Latent class analysis identified three groups: low cognitive and emotional relative deprivation , high cognitive and low emotional relative deprivation , and high cognitive and emotional relative deprivation . Adolescents with low income and without parental accompaniment were more likely to be assigned to Classes 3 and 2. Compared with Class 1, Classes 3 and 2 had significant positive predictive effects on physical aggression, relational aggression, and overall aggressive behavior. The classes of relative deprivation influenced both physical and relational aggression, but not verbal aggression. Based on these findings, demographic characteristics and latent classes of relative deprivation should be considered together when developing interventions for aggressive behaviors.Given the frequent occurrence of relative deprivation among adolescents and its negative effects, this study investigated relative deprivation among adolescents using a person-centered statistical technique ( Income inequality in China has escalated due to the general economic growth since 1978 . ExposurRelative deprivation theory divides personal relative deprivation into two components: cognitive relative deprivation, referring to one’s perceptions of deprivation, and emotional relative deprivation, referring to one’s feelings about deprivation. The emotional component of relative deprivation is an essential element based on the cognitive component. However, the emotional component might not always align perfectly with the cognitive component . For insThis study used LCA, a person-centered statistical method, to identify patterns in the cognitive and emotional dimensions of relative deprivation among adolescents and their relationship with diverse aggressive behaviors. LCA estimates class sizes based on a specific pattern of category probabilities for each item and assigns individuals to latent classes based on the highest membership probability. This analysis method has been widely used to identify latent groups in developmental psychology research because it enables the identification of specific patterns and alternative explanations for quantitative constructs ,13. We uRelative deprivation results from an individual’s subjective perception that she or he is in a disadvantaged situation based on upward social comparison with others in similar situations. Considering the high signification of peers during adolescence, individuals may unintentionally compare themselves to other adolescents . Family Finally, we proposed that gender may also play a significant role in relative deprivation. As the country is rooted in patriarchal traditions, for Chinese men, the importance of pursuing a career and taking responsibility for supporting a family has been historically underscored . For insThe prevalence of Chinese adolescents witnessing or experiencing violence has been reported to be 33.8% and 41.7%, respectively , making Despite previous research having established a link between relative deprivation and an increased risk of aggressive behavior among adolescents, different types of aggression  might have distinct causes. Hahn et al. , for insTo the best of our knowledge, this study is the first to investigate the relationship between the patterns of the cognitive–emotional dimensions of relative deprivation. This approach allows us to identify relative deprivation subgroups and their relationships with diverse aggressive behaviors and can inform intervention strategies to promote healthy development and prevent negative consequences. Therefore, this study aimed to support related research by using LCA to identify the effects of relative deprivation subgroups on different aggressive behaviors among adolescents.This study used LCA to identify relative deprivation subgroups. Furthermore, we examined the relationships between predictor variables and these subgroups in adolescents. Finally, multiple regression techniques were used to test the specificity of relative deprivation’s effects on various types of aggressive behavior. Accordingly, this study contributes to developing interventions to promote healthy adolescent development and prevent the negative consequences of relative deprivation.M age = 14.75 years, SD = 1.69) from six senior and high schools in two provinces of China responded to the anonymous questionnaire. Written informed consent was obtained from the adolescent participants’ teachers and parents/legal guardians before the survey. