of the dataset. Instead, we assume that the probability for finding an edge  by random chance is given by a hypergeometric distribution of the number of common neighbours  and . Letting Nz denote the total number of items in Z, Nx the number of neighbours of x in Z, and Ny the number of neighbours of y in Z, the hypergeometric probability distribution is given by While our approach provides a systematic way to predict edges in K, equal to or greater than the observed number Kx,y of common neighbours z of both x and y. Because this p-value test is applied to a large number of  edges in p-value less than where ror rate . Therefop-values give objective measures of the quality of predicted associations, from a user’s point of view it is often convenient to provide a simple and memorable quality scale. Therefore, we classify a predicted association as “Gold” if all of the individual data source p-values for this association are statistically significant.While the above consensus scores and p-values are statistically significant. Otherwise, it is classed as a “Bronze” association.A predicted association is classed as “Silver” if more than half of the data source X corresponds to one of the MF, BP or CC GO namespaces, and Y corresponds to one of the Pfam, CATH, or SCOP protein domain classifications. For each of the 9 combinations of X and Y, 3 data sources were selected to provide common neighbours (Z) of the items in X and Y, namely: (i) SIFTS providing curated PDB chain associations, (ii) UniProtKB/SwissProt (SP) providing curated UniProt entries, and (iii) UniProtKB/TrEMBL (TR) providing non-curated automatically annotated UniProt sequences.In this paper, the CODAC approach is applied to discover new weighted GO-domain associations. In our X and Y source datasets. All PDB chain IDs and UniProt ANs having identical sequences were clustered using the Uniref non-redundant cluster annotations [Flat data files of SIFTS June 2017), UniProt (June 2017), and InterPro (version 63.0) were downloaded and parsed using in-house Python scripts. Associations between PDB chains and GO terms, and associations between PDB chains and protein domains  were extracted from the SIFTS data. All CATH and SCOP domain families were transformed into their corresponding superfamilies, and all Pfam “repeat” and “motif” domain types were discarded. Associations between UniProt sequence accession numbers (ANs) and GO terms and AN-Pfam associations (as well as AN-CATH and AN-SCOP associations) were extracted from the UniProtKB/SwissProt and UniProtKB/TrEMBL sections of UniProt to give two datasets of UniProtKB/SwissProt associations and UniProtKB/TrEMBL associations, respectively. Then, using the evidence code of the GO term, the associations in the SIFTS, UniProtKB/SwissProt, and UniProtKB/TrEMBL datasets were divided into two groups, namely one group for which the GO term evidence code indicated manual curation, and one group for GO terms with evidence code “inferred from electronic annotation” (IEA). Here, the resulting 6 datasets are called SIFTS, SIFTS-IEA, SP, SP-IEA, TR, and TR-IEA. Thus, there are 6 input tripartite graphs for each of the 9 combinations of the , UniProtWe do not make any distinction between the various possible manual evidence codes. However, we note that the GO_REF field for IEA currently covers 12 annotations sources, namely InterPro2GO, UniProt Keywords2GO, UniProt Subcellular Location2GO, EC2GO, UniRule2GO, UniPathway2GO, Ensembl Compara, Ensembl Fungi, Ensembl Metazoa, Ensembl Plants, Ensembl Protists, and the Gene Ontology Consortium. Of these, the largest number of annotations come from InterPro2GO and UniProt Keywords2GO, which each provide around 169 million associations in UniProtKB. It should be noted that, only 34%, 4%, and 5% of the InterPro2GO annotations are GO-Pfam, GO-CATH, and GO-SCOP associations, respectively.For each of the 9 settings of this study, the weights assigned to each dataset have been optimised. The procedure is described in Methods (Algorithm 2) and is based on a ROC-plot analysis of the ranking of our Gold-Standard InterPro-based positive examples versus all other associations computed from all the datasets and considered as background. Then the best threshold is determined from the consensus scores calculated with the optimised set of weights, using the Gold Standard Training and Test subsets of positive and negative examples. Table O(s×c), where s is the number of sequences and c is the number of UniProt clusters. The scoring stage then scales as O(g×d), where g is the number of GO terms and d is the number of domains. Here, the largest calculation is to find GO BP-Pfam associations. This takes around 12 hours on one CPU core of an Intel Xeon E5-2630 2.40 GHz workstation with 128 Gb memory.Because we exploit existing UniProt cluster IDs to form clusters of similar protein sequences and to eliminate duplicate sequences, the computational cost in the initial data preparation stage scales as approximately Summaries of our calculated GO MF-domain, BP-domain, and CC-domain associations are shown in Tables The overlap between the GODomainMiner predictions and InterPro is shown in the last row of these tables . The high percentage of overlap between GODomainMiner and InterPro (from 91% to more than 99%) reflects the fact that our method is calibrated to recover as many as possible correct InterPro associations. Nevertheless it also shows that a small percentage of the InterPro associations have consensus scores below our calculated score threshold, revealing the role of human rather than data-driven knowledge in the definition of such associations.3 in Table Overall, our approach yields a total of 32,881 MF GO-Pfam associations , GODomainMiner predicts that each MF GO term and each Pfam entry are associated with an average of 5.2 domains and 4.0 MF GO terms, respectively, compared to averages of 3.9 domains and 1.3 MF GO terms in InterPro, respectively. For BP and CC GO terms we see similar enrichments from GODomainMiner compared with InterPro, with ratios of 5.4 versus 3.5 and 16.9 versus 6.8 associations per GO term, and 8.2 versus 1.17 and 4.5 versus 1.1 associations per Pfam, respectively. These results demonstrate that GODomainMiner produces a considerable enrichment in the number of annotations compared to InterPro. They also support the notion that many Pfam domains participate in different functions, either as singleton domains or as components of multi-domain proteins.The bar charts in Fig. 555p-values, it is interesting to see that the majority (68%) of our predicted MF GO-Pfam associations common with InterPro fall in this class. Overall, we calculate that 47% of the GODomainMiner MF GO-Pfam associations and 33% of the predicted BP and CC associations are of Gold quality. The quality of GO predictions for CATH and SCOP classifications also follow very similar paths  GODomainMiner associations overlap with InterPro, whereas only 55 (13% of InterPro set) SCOP2GO associations from dcGO are found in InterPro. This confirms the trend observed for GO-Pfam associations, in favor of a much better coverage by GODomainMiner than by dcGO, of the InterPro reference set.We also compared GO-SCOP associations predicted by GODomainMiner with the SCOP2GO database from dcGO and with InterPro. Overall, GODomainMiner calculates a total of 19,708 leaf GO-SCOP associations, compared to 2445 such associations in SCOP2GO and 422 in InterPro. Of these, 845 GO-SCOP associations are common to GODomainMiner and SCOP2GO. Also, 421 , 30 and 20 in the silver and bronze categories, respectively. From the 125 associations, 30 are already known in InterPro (21 from the gold category) and 95 are new (54 from the gold category). Manual checking of the MF GO terms and Pfam domain names led us to distinguish 5 situations  and 48 are new . For one Pfam domain only  the two GO terms are known already in InterPro. For 5 other Pfam domains, one of the GO terms is known in InterPro and the other one is new. New MF GO-Pfam associations generally give lower scores than known InterPro associations. However, in some cases this suggests an alternative substrate for the domain activity which may be interesting to investigate. For example, for Pfam domain Mqo , GO:0052589  activity) is found in addition to GO:0008924  activity). The remaining 24 Pfam domains all have new GO MF annotations that do not exist in InterPro. Interestingly, in some cases a different more general InterPro annotation exists, as in the case of PF07722 domain Peptidase_C2 which GODomainMiner associates with GO:0034722 (gamma-glutamyl-peptidase activity) and with GO:0033969 (gamma-glutamyl-gamma-aminobutyrate hydrolase) activity, whereas the InterPro annotation is simply GO:0016787 (hydrolase activity).It is natural to suppose that predicted GO-domain associations could help to annotate entire protein sequences. However, it does not automatically follow that GO-domain associations are directly transferable to sequences because the function of a particular protein can depend on, for example, its domain architecture, organism, cell type, and cellular location . TherefoIn any case, it is reasonable to expect that the difference in specificity compared to InterPro annotations will likely prevent many of the GODomainMiner annotations from being transferred directly to all proteins that match a given domain. However, there is no doubt that the newly discovered associations should contribute to the generation of new rules to annotate protein sequences. Nonetheless, the domain-level functional annotations predicted by GODomainMiner should first be subjected to further benchmarking in order to validate their usefulness. We recently participated in the 2017 round of the CAFA  community experiment , in whicWe have presented a systematic approach called CODAC for mining associations from datasets that can be represented as tripartite graphs. We have presented one implementation of this approach called GODomainMiner, for predicting associations between GO terms and protein domains. This was achieved by first collecting existing Pfam, CATH, and SCOP domain annotations of protein chains and sequences on one hand and MF, BP, and CC GO term annotations on the other. We then applied our method to find a list of direct associations between GO terms and domains. Considering only the most-specific GO terms, our approach yields an enrichment of about 15-fold in the number of GO-Pfam associations that currently exist in InterPro. A selected subset of one-to-one and many-to-one associations has been analyzed from a biological point of view, and these all appear to be highly meaningful and consistent with available knowledge. Nonetheless, there remains a need for the associations predicted by our approach to be validated more extensively, and we plan to test our approach thoroughly in the next CAFA community experiment.Additional file 1Supplementary figures. (PDF 93 kb)Additional file 2Supplementary tables. (PDF 36 kb)