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Shanghai Normal University (protocol code 076 on 6 November 2022). Ethics approval was obtained from the authors’ affiliated institution. In the sample, 46.31% of the participants declared a monthly per capita family income of ¥0–3000, and 12.88% were above ¥8000. According to the participants, 90.8% of their fathers and 87.0% of their mothers had an educational level above senior middle school. Considering that some parents work in areas other than where their homes are, 7.83% of participants reported that they lived with both parents, while 14.52% lived with only one parent.We used the random cluster approach to recruit participants. Questionnaires were anonymized to encourage honest reporting and avoid social desirability bias. A sample of 1196 adolescents . Example items include “Compared with my friends, my parents are generally less supportive of me than their parents are of them” and “I’m frustrated by my pocket money relative to my friends”. We used the average scores for these items to assess relative deprivation levels; higher scores indicated higher levels of relative deprivation. In this study, the Cronbach’s αs were 0.807 , 0.867 , and 0.879 .Relative deprivation was measured using the Adolescents’ Relative Deprivation Scale . It inclnot at all to 5 = absolutely like me). The average score of the five items was used to assess life satisfaction, and higher scores mean higher life satisfaction. In this study, Cronbach’s αs for the subscales  and the whole scale were 0.79, 0.51, 0.66, and 0.84, respectively.Aggressive behavior was measured using the 19-item Chinese version of the aggression questionnaire . The ChiWe used SPSS 26.0 to summarize the descriptive statistics and explore the associations between the study variables. Then, we used latent profile analysis to identify patterns of relative deprivation among adolescents in Mplus 7.4 . A serieTo examine the relationships between demographic characteristics and latent classes of relative deprivation, we used the most likely class membership as an observed variable in a multinomial logistic regression in SPSS. Specifically, after choosing the reference class, dummy variables were created for each of the other classes, which were then allowed to simultaneously examine various demographic variables. Finally, a multiple linear regression model was used to investigate the relationship between latent classes and aggressive behaviors.p < 0.001, η2 = 0.75]. Furthermore, each item in Class 1  had lower scores than those in Class 2 , and the scores for each item in Class 2 were lower than those in Class 3 . However, Class 2 had higher scores for cognitive relative deprivation than emotional relative deprivation.We used multinomial logistic regression to test whether demographic characteristics affected latent class assignment. The data are shown in B = 0.161, p < 0.001), relational aggression , and overall aggressive behavior . Compared with Class 1, Class 2 had positive predictive effects on physical aggression , relational aggression , and overall aggressive behavior . Furthermore, the predictive effects of Class 2  and Class 3  on verbal aggression were not significant.We performed multinomial linear regression using the mean whole scale and subscale scores for each class. The data are shown in This study is the first to examine the latent classes of relative deprivation based on cognitive–emotion components and their effects on various aggressive behaviors. Our analyses of the participants’ response patterns led us to identify three latent classes. The analyses of demographic characteristics revealed possible risk factors for relative deprivation. The findings revealed that the three latent classes had significantly different aggressive behavior levels. These findings highlight the individual differences in adolescent relative deprivation and provide useful reference data for developing effective intervention strategies.Adolescents in Class 1 had lower levels of both cognitive and emotional relative deprivation than those in other classes. Those in Class 3 had higher levels of both cognitive and emotional relative deprivation. Interestingly, in Class 2, which had high cognitive but low emotional relative deprivation, the scores for each item were in the middle compared with those of the adolescents in Classes 1 and 3. Relative deprivation theory proposes that the leading cause of relative deprivation is the belief that it is unfair for others to have a better social status than oneself . EmotionThe implications for interventions differ depending on the classes of relative deprivation. If the level of relative deprivation is moderate (Class 2), the interventions for cognitive strategies might be more efficient than those for emotional strategies. Thus, cognitive interventions could be developed to enhance the attentional filtering ability of Class 2 adolescents to reduce their cognitive relative deprivation . HoweverWe further explored the relationship between demographic characteristics and the classification of LCA. Objective status  provides a foundation for social comparison and can cause adolescents to assign themselves to a given relative position . Some stPrevious studies prove that relative deprivation can contribute to aggressive behavior ,4,38,39.Compared with individuals in Class 1, those in Classes 3 and 2 were linked to high physical aggression, relational aggression, and overall aggressive behavior. Previous research suggests that physical and relational aggression