Roasting millet grains at 140°C significantly (p < 0.05) increased the protein digestibility (22.3%–60.1%) and reduced protein (9.3%–7.8%), phytate (3.9–3.6 mg/g), and total sugar content (2.2%–1.9%). Germinating millet grains at room temperature for 48 hr resulted in millet flour with the best nutritional quality and was adopted for the production of millet‐based composite flour. Addition of vegetables and skimmed milk powder to germinated millet flour significantly (p < 0.05) increased the macro‐ and micronutrient contents and the functional properties of millet‐based composite flour. The study demonstrated that the use of skimmed milk powder and vegetables greatly improves the protein quality and micronutrient profile of millet‐based complementary foods. The product has the potential to make a significant contribution to the improvement of nutrition of children in developing countries.The aim of this study was to develop a nutrient‐enhanced millet‐based composite flour incorporating skimmed milk powder and vegetables for children aged 6–59 months. Two processing methods were tested to optimize nutrient content and quality of millet‐based composite flour, namely germination for 0, 24 and 48 hr and roasting at 80, 100, and 140°C. The amount of ingredients in the formulation was determined using Nutri‐survey software. Germinating millet grains for 48 hr at room temperature significantly ( After blanching, carrots were shredded using a hand grater with holes of a diameter of 0.6 cm. The shredded carrots and cowpea leaves were dried for 6 hr in an electric cabinet dryer  set at 55°C. Dried carrots and cowpea leaves were then milled into fine powders using a locally fabricated hammer mill. Pumpkin seeds were cleaned and roasted at 140°C for 20 min using an infrared Food oven . Dry pumpkin seeds were blended into a fine powder using an electric blender .2.5Germination time and roasting temperatures were varied as shown in Table Y = Response function, β0 = Constant (the intercept), β1 or β2 = Coefficient of the linear effects, X1 and X2 = Linear effects of germination time and roasting temperature, respectively.where 2.6Nutri‐survey software, 2007 was used to estimate the optimum amounts of skimmed milk and vegetable powders to add to millet flour in order to meet the protein (13 g/day) and energy  requirements for children of age 6–59 months WHO, . The det2.72.7.1The millet and millet‐based composite flours were analyzed for moisture, crude protein, crude fat, crude fiber, and ash contents according to the method described by AOAC . Carbohy2.7.2The amount of calcium, zinc, iron, magnesium, and copper in the composite flour was measured by an atomic absorption spectrophotometer according to the method of Hernández‐Urbiola, Pérez‐Torrero, and Rodríguez‐García . Calibray = intercept on y‐axis; D = Dilution factor; 10 =  a conversion factor from mg/kg to mg/100 g; V = volume of sample in ml.where 2.7.3The β‐carotene content of flour was determined according to Rodriguez‐Amaya and Kimura . CarotenA = absorbance; V = total extract volume; p = sample weight; A1cm1% = 2592 (β‐carotene extinction coefficient in petroleum ether). where where 12 = retinol activity equivalent conversion factor, 1 RAE = 12 μg β‐carotene.2.7.4A modified method of Ijarotimi and Babatunde  was used2.7.5Protein digestibility was determined by the porcine pepsin method as adapted by Gomez, Obilana, Martin, Madzvamuse, and Monyo .2.7.6Total sugars were determined according to the method described by Nielsen .2.8The bulk density, water, and oil absorption capacities of the millet flours were determined according to the method described by Onwuka .2.9The method described by Leach et al.  was used2.10Pasting characteristics of the porridge from the composite flour were determined with a Rapid Visco Analyzer  according to Ikegwu, Okechukwu, and Ekumankana . Peak vi2.11Percentage contribution to recommended dietary allowance was expressed as % of RDA.X is the amount of nutrient analyzed and Y is the RDA for a given nutrient/variable.where 2.12t test analysis of variance (ANOVA) using XLSTAT software version 2017 to determine variation between means. Significance variation was accepted at p < 0.05. Simple linear regression analysis was used to determine the influence of germination time and roasting temperature on the nutritional quality of the millet flour.All determinations were performed in duplicates and subjected to statistical two sample 33.1p < 0.05) increased the protein content, protein digestibility, and total sugar content of millet flour by 0.03%, 0.79%, and 0.07%, respectively, and reduced the total phytates by 0.005 mg/g. Roasting temperatures had a positive linear effect on the protein digestibility and a negative linear effect on the total phytates, protein, and total sugar contents of millet flour. An increase in roasting temperature significantly (p < 0.05) reduced the total phytates by 0.003 mg/g, protein content, and total sugar content of millet flour each by 0.01%. However, an increase in roasting temperature increased the protein digestibility of millet flour by 0.34%.The effect of roasting and germination on the nutrient composition of millet flour as predicted by the linear regression models is indicated in Table 3.2p < 0.05) increased the protein content (9.3%–10.6%), total sugars (2.2%–5.5%), and protein digestibility (22.3%–65.5%). Results in Figure p < 0.05) reduced total sugars (2.2%–1.9%), protein content (9.3%–7.8%), and phytates (3.9–3.6 mg/g) but increased protein digestibility (22.3%–60.1%). The study findings indicate that germination for 48 hr resulted in millet flour with a high protein content (10.6%), protein digestibility (65.5%), total sugars (5.5%), and low levels of phytates (3.7 mg/g) and was therefore taken as the optimal procedure to process the flour that was used in the formulation of the composite flour. The increase in the total sugars during germination of millet grains is due to the hydrolysis of starch into shorter chain sugars by amylases. The results of this study agree with those reported by other researchers. Coulibaly and Chen  increased the crude protein, crude fat, crude fiber, ash, and gross energy of millet‐based composite flour by 7.3%, 2.1%, 2.5%, and 1.6%, respectively. These results further indicated that moisture content and carbohydrate content of the millet‐based composite flour significantly (p < 0.05) decreased by 1.1% and 9.9%, respectively.Table p < 0.05) increase in the ash content of millet‐based composite flour is attributed to the addition of vegetables as they are reported to be rich in minerals. Findings from this study are in agreement with those of Mogra and Midha (p < 0.05) increased, as was to be expected from the increased fat content  in zinc (2.1–4.2 mg/100 g), copper (0.5–0.9 mg/100 g), and calcium (143.6–667.8 mg/100 g) contents was observed in the millet‐based composite flour. The results in Table p > 0.05). The findings also indicate that the vitamin A content of millet‐based composite flour was significantly (p < 0.05) higher (641 μg RAE/100 g) than that of millet flour (15.5 μg RAE/100 g). The increase in vitamin A content of the millet‐based composite flour is attributed to the addition of carrot and cowpea leaves. Findings from this study were in agreement with those of Sadana, Bakhetia, and Aggrawal  higher than those of millet flour but bulk density did not change (0.6 g/ml) on the addition of vegetables and skimmed milk powder. OAC of the millet‐based composite flour increased from 59.2% to 77.9%, while WAC capacity increased from 117% to 225%.Table OAC is the ability of flour protein to physically bind fat by capillary attraction, and it is of great importance as fats act as flavor retainer and increase the mouthfeel of foods . The starch granules substantially increased in size due to hydration of the amorphous phase causing loss of crystallinity and molecular order . At low temperatures (40–50°C), starch granules are less soluble in water due to the hydrogen bonds and crystallinity of the molecule, thus resulting in the low swelling power observed in both samples. Between 50 and 80°C, which is the porridge cooking temperature, the size of starch granules increased substantially, breaking the molecules and consequently leaching the amylose to form a three‐dimensional network and increased the paste's viscosity  lower than that of millet flour (67.9°C). The pasting temperature provides an indication of the minimum temperature required for cooking the flours  was significantly (4Germination of millet grains and incorporation of skimmed milk and vegetable powders resulted in a nutrient‐enhanced composite flour with improved functional properties. Scaling up production of this composite flour can contribute toward improving the nutrition of children in developing countries.The authors declare no conflict of interest.


Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult Nematostella vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly defined TSP superfamily by gene duplications, radiation, and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity; however, the repertoire of ECM proteins in nonbilaterians remains unclear. Thrombospondins (TSPs) are known to be well conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highly conserved C-terminal region of TSPs, we identify undisclosed new families of TSP-related proteins in metazoans, designated mega-TSP, sushi-TSP, and poriferan-TSP, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-TSPs, the only form identified in ctenophores, are typically >2,700 aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM,  Capsaspora owczarzaki that represent the closest unicellular relatives of metazoans (The extracellular matrix (ECM) of metazoans holds great fascination because of its roles in support of metazoan multicellularity and organismal complexity. In vertebrates, ECM is highly tissue specific and typically composed of around 150–300 proteins, along with carbohydrate components . Comparietazoans .Drosophila, where they have roles in body axis maintenance and developmental ECM organization, respectively -like and thrombospondin type 1 domains (TSRs) are each widely represented in other categories of extracellular proteins  and are Nematostella vectensis, identified four transcribed paralogs. In phylogenetic trees based on the conserved TSP C-terminal region, one of these, Nv85341, has an unexpectedly close phylogenetic relationship with a thrombospondin of Ciona intestinalis termed TSP-DD  and is secreted from cells as a monomer. At the time of its identification in 2010, protein sequence orthologs of TSP-DD were restricted to invertebrate deuterostomes . We report here on previously undisclosed categories of TSP-related proteins, which we have designated mega-thrombospondin (mega-TSP), sushi-thrombospondin (sushi-TSP), and poriferan-TSP. All the predicted proteins are clearly related to TSPs by inclusion of the characteristic TSP C-terminal region domain architecture and differ in other domains and their phylogenetic distributions within the Metazoa. We present the first systematic evaluation of these proteins, their phylogenetic relationships with canonical TSPs, and the first analysis of biological function of a mega-TSP. These data illuminate the existence of an unappreciated TSP superfamily and lead to a new evolutionary scenario for the emergence of the canonical TSPs with implications for understanding of early metazoan evolution.The apparently anomalous identification of a possible TSP-DD-like polypeptide in N. vectensis TSP85341 (Nv85341) and then with other representative TSPs. These led to the identification of further predicted TSP-related protein sequences in multiple cnidarians. Because some of these sequences are predicted as much longer polypeptides than a canonical TSP , the complete nucleotide sequence of the open reading frame (ORF) of Nv85341 was obtained by DNA sequencing from cDNA prepared from RNA from 2-month-old juvenile N. vectensis polyps. Similarly, a related ORF of Hydra magnipapillata  was confirmed and extended from a transcriptome database . The full ORF has 99% identity to XP_012565470 of H. vulgaris. The complete N. vectensis and H. magnipapillata protein sequences and closely related partial sequences from other cnidarians were then used to query genomic and transcriptomic databases at NCBI and other repositories, which led to identification of further categories of proteins.Comparative genomic and transcriptomic searches were carried out initially with the predicted partial protein sequence of Sycon ciliatum  or craniates . We designated these proteins “mega-thrombospondins”  because of their very large size  and because the discoidin domain is not present in the earliest emerging forms (see below). In most species, a single mega-TSP was identified; some species encode two distinct gene products A. We alsciliatum .These studies also disclosed variation in the number of TSP-related proteins per species in poriferans, albeit that some identifications were uncertain due to ORFs that are incomplete for the N-terminus. The poriferans examined and the full sets of TSP-related proteins and candidate TSP-related proteins identified are summarized in The domain architecture of EGF-like domains, TSP type 3 repeats and a single L-type lectin-like domain  domain at the C-terminus is a defining feature of canonical TSPs . Domain Leucosolenia complicata mega-TSP, a “tyrosine kinase ephrin A/B receptor-like” domain (IPR01641) in addition to a cadherin-like domain: it can be noted that immunoglobulin-like and cadherin domains belong to the same “Greek key,” beta-sandwich fold group (H. magnipapillata mega-TSP (HmMT) also includes a beta-barrel PA14 domain (InterPro011658)  B. In mego011658) B. Thus, Oscarella carmela, has tandem TSP-C/Con A-like and Con A/MAM-like domains proximal to the C-terminus, whereas sushi-TSPs from Orbicella faveolata and Acropora digitifera (both cnidarians) include three Con A-like domains at the C-terminus  that also lack a coiled-coil domain and are distinguished by the presence of three or more tandem sushi/short consensus repeat domains in the N-terminal region and a single EGF-like domain. In common with the mega-TSPs, the sushi-TSP of the sponge, Amphimedon queenslandica , and Xestospongia testudinaria . The full-length sequences identified from the demosponges slandica , Halicloudinaria   was also modeled on PDB 3FBY (of 46% identity) and the second Con A-like domain on PDB 4DQA (of 21% identity). Again, the model had good correspondence to the TSP domain structure , the type 3 repeats and first ConA-like domain had 49% sequence identity with 3FBY and corresponded well with this structure . In the 1UX6 structure, these residues constrict the entrance to a short loop with D902, S903, D904, G905, and D906, and these three aspartates chelate a calcium ion. In the HmMT model, the analogous loop is extended and the negatively charged residues do not appear to be in favorable positions to support calcium binding B. In NvMWith regard to the conserved DDD motif in the L-lectin/Con A-like domain of TSPs, the corresponding residues in HmMT are GID (aa312–314 of the model). Although D314 of HmMT overlays D1003 of 1UX6, N337, of HmMT overlays Q1027, and the HmMT model provides two glutamates (E311 and E336) in this vicinity, on balance, it appears that the required orientation of charges for calcium binding is unlikely to be achieved at the single aspartate residue C. In theNematostella vectensis is well developed as a laboratory model for the anthozoan class of cnidarians , the ectodermal layer was negative and the NvMT transcript was detected specifically as faint expression in the aboral endoderm , lower expression in blastulae, increasing expression throughout the gastrula (24h–48hpf) and planula (48h–120hpf) stages, and maintenance of higher expression in juveniles (from 120hpf onward)  of HmMT transcript during head regeneration after transection showed a small initial decrease, recovery to similar abundance between 6 and 18 h, and increased abundance between 24 and 48 h followed by a decline (The elevated expression of  decline E. The 24 decline .HmMT expression was silenced by electroporation of short interfering RNAs (siRNA). For these experiments, a transgenic Hydra line expressing green fluorescent protein (GFP) in ectodermal cells and red fluorescent protein (RFP) in endodermal cells  are more closely related to each other than to any of the additional Con A-like domains  online. Among the hundreds of proteins in the metazoan extracellular milieu, TSPs have been considered a unique form of matricellular and ECM protein. Here, we identify, by comparative genomics, domain composition and molecular modeling, multiple forms of TSP-related proteins  that all share a domain region equivalent to the C-terminal region of TSPs. These identifications provide the first evidence for evolution of a TSP superfamily. Whereas secreted TSP-like proteins are not present in the close relatives of metazoans , we findPotential molecular activities of mega-TSP and sushi-TSP were explored by analysis of molecular structures and motifs. The C-terminal regions of canonical TSPs have roles in binding cell surface receptors as well as other ECM proteins  and we smega-TSP transcripts were examined in N. vectensis and H. magnipapillata, each of which has distinct advantages for study of developmental stages (Nematostella) or regeneration of adult polyps (Hydra). In situ hybridization demonstrated widespread and dynamically regulated expression of NvMT during development, with transcripts confined to the endoderm until late stage polyps, with graded expression increasing toward the distal ends of tentacles. Ectodermal expression was prominent in the mesenteries of adult N. vectensis polyps, and ectodermal expression was conserved in the body wall of Hydra polyps. Distinctions in the patterns might reflect subfunctionalization of NvMT due to the greater number of TSP superfamily members expressed by N. vectensis  (H. vulgaris TSP (XP_002164610), also human TSP1 (NP_003237.2), human TSP5 (NP_000086.2), and C. intestinalis TSP-DD (NP_001265897.1) (Mnemiopsis leidyi (research.nhgri.nih.gov/mnemiopsis/)   with the genome-predicted protein sequence, XP_012565470. The GenBank TSA is GAOL01026196.1 (H. magnipapillata Seq37942.0 transcribed RNA sequence). The domain composition of all validated protein hits was ascertained via CDD (http://www.cbs.dtu.dk/services/) , of 1639928) , H. vulg65897.1)  as queryiopsis/) , Pleurob_Models) , and NCBrniensis ; the porslandica , Ephydat carmela  at http:agen.org ; corals gitifera  and Stylstillata ; Lingulaiculatus ; acorn wra flava  and Japaa fucata  at marina fucata ; Japanesr.edu.sg , and chovicollis  and Salp rosetta , and theczarzaki  at NCBI.czarzaki  (ebi.ac. via CDD  and Intervices/) . Inspectrvices/) .Data sets included the TSPs listed above and TSP-related proteins from the set in https://toolkit.tuebingen.mpg.de/#/) were used to provide model segments based on the following: HmMT residues 108–359 (SDAIT to LTELW), modeled on pdb 4V2D (www.gromacs.org) . The HmMT model shared 42% sequence identity with pdb 3FBY, the C-terminal region of human cartilage oligomeric matrix protein, also known as thrombospondin-5 (TSP-5)  , with reacs.org)  and Modeacs.org)  (https:/ (TSP-5) , betweenhttp://www.cgl.ucsf.edu/chimera). Procheck (http://www.ccp4.ac.uk/dist/html/procheck_man/index.html).All the resulting models were read into the Sybyl suite of software  where they were inspected. Cysteine residues likely to form disulfides were oriented within optimal reach of each other, oxidized, and disulfide bonds formed. The models were then minimised and overlaid for comparison with two TSP structures from pdb, neither of which had been used in model building: 1UX6   at 18 °C and fed two to three times a week with freshly hatched Artemia salina nauplii. Before experiments, animals were starved for 24 h. For qPCR, RNA was prepared as described in H. vulgaris strain expressing ectodermal GFP (actin::GFP) and endodermal RFP (actin::RFP)  was added to the cuvette. Twenty four hours after electroporation, living animals (n = 20) were transferred into a new Petri dish containing hydra medium and kept under normal hydra conditions. The feeding routine was restarted 2 days after electroporation. For the regeneration experiments, animals were allowed to recover for 7 days and fed at least four times. The animals were cut at 50% body length using a sterile scalpel blade . After three washing steps in hydra medium for 10 min at 18 °C, the head and foot regenerates, respectively, were kept separately in Petri dishes containing hydra medium. Regeneration was documented after 72 and 96 h, using a Nikon SMZ25 stereomicroscope equipped with a Nikon DS-Ri2 high-definition camera.in::RFP) , providein::RFP) . 3 μM siN. vectensis polyps descended from clones CH2 and CH6 from the University of California Davis Bodega Bay Marine Laboratory were raised as described (Nematostella polyps and embryos in the Heidelberg laboratory were kept in 1/3 seawater (Tropic Marine) at 18 °C in the dark and fed once or twice a week with freshly hatched Artemia nauplii.A colony of mature escribed . NematosN. vectensis genomic sequence. The oligonucleotide primer pairs (synthesized by Eurofins) used were 5′-aacttttaaaacacggcatgg-3′ and 5′-ggcagttgatcaaagggtgt-3′; 5′-ttgcgccacaacaagctat-3′ and 5′-tcgaatgtaggacagttggttc-3′; 5′-tcaagaataatcgcctgacca-3′ and 5′-aaaagcatcttggccgatgt-3′; 5′-acgccagggacctagatttt-3′ and 5′-gattgcgtctgtgtacccct-3′; 5′-tctacagtggccccacctac-3′ and 5′-gtcgcctcatagtccagctc-3′; 5′-ccacgatggctacatcacac-3′ and 5′-atctggacagctctcgcatt-3′; 5′-gtgcttcccaggagtgaagt-3′ and 5′-caccatcgctatcagtgtcg-3′; 5′-gtgcttgcgacactgatagc-3′ and 5′-tcctttgatcccaacattga-3′; and 5′-ggagaaaagaaaacatcaatttcg-3′ and 5′-cgtgtacggtctataaatctctgg-3′. The polyA+ RNA template for reverse transcription/polymerase chain reaction (RT-PCR) was made from 2-month-old juvenile N. vectensis polyps with an RNeasy Mini Kit (Qiagen) and was treated with DNase I (Life Technologies) before use. RT-PCR was carried out using a One-Step RT-PCR Kit (Qiagen) and the following amplification parameters: 1 min at 94 °C, 1 min at 50 °C, and 1.5 min at 72 °C, for 40 cycles in a MJ Mini cycler. PCR products were subcloned into pCRII with a TOPO PCR Cloning Kit (Life Technologies) and the inserts were sequenced on both strands at UC Davis Sequencing. When assembled, the nine overlapping cDNAs correspond to an 8,509-bp nucleotide sequence encoding a 2,804 aa ORF. The full sequence is deposited as GenBank accession MF962901.Cloning and sequencing of a partial cDNA for NvMT was reported in N. vectensis were raised and prepared for in situ hybridization as described  corresponding to the antisense strand of eviously . For hybHydra polyps were embedded in 1.5 ml PBS containing 0.5% gelatin, 30% bovine albumin-fraction V (Carl Roth) and solidified with 105 μl glutaraldehyde (Sigma). Cross sections (100 μm) were cut from the midgastric region with a VT1000 S vibrating blade microtome (Leica) and mounted on microscope slides in Mowiol 4-88(Carl Roth).Freshly in situ-stained Images were captured with a Nikon Eclipse 80i or E800 photomicroscope using NIC filters and a Nikon Digital Sight DS-U1 color camera and processed with Adobe Photoshop CS6 and ImageJ.Molecular Biology and Evolution online.Supplementary_Material_msz060Click here for additional data file.