have a consistent underlying motivation  and are This study has several limitations. First, the LCA results, based on observation data, are restricted to the characteristics of adolescents in China. Although existing research suggests that relative deprivation is a consistent predictor of individual development across different cultures, cultural differences are associated with the effects of relative deprivation . Future Moreover, this study can help improve future research. Using our LCA-based findings as reference data to develop interventions could give researchers a more holistic account of individual differences in relative deprivation among adolescents. Furthermore, our findings based on demographic characteristics highlight the potential risk groups of relative deprivation among adolescents. We also explored how and toward whom the latent classes of relative deprivation affect physical, relational, and verbal aggression behavior. These findings offer valuable insights that can inform targeted recommendations for interventions aimed at addressing the adverse effects of different subgroups of relative deprivation.Since the prevalence of relative deprivation among adolescents worldwide, identifying patterns of relative deprivation and examining their causes and outcomes could provide critical implications for theory and practice. In this study, we used LCA to identify three classes of relative deprivation based on cognitive and emotional relative deprivation: “high cognitive and emotional relative deprivation class” (Class 3), “high cognitive and low emotional relative deprivation” (Class 2), and “low cognitive and emotional relative deprivation class” (Class 1); this suggests that the heterogeneity of relative deprivation among adolescents exists. Moreover, the desynchronization of the cognitive and emotional relative deprivation class was observed within classes, supporting the notion that emotional relative deprivation does not always align with cognitive relative deprivation. Therefore, interventions aimed at decreasing adolescents’ relative deprivation should consider both cognitive and emotional relative deprivation. Interventions must be designed to target cognitive or emotional components.In addition, we found that a lack of parental accompaniment and low family income can increase the risk in Classes 2 and 3, highlighting the influence of both subjective and objective resources on adolescents’ relative deprivation. Therefore, parents who leave their adolescents at home due to work obligations could use technological devices to keep in touch with adolescents and meet their belonging needs . FurtherFinally, previous research has examined the influence of relative deprivation on aggressive behaviors ,24. In t


Saccharomyces cerevisiae genome annotations, we analyzed the domain gain and loss events in the predicted proteomes of 9 non-human primates (NHP) and 20 non-S. cerevisiae fungi (NSF) as annotated in the Uniprot and Interpro databases. Our approach allowed us to quantify the impact of errors on estimates of protein domain gains and losses, and we show that domain losses are over-estimated ten-fold and three-fold in the NHP and NSF proteins respectively. This is in line with previous studies of gene-level losses, where issues with genome sequencing or gene annotation led to genes being falsely inferred as absent. In addition, we show that insistent protein domain annotations are a major factor contributing to the false events. For the first time, to our knowledge, we show that domain gains are also over-estimated by three-fold and two-fold respectively in NHP and NSF proteins. Based on our more accurate estimates, we infer that true domain losses and gains in NHP with respect to humans are observed at similar rates, while domain gains in the more divergent NSF are observed twice as frequently as domain losses with respect to S. cerevisiae. This study highlights the need to critically examine the scientific validity of protein annotations, and represents a significant step toward scalable computational fact-checking methods that may 1 day mitigate the propagation of wrong information in protein databases.Protein annotation errors can have significant consequences in a wide range of fields, ranging from protein structure and function prediction to biomedical research, drug discovery, and biotechnology. By comparing the domains of different proteins, scientists can identify common domains, classify proteins based on their domain architecture, and highlight proteins that have evolved differently in one or more species or clades. However, genome-wide identification of different protein domain architectures involves a complex error-prone pipeline that includes genome sequencing, prediction of gene exon/intron structures, and inference of protein sequences and domain annotations. Here we developed an automated fact-checking approach to distinguish true domain loss/gain events from false events caused by errors that occur during the annotation