To identify the specific domains of the presynaptic protein synapsin targeted by recently described autoantibodies to synapsin.Sera of 20 and CSF of two patients with different psychiatric and neurological disorders previously tested positive for immunoglobulin (Ig)G antibodies to full-length synapsin were screened for IgG against synapsin I domains using HEK293 cells transfected with constructs encoding different domains of rat synapsin Ia. Additionally, IgG subclasses were determined using full-length synapsin Ia. Serum and CSF from one patient were also screened for IgA autoantibodies to synapsin I domains. Sera from nine and CSF from two healthy subjects were analyzed as controls.1 in two sera and one CSF, IgG2 in none of the samples, IgG3 in two sera, and IgG4 in eight sera. One patient known to be positive for IgA antibodies to full-length synapsin had IgA antibodies to the D-domain in serum and CSF.IgG in serum from 12 of 20 IgG synapsin full-length positive patients, but from none of the healthy controls, bound to synapsin domains. Of these 12 sera, six bound to the A domain, five to the D domain, and one to the B- (and possibly A-), D-, and E-domains of synapsin I. IgG antibodies to the D-domain were also detected in one of the CSF samples. Determination of IgG subclasses detected IgGAnti-synapsin autoantibodies preferentially bind to either the A- or the D-domain of synapsin I. By using a combination of immunoprecipitation methods, mass spectrometry, immunohistochemistry of brain sections from wild type and synapsin-knock mice, as well as cell-based assays and biochemistry we recently identified the presynaptic vesicle-associated protein synapsin as a novel target of antineuronal autoantibodies in serum and CSF samples from a patient with limbic encephalitis . In a foSynapsin I and II are major synaptic vesicle proteins of the adult brain, regulating the availability of synaptic vesicles and thus affecting synaptic transmission ; 4; 5. SThe pathophysiological relevance of antibodies to synapsin remains to be clarified. Our finding that antibodies to synapsin are associated with different clinical phenotypes raises the question whether this may be related to distinct functions of synapsin and to the recognition of different domains of synapsin by autoantibodies to synapsin. To better understand the potential pathophysiological relevance of antibodies to synapsin, we here characterized in molecular detail the specific domains of synapsin targeted by synapsin autoantibodies.The study was approved by the institutional review board of Charité– Universitätsmedizin Berlin  and the regional committee for medical and health research ethics, central Norway (2011/137). All participants provided written informed consent.Patient sera investigated in this study were previously shown to contain antibodies to full-length rat synapsin Ia and included sera from 16 patients positive for IgG autoantibodies to rat synapsin Ia identified from a total of 207 patients admitted to acute psychiatric inpatient care at the Department of Psychiatry, St. Olav’s University Hospital, Trondheim, Norway, between September 2011 and March 2012 . AdditioThe expression constructs for the various synapsin domains have been described earlier . In brieA monoclonal mouse antibody against the D-domain of synapsin I #106011) and a polyclonal rabbit antibody against the A-domain of synapsin I/II (#106002) were purchased from Synaptic Systems . Rabbit polyclonal antibodies against the C-domain of synapsin I/II and the E-domain of synapsin Ia were developed by one of us (F.B.), as described earlier [ and a po1, IgG2, IgG3 and IgG4 antibodies . Detection of immunoglobulin A antibodies in the patients’ CSF or serum was performed by using a Texas Red 594 coupled goat anti-human IgA secondary antibody .Mouse antibodies were visualized using Alexa Fluor 594 goat anti-mouse IgG . An Alexa Fluor 488 goat anti-guinea pig IgG (#A11073) was also from MoBiTec. Alexa Fluor 594 goat anti-rabbit IgG  was applied to detect bound rabbit antibodies. Bound human antibodies of the immunoglobulin type G  were visualized by incubation with Alexa Fluor 594 labeled goat anti-human IgG directed against the Fc fragment . Visualization of human IgG subclass-specific antibodies was achieved using Alexa Fluor 647 labeled goat anti-human IgGHuman embryonic kidney (HEK) 293 cells were cultured in 24-well multiplates to 70% confluency and transfected with 0.5–1 μg plasmid cDNA per well for 24 hours using Lipofectamine . Transfected cells were fixed with ice-cold 80% methanol for 20 minutes at -20°C . Cells were subsequently permeabilized with 0.1% Triton X-100. Thereafter, cells were incubated with either the domain specific primary antibodies at the indicated concentrations or with patient or control serum or CSF at a dilution of 1:320.-1/anti-synapsin titer serum-1)/. AI values above four were considered as evidence of an intrathecal antibody synthesis [HEK 293 cells transfected with rat synapsin Ia were processed as described above and incubated with increasing dilutions  of patient CSF or serum to determine titers of anti-synapsin IgG in a paired CSF/serum sample. Alexa Fluor 594 goat anti-human IgG served as secondary antibody. Antibody index (AI) for IgG antibodies to synapsin Ia was calculated as previously described by the formula: AI = . Among these seven sera, only one serum was also positive for the BC fragment and none was positive for the single C-domain. Thus, sera of six patients were likely exclusively positive for the A-domain  that did not react with the ABC fragment exhibited reactivity to either the DE fragment and the D-domain (three), the DE fragment alone (one) or the D-domain alone (one). None of these five sera showed reactivity towards the E-domain alone, indicating that the D-domain was the targeted antigenic region of synapsin Ia. A CSF sample available from a patient whose serum reacted with the DE fragment and the D-domain likewise reacted with the DE fragment and the D-domain. The results of the cell-based assays for this patient with a CIS are shown in Of note, all patient and control samples were also tested for IgA reactivity to the ABC and DE fragments. All samples remained negative, thereby confirming our previous results obtained for full-length synapsin . As a fuWe also analyzed serum and CSF from our index patient, who had high titers of anti-synapsin IgA and IgG in serum and CSF . Both se1, IgG2, IgG3 and IgG4  out of 20 patients exhibited immunoreactivity to fragments of synapsin Ia and had a mean ± SD (range) age of 42,9 ± 19,8 (20–77) years. Controls (nine) had a mean ± SD (range) age of 38,6 ± 17,2 (18–65) years.1, IgG3 and, with highest prevalence, IgG4 were detected.Despite the apparent clinical heterogeneity, a preference in epitope specificity of anti synapsin autoantibodies in psychiatric and neurological patients exists. Of the 13 patients showing a clearly detectable immune reaction to one of the investigated synapsin fragments six sera exhibited binding of IgG to the A-domain. The same number was detected for the D-domain. Binding to the B-domain and E-domain was shown for one individual patient. Concerning IgG subclasses, IgGin vivo model [Given the fact that synapsin is a ubiquitous presynaptic protein, occurrence of autoantibodies in a variety of psychiatric and neurological disorders is conceivable. Subject to functional evidence, it remains unclear if the existence of autoantibodies to synapsin in serum or CSF has a pathogenic relevance or represents an epiphenomenon. In addition, it remains to be elucidated whether the antigenic epitope presentation of synapsin domains relies on the extracellular presentation of synapsin in the course of an ongoing neuronal repair mechanism as proposed in an vo model , a specivo model . In contvo model ; 17; 18 vo model ; 20. Forvo model  or a redvo model . Internavo model . In the vo model .When comparing the clinical diagnoses, antibodies to the D-domain were observed in a somewhat broader spectrum of diseases  than antibodies to the A-domain, that dominated in bipolar disorder patients (four out of six patients). The relatively small number of patients, however, prevents us from drawing definite conclusions on the association of specific diseases with antibodies to distinct synapsin domains.In addition to our previous prevalence study that addressed the presence of antibodies in serum we had one additional CSF sample available for testing CIS_0117). As shown for IgA antibodies of the index patient with limbic encephalitis this CSF also showed IgG reactivity to the D-domain. Future studies are needed to show whether the D-domain is the preferred target of CSF antibodies. Noteworthy, for both available CSF samples with antibodies to synapsin an intrathecal synthesis of immunoglobulins could be detected in this study and [17. As shThe observation that seven out of 20 sera reacting with full-length synapsin Ia did not react with distinct synapsin domains might be explained by the fact that a certain conformational epitope structure can be provided only by the full-length protein. Missing homology between human and rat sequences can be ruled out to account for this observation since all 20 sera reacted with full length rat synapsin. Alternatively, anti synapsin antibodies may exhibit a lower affinity to the expressed domains than to the full-length protein and therefore escaped detection in our assay, that was standardized to yield a high signal to noise ratio based on a 1:320 dilution of the applied patient samples. Antibody titers to full length protein were initially analysed for all individual sera [1 (two patients), IgG3 (two patients) and, most frequently, IgG4 (eight patients) in our cohort. Despite being the last prevalent subclass detected in serum [4 antibodies therefore seem to be the dominant, albeit not exclusive, subclass in synapsin autoimmunity, at least for the relatively few cases described so far. So, the ability to fix complements seems not to be necessary for the immune reaction in the majority of patients. Antibodies of the IgG4 subclass have already been described for LGI1 and Caspr2 encephalitis [4-related systemic disease, able to cause pancreatitis and infection of other organs [Of the four IgG subclasses described in the literature we were able to detect IgGin serum  IgG4 antphalitis ; 27, andphalitis . Moreover organs , 30.In summary, the present study adds data to further characterize the recently identified antineuronal antibodies against synapsin in molecular detail. Functional cell culture and animal studies investigating the pathological potential of these antibodies by electrophysiological and biochemical methods are under way to shed light on the putative pathogenic impact of these antibodies.