process. Using genome-wide ortholog sets and taking advantage of the high-quality human and  Extensive amounts of data from next-generation sequencing have led to the accumulation of information that provides insight into the evolutionary landscape of related proteins . ComparaThe most common changes observed in both ortholog and paralog pairs involved loss/gain of domains, while domain shuffling and segment duplication/deletion are less frequent . Howeverhttp://www.genomesonline.org/cgi-bin/GOLD/index.cgi). The subsequent genome annotations of gene intron/exon structures are also of highly diverse quality. For model genomes, for example from human, mouse or the yeast Saccharomyces cerevisiae, extensive transcriptome data is available allowing more accurate gene identification  respectively from the UniprotKB database, and evaluate all potential domain gain and loss events using the well-studied human and S. cerevisiae proteomes as a reference. However, the method should be easily applicable to other ortholog sets, where it can be used to filter the potential domain events that are due to annotation errors, thus highlighting candidates for true evolutionary novelty.This article investigates the effects of systematic errors on the ability to identify true domain events. We developed an automated fact-checking method that identifies errors at three main levels: genome sequencing/assembly, gene prediction, and domain identification. The protocol relies on the availability of high quality protein sequences from a model species, which we call the reference species, and takes advantage of the single copy (or one-to-one) orthologs widely used for evolutionary studies and functional inferences . We applS. cerevisiae proteins were identified in the Uniprot database version 2022_03. For each protein, one-to-one orthologs for 9 NHP and 21 NSF species were then extracted from the OrthoInspector database (v3) (Human and ase (v3) . For allase (v3) , using tFor all genomes, we also collected BUSCO complete metrics as provided in . BUSCO iS. cerevisiae reference protein as appropriate, identifying sequences that had either missing or additional domains. The domain content of a protein was defined as the list of distinct Pfam-A domains. Only distinct domains were taken into account, ignoring duplication events that create new copies of the same domain. Architectures with either different domain counts or order were considered to have the same domain content  was used to search each protein sequence against the Pfam HMM-profiles with an E-value threshold of 0.01 and the gathering (GA) threshold option.For all ortholog pairs with different domain contents, the domain annotations were re-computed using the same protocol. Pfam HMM-profiles (version 35.0 November 2021) were downloaded from S. cerevisiae protein sequence. A new predicted protein sequence was then extracted from the prosplign alignment using an in-house script. To evaluate the new predicted sequences, we searched the Pfam HMM-profiles (version 35.0) against the sequences using the HMMER tool (version 3.3.2) with E-value threshold of 0.01 and the gathering (GA) threshold option.For each orthologous sequence with a different domain composition, the prosplign algorithm implemented in the NCBI Genome Workbench  was usedThe genomic sequence coding for the ortholog proteins (NHP/NSF) were parsed to identify undetermined nucleotides, represented by ‘N’ characters.To investigate whether missing domain (MD) or additional domain (AD) events occurred predominantly at the N/C-termini of proteins rather than internally, we determined the ordinal positions of true MD/AD events with respect to the domain architecture. To do this, we considered only ortholog pairs where both proteins had at least two domains. For each protein, the annotated domains were ordered from the N-terminus to the C-terminus, and the position of the MD/AD was classed as either N-terminal, C-terminal or internal.https://ftp.ebi.ac.uk/pub/databases/Pfam/current_release/Pfam-A.clans.tsv.gz.The domain entries in Pfam are grouped hierarchically into clans if they are considered to have arisen from a single evolutionary origin, assessed by the presence of related 3D structures, related functions, significant matching of the same sequence to HMMs from different families and profile–profile comparisons. We retrieved the clan classification for all Pfam-A domain entries from the Interpro resource at S. cerevisiae) and non-reference species (NHP/NSF), (i) for NHP, we downloaded the species tree from the Ensembl database, and (ii) for NSF, we retrieved a recently published genome-level phylogeny (http://cran.r-project.org/package=caper) v0.5 package for R. Following standard practice, delta (δ) and kappa (κ) were set to 1 while the maximum-likelihood value of lambda (λ) was estimated and used to transform branch lengths.To estimate the phylogenetic distance between the reference species , but proteins are generally more complex, with multi-domain