Gloeobacter, Prochlorococcus, and Cyanobium). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria Synechococcus and Synechocystis. In silico comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.Orthocaspases are prokaryotic caspase homologs – proteases, which cleave their substrates after positively charged residues using a conserved histidine – cysteine (HC) dyad situated in a catalytic p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis  or oxygenic photosynthesis (all sequenced cyanobacteria, except  Metazoan caspases (cysteine-aspartic proteases), playing essential roles in programmed cell death, are synthesized as inactive zymogens, which for activation are cleaved into a catalytic, large (p20) and regulatory, small (p10) subunit . The famTrypanosoma brucei, TbMC1 and TbMC4 contain HS and YS substitutions . Their possible function in prokaryotic organisms is discussed.To better understand the distribution of pseudo-orthocaspases, we performed a comprehensive hmmbuild from HMMER v3.1b21 and the first 457 columns of the Pfam Peptidase_C14 (PF00656) seed alignment, downloaded from https://pfam.xfam.org/. The p20 HMM was used in an hmmsearch versus UniProt release 2018_10 , downloaded from https://www.uniprot.org/ on 12 November 2018. Hits with a best one domain E-value lower than 0.0001 were accepted. Further filtration was then performed by alignment to the p20 HMM using hmmalign, followed by removal of sequences with a gap in the His or Cys catalytic dyad positions. Finally, sequences with a p20 domain length not falling within a range of two standard deviations from the mean of the remaining sequences, i.e., 101–200 amino acids, were also removed. For identification of p20-containing proteins in organisms of genera Synechocystis, Synechococcus, Prochlorococcus, and Cyanobium we performed a DELTA-BLAST protein homology search2 using the previously identified Synechocystis sp. PCC 6803 p20 domain (Caspase-homologs including orthocaspases were identified using a p20 domain profile hidden Markov model (HMM) constructed using 0 domain  as a quehmmsearch with four additional HMMs was performed versus the sequence dataset, aiming to identify metacaspases and paracaspases via domain architecture. The p10 domain was identified using a p10 HMM constructed with hmmbuild from the last 211 columns of the Pfam Peptidase_C14 seed alignment. Paracaspases were identified by having at least one Pfam Immunoglobulin (Ig) (PF00047), Ig_2 (PF13895), or Ig_3 (PF13927) domain beginning its alignment before the start of the p20 alignment. We applied domain cutoff scores of 30.0 for p10, 21.8 for Ig, 27.0 for Ig_2, and 30.0 for Ig_3. Since a few proteins had multiple p20 domains, a cutoff score was implemented for those as well. First, the maximal p20 score in each sequence was extracted, then the cutoff was set to the minimal value among those maxima, i.e., 35.2. The p20 and p10 domain alignment positions were compiled into a domain architecture. Sequences with a p10 domain starting after the start of the p20 domain were classified as type I metacaspases if the distance between the domains was 66 amino acids or shorter, and otherwise as type II metacaspases. The 66 amino acid limit was based on known interdomain distances , using hmmalign versus the p20 HMM with the --trim option, followed by gap removal with seqmagick v0.6.24. The p20 domains were aligned using MAFFT v7.271 , situated on the surface of the domain , we first tried to identify paracaspases by searching for the presence of immunoglobulin-like (Ig) domains within the identified sequences. Neither Archaea nor Bacteria contained sequences encoding paracaspases, confirming their absence in prokaryotes. The presence of a p10 domain, located either N- or C- terminal to the p20 domain, defines metacaspases. Sequences with high similarity to the p10 domain could be identified in 11 archaeal and 1,121 bacterial sequences, representing 14.9% and 10.1% of all p20-containing sequences, respectively . No typeAll remaining sequences lacking the p10 domain were termed orthocaspases. Similar to bacterial orthocaspases , also arThe presence of the HC dyad within the p20 domain is an important prerequisite for proteolytic activity. Analysis of all archaeal meta- as well as orthocaspases revealed the presence of this conserved functional dyad in all 93 sequences . HoweverSince pseudo-metacaspases represented only 0.36% of all bacterial sequences with homology to the p20 domain, they were statistically irrelevant for further analysis. Bacterial pseudo-orthocaspases, however, represented a significant fraction of 8.6% of all bacterial caspase homologs, warranting further analysis. While 90% of all pseudo-orthocaspases were found in organisms belonging to either Alphaproteobacteria (582 sequences) or Cyanobacteria (193 sequences) , only feKtedonobacter racemifer DSM 44963 and Ktedonobacter sp.), while the third pseudo-orthocaspase was found in the class Anaerolineae (strain Anaerolineaceae bacterium 4572_78). Both classes belong to non-phototrophic Chloroflexi.Although Alphaproteobacteria were found to be rich in metacaspases, 80.4% of alphaproeobacterial sequences containing a p20 domain lacked the p10 domain and were classified as orthocaspases . Among tMore detailed analysis of the substitutions of the HC dyad in pseudo-orthocaspases of Rhizobiales showed the most common dyads to be Tyr – Ser (YS), Tyr – Ala (YA) or Phe – Ser (FS). While often both amino acid residues were substituted, also Gln – Cys (QC) and His – Ala (HA) combinations seem to be common. We further observed that each of the substitution variants corresponded to a certain overall protein domain architecture . While pAnabaena, Calothrix, Coleofasciculus, Lyngbya, Moorea, and Nostoc. However, as only few species within these genera contained metacaspases, metacaspases most likely have been acquired via horizontal gene transfer during the evolution of these complex cyanobacteria.Although cyanobacteria were thought not to contain metacaspases , we idenMicrocystis, unicellular nitrogen-fixing Gloeocapsa, to filamentous nitrogen-fixing Nostocales. Their catalytic dyad was most often substituted by YS and no other domains were identified  detected only in some filamentous cyanobacterial species, pseudo-orthocaspases with an YN dyad could be found in all analyzed organisms of these genera. It should, however, be noted that YN variants were found to be absent in unicellular nitrogen-fixing Cyanobacteria  as well as filamentous non-heterocystous Cyanobacteria , thus suggesting specific function of the YN variant in organisms with both properties: multicellularity and nitrogen-fixation. Only multicellular nitrogen-fixing cyanobacteria therefore contain two distinct pseudo-orthocaspase types, one with an YN substitution and the other with an YS substitution.Within the set of cyanobacterial pseudo-orthocaspases a large group of sequences contained YS and YN pseudo-catalytic dyads  and lackTrypanosoma brucei: Cys92, Asp95, Ser159 and Asp211  of d Asp211 ; the twod Asp211 . Highestd Asp211 , represe sp. T09  contain The two well-conserved Asp residues  are locaThe caspase-hemoglobinase is one of the most conserved folds, present in proteins of prokaryotic as well as eukaryotic organisms. It is composed of four beta-sheets surrounded by three alpha-helices that form a compact globular domain . This foIn this study we were able to identify the presence of not only orthocaspases, but also metacaspases in prokaryotes. Although metacaspases were earlier reported to be rather scarce in Alphaproteobacteria and absent in Cyanobacteria , we coulTrypanosoma brucei TbMC4, in which YS replaces the catalytic dyad , Prochlorococcus and Cyanobium and (ii) in Synechocystis sp. PCC 6803 as well as in Microcystis aeruginosa PCC 7806 genes encoding these pseudo-orthocaspases are strongly light-regulated  photosynthetic organisms carry pseudo-variants, an observation further supporting our hypothesis that pseudo-orthocaspases are involved in photosynthetic processes in Prokaryotes. However, pseudo-orthocaspases are absent in green- and purple-sulfur bacteria and only three pseudo-orthocaspase sequences within green non-sulfur photosynthetic bacteria (including the phylum Chloroflexi) were detected. The photosynthetic reaction centers (RC) of anoxygenic photosynthetic prokaryotes have a common origin, but diverged in the course of evolution e.g., . RCII ofGloeobacter, contains only the true variant, appearing to have diverged before the emergence of the distal YS pseudo-orthocaspases , an inactive caspase-8 homolog in humans, has been shown to act as dimerization partner of caspase-8  Various pseudo-variants are scattered throughout phylogenetically loosely related organisms , suggestcaspases . Enzymescaspases . The pseterparts . The pseaspase-8 . While tproteins .Arabidopsis thaliana was found to be independent of the presence of putative catalytic residues (in vitro as well as in vivo characterizations are necessary for our understanding of the function of the p20 domain in the various cellular environments they occur: from a simple unicellular bacterium to humans.The high conservation of pseudo-orthocaspases in Cyanobacteria and their putative involvement in processes of oxygenic photosynthesis on one hand, with their absence in eukaryotic Chlorophyta and Streptophyta  on the oresidues . FurtherAll datasets generated for this study are included in the manuscript and/or the All authors designed the study. MK and JA-S analyzed the data and drafted the manuscript. All authors revised 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.


It was clarified by this study that limited ductility of hexagonal Mg alloys could be overcome by activating unusual slip systems (<c + a> dislocations) in fully recrystallized UFG microstructures.Recently, we have found that fully recrystallized ultrafine-grained (UFG) microstructures could be realized in a commercial precipitation-hardened Magnesium (Mg) alloy. The UFG specimens exhibited high strength and large ductility under tensile test, but underlying mechanisms for good mechanical properties remained unclear. In this study, we have carried out systematic observations of deformation microstructures for revealing the influence of grain size on the change of dominant deformation modes. We found that plastic deformation of conventionally coarse-grained specimen was predominated by {0001} <11–20> slip and {10–12} <10–11> twinning, and the quick decrease of work-hardening rate was mainly due to the early saturation of deformation twins. For the UFG specimens, {10–12} <10–11> twinning was dramatically suppressed, while non-basal slip systems containing < However, so far, the applications of Mg and Mg alloys are substantially restricted and most of them are used in cast products. One of the reasons to hinder their widespread applications is the low strength, which is much lower than that of other metallic materials widely used. Another critical shortcoming is their poor ductility and formability. Unlike other metallic materials, such as steels and Al alloys having cubic crystal structures, Mg has a hexagonal close-packed (HCP) crystal structure with an axial ratio (c/a) of 1.624. As a result, only {0001} <11–20> basal slip system having <a> Burgers vector is easily activated at room temperature. However, basal slip can realize only 2-dimentional deformation in each grain, which is far less than the 5 independent slip systems required for free deformation in polycrystalline materials in von Mises criterion, resulting in limited cold-forming capability as well as pronounced mechanical anisotropy in Mg alloys8. Deformation twinning is another important deformation mechanism in addition to basal slip, which can accommodate strains along the c-axis during plastic deformation10. However, the deformation twins usually induce the formation of cracks or voids at grain boundaries they impinge, leading to failure11. Therefore, a lot of efforts have been paid to improve the strength and ductility/formability in Mg alloys.As the lightest metal, magnesium (Mg) and its alloys have long been considered as candidate materials to be used in aerospace and automobile industries12. However, high temperature processes cost and may lead to grain coarsening. Furthermore, intermediate annealing is often required during multi-pass high temperature deformation to avoid cracks, which increases the processing cost furthermore.Processing at elevated temperatures is one possible solution for improving the formability of Mg alloys, since multiple slip systems in addition to basal slip can be activated at elevated temperatures because of the temperature dependence of critical resolved shear stress (CRSS) for each slip system15. For instance, Mukai et al.15 found that the grain size of a commercial AZ31 Mg alloy could be refined to 1 μm after heavy deformation by equal channel angular pressing (ECAP) for 8 passes (effective strain ~9.2) at a temperature of 200 °C. Moreover, Mukai et al.16 have also demonstrated that the ductility in AZ31 and WE43 Mg alloys was significantly enhanced by the grain refinement. Recently, we have reported that the average grain size of a precipitation hardened Mg alloy (ZKX600) could be refined to 100 nm after SPD using high pressure torsion (HPT) by 1 rotation (360°) at room temperature. Furthermore, fully recrystallized ultrafine grained (UFG) structures which have not been achieved by other researchers were also obtained in the HPT processed material after subsequent rapid annealing17.On the other hand, Mg alloys can be deformed to ultra-high strains without cracking by severe plastic deformation (SPD) at relatively low or even ambient temperature18. In contrast, fully-recrystallized UFG ZKX600 alloy specimens exhibited both enhanced strength and ductility compared to those of the starting material having conventional coarse grained microstructure17. However, detailed strengthening and toughening mechanisms have not been clarified yet. Thus, the main purpose of this paper is to reveal the influence of grain size on the change of deformation mechanism in the ZKX600 alloy. We carefully observed the deformed microstructures of the specimens having two typical grain sizes and found that the dominant deformation mechanisms  have been significantly changed in the UFG specimen.It has been known that plastic instability happens in most UFG materials at early stages of tensile deformation because of highly enhanced yield strength and limitation of strain hardening in UFG structures, and thus it is difficult to manage both high strength and enough ductility in bulk UFG materials. For example, UFG Al and IF steel having sub-micrometer grain sizes exhibited quite early plastic instability, so that their uniform elongation is limited within a few percents1 phase, whose structure was a hexagonal structure as reported previously in various Mg-Zn based alloys19. Statistical length distribution of the rod-shaped precipitates is shown in Fig. As mentioned in the last section, the ZKX600 alloy is a typical precipitation hardened Mg alloy. The change of nano-precipitates during the present processing was investigated by transmission electron microscopy (TEM). Figure 1 phase was refined by HPT. The histogram of the length of the precipitates  scanning transmission electron microscopy (STEM) mode Fig. . Since ttes Fig.  indicateage Fig.  showed bage Fig. . The con17. Thus, in the present study, the specimen HPT processed by 360° (corresponding shear strain: γ = 23.55) was annealed at various temperatures for different holding periods, and then the specimens with recrystallized grain sizes ranging from sub-micrometer to several tens of micrometers were obtained. The EBSD inverse pole figure (IPF) maps and corresponding grain boundary (GB) maps of several representative specimens are displayed in Fig. It has been clarified by our previous study that saturated UFG microstructure could be realized after HPT by 360° rotationet al.21 have studied the texture evolution in a ZK60A Mg alloy during HPT and they found that a significant difference in the evolution of texture between the overall texture taken throughout the disc and the local texture at the disk edges up to at least 5 turns of HPT. That means, the selection of the measured area is critical for texture analysis in HPT processed Mg. In the present study, local areas corresponded to the center of the gauge part of the tensile specimens  of the 360° HPT processed specimen (d = 0.1 μm) reached 311 ± 3 MPa. However, premature fracture took place at a very limited total elongation (TE) of 1.2 ± 0.1% . The low ductility was primarily due to their very high YS and low work-hardening capability, which was caused by their very small grain sizes and high density of dislocations retained in the microstructure26. Interestingly, fully recrystallized UFG specimens could manage both high strength and large ductility. Particularly, the specimen with the finest recrystallized grain size (d = 0.77 μm) exhibited superior ductility (uniform elongation (UE)~20.5 ± 1.0%, TE~26.1 ± 1.4%), without sacrificing high ultimate tensile strength . With increasing the grain size, the strength decreased gradually, but the change in tensile elongations was not significant. For example, the UE and TE were maintained at 22.7 ± 1.8% and 28.0 ± 1.3%, respectively, in the specimen having a mean grain size of 6.11 μm. Strength and ductility decreased simultaneously by further increase of the grain size. The YS and TE decreased to 90 ± 5 MPa and 20.3 ± 2.0%, respectively, in the specimen having a coarse grain size of 23.3 μm. It is worth pointing out that, in addition to grain growth, coarsening of precipitates (β′1 phase) may take place during the long-time annealing treatment at high temperatures, which can also result in the decrease of the tensile strength.Nominal stress-strain curves of the as-deformed and the as-annealed specimens are displayed in Fig. Θ = dσ/dε, where σ and ε are the true stress and true strain, respectively), since uniform elongation in tensile tests is determined by the plastic instability condition . The Θ-ε curves and σ-ε curves of several representative specimens are plotted in Fig. Θ decreased quickly at very beginning of plastic deformation, followed by an increase of Θ upon further deformation, resulting in a V-shaped work-hardening curve at small strains (below 0.03). After that, the Θ value became higher than that of the coarse grained specimen (d = 23.3 μm), leading to relatively larger uniform elongation. As the grain size increased, the V-shaped valley became less and less obvious and eventually disappeared.It is well known that the large tensile ductility is often derived from the high work-hardening rate  and a coarse grained specimen (d = 23.3 μm) at various tensile strains were analyzed by a digital image correlation (DIC) technique29. As shown in Fig. It is considered that the V-shaped change of the work-hardening rate at small strains in the UFG specimens was related to the discontinuous yielding phenomena involving Lüders deformationd = 23.3 μm) were selected for detailed investigations on deformed microstructures. First, we focused on deformation twinning. Figure e = 0) was rather random. Besides, only a few {10–12} <10–11> twins (ftwin = 0.1%) were observed, which were considered to be introduced by mechanical polishing. At a tensile strain of 0.03, a fairly large number of lenticular-shaped deformation twins (ftwin = 8.8%) were formed. They were all {10–12} <10–11> twins, whose CRSS is significantly lower than other twinning systems in Mg alloys30. Since the {10–12} <10–11> twin rotates the lattice by 86°, it can effectively accommodate the extension strain along c-axis32. In addition, most of the twins appeared in blue or green colors in the partitioned EBSD-IPF maps  decreased with increasing tensile strain, supported such a tendency. Further deformation to a strain of 0.08, the area fraction of {10–12} <10–11> twins dramatically increased to 16.4%, and mutual intersection of twins were frequently observed. It is interesting that some grains were completely replaced by twins. However, at a strain of 0.15, which was almost twice as that of the previous stage (0.08), there was only a slight increase of the twined area (18.6%), indicating a saturation of deformation twinning around this strain level.A CG specimen  was used to distinguish the types of dislocations 34. Figure g) in Fig. g·b = 