architectures. Furthermore, some of the genome assemblies are incomplete, as estimated by their contig N50 metric of <100 Kb compared to >10 Mb for the higher quality genomes or 55 Mb for the human genome. The contig N50 is defined as the length of the shortest contig for which longer and equal length contigs cover at least 50% of the assembly. The NSF represent more divergent species (mean sequence identity ranging from 24.5% to 41.8% with S. cerevisiae). However, the genomes are smaller and gene prediction is simpler since the genes are less intron-rich.We applied our protocol to two data sets containing: (i) 9 NHP proteomes  which weS. cerevisiae) and the corresponding non-reference proteomes . Orthologs were extracted from the Ortholnspector database and the sequences that were cross-referenced in the Ensembl genome database were retained. In order to avoid redundancy and bias in the data sets, only one-to-one orthologs were considered. For the primate dataset, this resulted in 19,251 human proteins  that had at least one-to-one ortholog, and a total of 152,240 orthologous NHP sequences  were retained and a total of 40,410 orthologous NSF sequences  compared to human proteins (97%), due to the higher divergence of the selected species in the fungi dataset. The number of orthologous NSF sequences retained for each species ranges from 1819–2,889, with the exception of the two obligate unicellular parasites (Nosema ceranae and V. culicis) that share less than 500 orthologs with S. cerevisiae, and A. macrogynus that has a higher level of gene duplications resulting in a larger proportion of one-to-many orthologs and only 402 one-to-one orthologs.We then identified all ortholog pairs between each reference proteome  presenting an AD. The Microsporidia parasites N. ceranae and V. culicis had a smaller number of potential AD (15 and 14 respectively) as might be expected since they are obligate unicellular parasites with a smaller proteome (467 and 491 protein orthologs respectively), but a much larger number of MD (178 and 191 respectively). For the remaining NSF, the MD/AD ratio varied from 0.5 (i.e. AD are twice as frequent as MD) for Spizellomyces punctatus to 1.6 (i.e. MD are more frequent than AD) for E. lata and A. macrogynus. No correlation was observed between the MD/AD ratios and the NSF clades, but the overall frequency of events per ortholog was generally lower for the Ascomycota, with on average 14% of orthologs with domain events, compared to Basidiomycota, Blastocladiomycota and Chytridiomycota, with 19%, and Microsporidia with 41%.For the fungi dataset , a totalWe used our fact checking approach  to evaluS. cerevisiae), we identified 1713 (38%) and 2008 (50%) MD events for NHP and NSF respectively linked to inconsistent domain annotations. These false positive MD events include 1,147 and 1,614 cases for NHP and NSF respectively, where the MD was identified in the orthologous sequences by our protocol. In other words, while the NHP/NSF protein was not annotated with the domain in the Interpro database, increasing the hmmsearch E-value allowed us to identify the MD. As an illustrative example, the human protein P55318 (FOXA3_HUMAN: transcription factor forkhead box protein 3) and the macaque ortholog F6Q3J5 both contain a central forkhead domain (PF00250), but the forkhead N-terminal region (PF08430) is not annotated in the macaque sequence. The N-terminal region was found in the human sequence with an hmmsearch E-value = 3.9 × 10−10, and in the macaque sequence with E-value = 1.6 × 10−8. This domain is also identified by a new Deep Learning method  is annotated with the Interpro domain “Zinc finger C2H2-type” (IPR013087), which combines signatures from two member databases SMART (SM00355) and Pfam (PF00096). The annotation file retrieved from Interpro contains both signatures, even though the Pfam signature does not match this specific protein with an E-value below the threshold.More surprisingly, we discovered 566 and 394 cases for NHP and NSF respectively, where the MD was not detected in the reference sequence by our protocol. Thus, the protein is annotated with the domain by Interpro, but is not detected by hmmsearch with an E-value threshold of 0.01. As an example, the Next, we focused on the identification of gene/isoform prediction errors for the remaining 2749 NHP and 1979 NSF potential MD events that could not be explained by domain annotation issues. After applying a simple correction protocol based on alignment of the reference protein sequence with the genomic sequence of the ortholog (see Methods), the MD was identified in 1368 NHP and 464 NSF orthologs. An illustrative example is shown in Finally, in step 3, we identified genome sequence issues, where 970 NHP domains and 272 NSF domains had a corresponding genome sequence containing undetermined characters (N’s). As a consequence, it is difficult to determine whether the missing domain is present or not.After the three stages of error detection , Table 6S. cerevisiae), 716 and 1,622 