0” criterion, dislocations having <c> component are invisible in Fig. a> type dislocations should disappear in Fig. (a)) were observed. These dislocations were most likely basal dislocations having <a> component in Burgers vector. On the other hand, Fig. c> component in Burgers vector were still not activated. It should be noted that the activation of non-basal dislocations have been reported in some Ca-containing Mg alloys 36. However, those alloys were mostly simple solid solution Mg alloys. In contrast, the distribution of Ca in precipitation hardened Mg alloys were inhomogeneous. For example, in Mg-Zn based alloys, Ca precipitates out with Zn, forming nano-precipitates37. Thus, the role of Ca in the present quaternary alloy is somehow complicated and further investigations on this issue should be done in the future.The surface of the tensile specimen became very rough at higher strain levels and it was difficult to determine the activated slip systems through the EBSD assisted slip trace analysis. Instead, we carried out TEM observations of the CG specimen deformed to a tensile strain of 0.10. The “).Therefore, it is considered that the plastic deformation of the CG specimen was predominated by the {10–12} <10–11> twinning and {0001} <11–20> basal slip. The quick decrease of work-hardening rate was mainly due to the rapid growth and saturation of deformation twins, as indicated by the deformation microstructures observed Figs –7.7d = 0.98 μm), in the same way as those for the CG specimen  was totally free of twins, suggesting that the grain-refinement strengthening inhibited deformation twinning even during mechanical polishing. At a tensile strain of 0.04, only 0.3% of deformation twins were formed, which was significantly lower than that of the CG specimen (8.8%) at a similar tensile strain (e = 0.03). Again, these twins were exclusively {10–12} <10–11> twins. With the increase of tensile strain, the area fraction of twins increased gradually. Even at a strain of 0.14, however, the fraction of twins was only 1.5%, which was again far less than that in the CG specimen (18.6%) deformed to a similar strain.Identical area EBSD observations of deformation twinning was also conducted on the UFG specimen  exhibited very large room temperature tensile elongation of 0.47, which was mainly due to the cross-slip to non-basal planes induced by plastic compatibility. In contrast, Cepeda et al.39 reported reduced tensile ductility in the pure Mg when grain size was refined from 36 μm to 5 μm. Their slip trace analysis suggested a transition from the non-basal slips dominated deformation in the coarse grained (36 μm) specimen to the basal slip dominated deformation in the fine grained (5 μm) specimen, which was totally opposite to the result reported by Koike et al.38. Thus, we focused on investigating the active slip systems in the UFG specimen (d = 0.98 μm).Figure 20 zone axis by a minimum tilting. As indicated by the SAED pattern inserted in Fig. 38. Figure (a), from which profuse dislocations aligned roughly parallel to the basal trace (the broken line in (b)) were observed. These dislocations were most likely basal dislocations. Besides, some curved dislocation segments (indicated by red arrows) that were not parallel to the basal trace were also observed, indicating some non-basal slip systems were also activated. In order to know the Burgers vector of these dislocations, this grain was further observed from near the20 zone axis under two beam conditions. The operation vector (g) in Fig. a>. We also checked many other grains and they all exhibited similar dislocation configurations. Thus, we can conclude that the non-basal slip system having <c> component such as {11–22} second-order pyramidal slip was not activated at this strain level. The dislocation segments that were not parallel to the basal trace  were shown in Fig. c> component. These dislocations were further observed at higher magnification under a weak-beam dark field (WBDF) condition  operation40. The diffraction contrast (dislocation segments having bright contrast) in Fig. c> component were activated at this strain level in the UFG specimen. Therefore, we can conclude that deformation twinning was significantly suppressed throughout the entire deformation process in the UFG specimens, whereas the non-basal slip systems having <c> component Burgers vectors were activated to realize three-dimensional plastic deformation. The activation of the unusual slip systems is considered to contribute to the enhanced work-hardening rate in the UFG specimen, as is discussed in the next section.The UFG specimen was further observed at a tensile strain of 0.095 by TEM. Figure ion Fig.  through ), the relationship between the work-hardening behavior and the dominated deformation modes at various strain levels in the CG and UFG specimens is schematically revealed in Fig. 45. Afterward, the subsequent quick decrease of work-hardening rate is due to the deceleration and saturation of deformation twinning at the high strain level, as confirmed in Fig. a> component dominated the plastic deformation at the early stage. Subsequently, <c + a> dislocations were activated, which played a crucial role in strengthening the material, especially in producing deformation along the <c> axis. Additionally, activation of dislocations having totally different Burgers vectors would enhance interactions between dislocations, leading to an increase of work-hardening46, which was supported by the high density of tangled dislocations observed in Fig. Based on the systematic observations of deformation microstructures Figs –10, the 10et al.47 reported that a bulk specimen of pure Mg with a mean grain size of about 1 μm could be compressed to a large strain of 85% without fracture. Their observation of deformation microstructures and strain rate jump test suggested that intergranular deformation modes such as grain boundary sliding were responsible for the good formability of the UFG pure Mg. However, for the highly alloyed Mg-Zn-Zr-Ca alloy studied in the present study, the situation is greatly different. As shown in Fig. It should also be noted that, in addition to the activation of unusual slip systems, some other mechanisms such as grain boundary sliding are also expected to be important in UFG Mg, which could improve the ductility or formability of Mg. For example, Zeng c> component could be activated when the grain size was refined down to sub-micrometer scale. Consequently, synergetic improvement of strength and ductility was realized in the UFG specimen. The reason for the activation of unusual slip systems is still unclear. It is noteworthy, however, that UFG materials that can manage both high strength and large ductility have been recently found in particular alloys such as Cu-Al49 and high-Mn steels51, although it was considered that ultra grain refinement generally leads to the decrease of uniform elongation owing to early plastic instability. For example, in Cu-Al alloys having single-phased FCC structure with very low stacking fault energies, deformation twinning was found to be enhanced in fully recrystallized UFG microstructures49, which was totally opposite to the well-known grain size dependence of deformation twinning, i.e., deformation twinning becomes difficult to occur with decreasing the grain size. All those results suggest that there is an unknown mechanism to enhance unusual deformation modes in fully recrystallized UFG structures.It is an important and new finding that unusual slip systems having <d = 23.3 μm) was predominated by {10–12} <10–11> twinning and {0001} <11–20> basal slip, and the quick decrease of work-hardening rate at the later stage of plastic deformation was due to the deceleration and saturation of deformation twinning, (ii) deformation twinning was dramatically suppressed in the UFG specimen (d = 0.98 μm), while non-basal slip systems having <c> component Burgers vectors were activated successively. Particularly, the operation of the <c> component dislocations was responsible for the enhanced work-hardening rate. The present results showed a new insight that hexagonal Mg alloys can be very ductile by activating unusual deformation mechanisms (<c + a> dislocations) through realizing fully recrystallized UFG microstructures.In summary, the change of mechanical properties and deformation mechanisms in fully recrystallized UFG specimens were systematically investigated. We found that: (i) the plastic deformation of the coarse grained specimen  alloy produced by induction melting in Ar was used as the starting material. The as-cast alloy was provided for a multi-step thermo-mechanical processing, including solution treatment (ST), HPT to various rotation angles, and subsequent annealing treatment. The details of processing can be found in our previous publication4)2, 500 ml methanol and 100 ml 2-butoxy-ethanol at a voltage of 65 V and temperature approximately −50 °C.Microstructures of the specimens having various grain sizes were examined using scanning electron microscopy (SEM) and electron backscattering diffraction (EBSD). The specimens for SEM and EBSD analysis were mechanically polished using a fine grade sandpaper followed by a final polish using a colloidal silica suspension . EBSD results were analysed by TSL OIM software. Dislocation structures were observed by transmission electron microscopy (TEM). TEM specimens were firstly mechanical polished to 100 μm in thickness and then twin-jet electropolished using a solution of 5.3 g LiCl, 11.6 g Mg(ClO−4 s−1. The selected positions for microstructure observations and tensile tests as well as the detailed dimensions of the tensile specimen are shown in Fig. Mechanical properties of the specimens having various grain sizes were characterized by tensile test with an initial strain rate of 8.3 × 10Supplementary Information


No significant difference was noted between the compression of the ND and LD cups, nor between LD cups and LD augments. The LD cup displayed significantly lower elastic distension than the ND cup, most likely due to increased stiffness from the affixed augment and screw fixation.Acetabular cup deformation may affect liner/cup congruency, clearance and/or osseointegration. It is unclear, whether deformation of the acetabular components occurs during load and to what extent. To evaluate this, revision multi-hole cups were implanted into six cadaver hemipelvises in two scenarios: without acetabular defect (ND); with a large acetabular defect (LD) that was treated with an augment. In the LD scenario, the cup and augment were attached to the bone and each other with screws. Subsequently, the implanted hemipelvises were loaded under a physiologic partial-weight-bearing modality. The deformation of the acetabular components was determined using a best-fit algorithm. The statistical evaluation involved repeated-measures ANOVA. The mean elastic distension of the ND cup was 292.9 µm (SD 12.2 µm); in the LD scenario, 43.7 µm (SD 11.2 µm); the mean maximal augment distension was 79.6 µm (SD 21.6 µm). A significant difference between the maximal distension of the cups in both scenarios was noted  = 11.404;  Total hip arthroplasty (THA) is considered to be one of the most successful operations performed in orthopedic surgery and the treatment of choice for end-stage osteoarthritis of the hip . ConsequCementless acetabular components have achieved widespread acceptance in THA as a result of their improved and reliable long-term results . PrimaryThe goal of our study was to evaluate and compare the elastic deformation of in-vitro titanium press-fit cups in two revision THA scenarios during subjection to cyclic gait loading for varying time periods. One sample group consisted of cadaver bone implanted with only a revision Gription cup  (GC), whereas the second group was implanted with a revision Gription cup (GCS) and Gription augment (GAS) construct (Depuy/Synthes) both fixed to the pelvis with screws. We considered the diametrical change of the component rim during cyclic loading to relate to and reflect elastic deformation of the component that could potentially influence stable seating of the implant and the bone/implant apposition. This could contribute to micromotion on the implant-bone interface and between the cup and modular liner, respectively, which in turn might affect the osseointegration of the implant or influence the backside wear of the liner.This study was approved by the local ethics committee .Six fresh frozen cadaver hemipelvises were thawed at room temperature, dissected free of soft tissue. BMD measurements were done on attached femoral neck fragments prior to their removal from the hemipelvises. BMD was evaluated using Dual X-ray Absorptiometry (DXA)  and AP radiographs were also obtained on all specimens for pre-operative planning, and to exclude relevant pathology.2 (range 19.0 to 28.9 kg/m2). The mean bone mineral density, BMD, was 0.8 g/cm2 (range 0.641 to 0.924 g/cm2). Cadaver pelvises with a BMD below 0.6 g/cm2 were excluded.The average donor age was 78 years (range 51 to 96 years); the average donor mass was 66 kg (range 49 to 86 kg); and the average donor height was 171 cm (range 147.3 to 177.8 cm) resulting in a mean BMI of 23 kg/mWe compared two component scenarios that are frequently used in revision surgery.® Multihole with Gription® coating, DePuy Synthes Companies, Warsaw, IN, USA) was implanted press-fit in six hemipelvises without bone defects according to manufacturer recommendations as described in previous studies .,23.22,23The data were evaluated descriptively using the arithmetic mean, standard deviation, minimum and maximum. A repeated-measures analysis of variance (ANOVA) was performed to test for significant differences for the parameter of deformation (primary cup vs. cup with screws and cup with screws vs. augment). Prior to data analysis, the normal distribution of the data was evaluated using a Shapiro–Wilk test, which was chosen over other statistical tests of normal distribution since a prior study has shown that this test provides more power given a known significance than other tests of normal distribution . SubsequThe results are displayed below in Comparison of the compression and distension deformation between primary and revision cup:p = 0.698. There was no statistically significant difference for the compression cup deformation between the primary and revision groups, F = 1.740, p = 0.217, yet a tendency for higher elastic deformation of the ND cup (GC) can be found.The cyclical loading showed no statistically significant effect under the standardized loading conditions with regards to the compression deformation, F = 0.484, p = 0.666. The repeated measures ANOVA with Greenhouse-Geisser correction determined that the difference in distension deformation of the cups in scenario 1 (No Defect) and scenario 2 (Large Defect) was statistically significant, F = 11.404, p = 0.007.The duration of load  had no significant influence on the distension deformation, F = 0.368, Comparison of the compression and distension deformation between the revision cup and augment:p = 0.367. There was no statistically significant difference for the compression deformation between the revision cup and augment, F = 0.015, p = 0.904.Under the standardized loading conditions the cyclical loading showed no statistically significant effect with regards to the compression deformation, F = 1.048, p = 0.340. The repeated measures ANOVA with Greenhouse-Geisser correction determined that there was no statistically significant difference between LD cup (GCS) and augment (GAS) with regards to the distension deformation of each, F = 0.389, p = 0.547.The duration of load (the later load cycles) had no significant influence on the distension deformation, F = 1.104, The results are displayed in A large body of existing research has played an important role in the current success of both primary and revision THA, and resulted in a steadily increasing number of procedures and a decreasing age of patients ,26. LoosIn contrast to studies of micromotion and other contributing factors leading to implant failure, implant deformation has received relatively little research attention. Existing studies have focused almost exclusively on deformation changes that occur during the process of implantation, and have disregarded deformation occurring afterward. The deformation of press-fit acetabular cups into 1–3 mm under-reamed sockets has been documented in several prior studies ,37. PrioDuring forceful press-fit implantation (previously measured at 400 N with porous titanium acetabular cups in-vivo) ,37, the The focus and methodology of our current study differ in several respects from all prior studies of deformation. Our study does not evaluate deformation occurring from the process of implantation, but looks at elastic deformation occurring as a result of cyclic loading applied several days post-implantation. We utilized cadaver bone rather than the synthetic bone to permit the most realistic testing scenario, and measurements were made several days after implantation to minimize the elliptical distortion of the implantation process since it has been demonstrated that for several days post-implantation there is a visco-elastic relaxation of the pelvic bone and implant that reduces the deformation of titanium components . We focuOur results showed that all our study specimens displayed some elastic deformation during cyclic loading, but there was a distinct difference between the two groups. We found statistically significantly more elastic deformation in Scenario 1  than in Scenario 2 (revision cup plus augment and screws implanted into a Paprosky 2b defect). Values for the revision cup with screws and values for the augment with screws were not significantly different from each other, indicating that cup plus augment and screws tend to function as a unit.For all specimens, maximal elastic deformation (compression and distension) was reached after approximately 50 cycles of loading with no significant additional deformation noted with increased cycles.We hypothesize that the addition of screws and screws plus augment effectively increases the rigidity of the construct, which is consistent with findings of prior studies on deformation that occurred secondary to implantation, which showed that rigid CoCr cups deformed less than titanium cups . AdditioOur results are clinically significant in several ways. The use of ancillary screws decreased the degree of elastic deformation of the implant rim, allowing better bone/implant apposition, reduced gap areas, and potentially improved osseointegration. The presence of an augment did not negatively impact the degree of elastic deformation, and the cup/augment construct effectively functioned as a unit. Our findings also indicate that cyclic loading that mimics normal gait is associated with increased elastic deformation and validates prior findings ,35. We pCup diameter and wall thickness have repeatedly been shown to affect cup deformation. Large (jumbo) porous cups are an alternative option to cup/augment combination in revision THA associated with deficient bone stock and acetabular defects, and their large diameter and thin walls predispose to increased deformation, although ancillary screw fixation may limit this deformation. Oversized porous cups have been reported to have an increased risk of dislocation of multifactorial cause ,8,44. DeOur study has several limitations. The cohorts were small, and only two revision constructs were assessed, the first without substantial osseous defect, and the second using a substantial (Paprosky 2b) bony defect.The optical markers covered only half the implant rim, since the remaining rim was concealed from the optical cameras by the moving prosthesis neck—we assumed that the cup rim was symmetrical. Our methodology utilized the optical marker data to derive a “best-fit circle” and the change of circle diameter was considered to reflect elastic deformation. Therefore, our results are not a direct measure of deformation or bone/implant gap regions, but serve only as an indirect indicator, and can only be used as a means of comparison between our two study groups. This is the first study to utilize this methodology and evaluate deformation during loading, and there are therefore no currently existing comparable data for verification.We utilized cadaver bone since the polyurethane models were validated only for deformation occurring during implantation  and we c2 (range 0.641 to 0.924 g/cm2) and values beyond this range may have different results.There is tentative evidence that hardness of bone, related to BMD, necessitates increased compressive force for implantation that may affect results. Our cadaver sample was from older patients with a mean BMD of 0.8g/cmIn our experimental set-up, only Paprosky 2b defects were examined, and defects of other grades may have different results.Multi-hole cups have been shown to present more deformation than single-hole cups during implantation , which aVariations in surgical technique cannot be excluded although the same highly experienced surgeon (RGB) performed all implantations with supplied manufacturers tools.In conclusion, our in-vitro study utilizing revision constructs in cadaver bone is the first to compare the elastic deformation occurring at the rim of two different implant constructs during cyclic loading that replicates the limited loading of normal gait as experienced in the early postoperative period. Our results show that the use of adjunctive screws significantly decreases the degree of elastic deformation under these conditions, and the inclusion of an augment does not adversely impact the degree of elastic deformation.