cases for NHP and NSF respectively were identified as false positives due to inconsistent domain annotations. These include 183 and 382 cases where the potentially gained domain was not identified in the NHP and NSF ortholog sequence respectively, as well as 533 and 1,240 cases where the AD was found in the reference sequence (human/S. cerevisiae) by our protocol. As an example, protein H2P1R9_PONAB is an ortholog of the human sequence Q96LM9 (CT173_HUMAN), an uncharacterized protein with an annotation score of 2/5 in Uniprot. Both proteins are annotated in Interpro as belonging to the GT29-like superfamily (IPR038578), but CT173_HUMAN has no Pfam annotations. H2P1R9_PONAB is annotated with a glycosyltransferase family 29 domain (PF00777) in the Interpro database and the domain is also identified using hmmsearch with an E-value = 2.0e-10. Using hmmsearch, PF00777 is also found in the CT173_HUMAN sequence with E-value = 3.2e-08. After the first step, 529 NHP AD events and 2161 NSF AD events remained as potentially true domain events.The first step is the identification of inconsistent protein domain annotations, as described in the previous section for MD. For the 1233 NHP orthologs and 3785 NSF orthologs with potential AD events compared to the reference (human/S. cerevisiae) in the Ensembl database, and checked whether an isoform was present that contained the AD. This step is only pertinent for the primate data set, since none of the S. cerevisiae genes corresponding to the potential AD events had known isoforms. For the NHP proteins, a total of 120 potential AD events were due to inconsistent isoform annotations.In the second step, we searched for alternative isoforms in the reference species . Gene prediction or isoform errors were the second most frequent causes of false positive domain events .N. ceranae and V. culicis from the NSF dataset, due to the bias in the MD/AD ratio for these parasitic fungi. First, we used the contig N50 as an estimate of genome sequence/assembly completeness , suggesting that the fact-checking protocol was successful in removing this bias. However, the correlation between N50 and the number of AD events is more complex. For NHP, the correlation is increased for true AD events  compared to potential AD events . For NSF, no significant correlations were found for the AD events before or after fact-checking.To further investigate the causes of the false domain events, we compared the rates of MD and AD events observed before and after the fact-checking protocol with two measures of genome and gene annotation quality. It should be noted that we excluded the two microsporidia species ess from  and compess from  and Speaess from . For botWe then used the BUSCO complete metric  as a comS. cerevisiae). Intuitively, we would expect more true domain events in species that are more phylogenetically distant. However, it may also be true that identification of domain events in more distant species is more difficult, giving rise to more errors. We estimated the phylogenetic distance from publicly available genome-scale trees (see Methods) and performed a phylogenetic generalized least squares (PGLS) regression analysis to account for the phylogenetic relatedness of the species  and true MD , but not for potential AD  or true AD . For NSF, no significant correlations were observed, except for true MD . Thus, our fact-checking protocol generally increases the correlation between phylogenetic distance and the number of predicted events, at least for MD, supporting the hypothesis that true MD events occur more frequently in more distantly related species. Again, the correlation for the number of AD events is more complex, and potential reasons for this are discussed below.We also compared the frequency of domain events before and after fact-checking to an estimate of the phylogenetic distance between the non-reference proteomes and the corresponding reference  = -5.70, p = 0.0005). However, we conclude that this difference is due to annotation errors, since after filtering of the false positives, MD and AD rates are the same (0.27% of orthologs with MD and 0.26% of orthologs with AD: paired t-test t (10) = -0.10, p = 0.92). Similar rates of MD and AD might be expected, since the absence/presence of a domain was arbitrarily defined to refer to the NHP protein compared to the human reference.For the NHP dataset, the potential MD events inferred from the public database annotations are more frequent than AD events , filtering of false positives led to more frequent AD in NSF with respect to S. cerevisiae than MD  = 2.61, p = 0.02). One reason for the higher rate of AD compared to MD may be the higher diversity of fungal genomes due to their diverse lifestyles and specific adaptations to natural or laboratory ecosystems  that were retained as true events in the primate dataset. Our initial error detection protocol identified AD and MD at the level of Pfam protein families. However, many Pfam families are related and can be classified hierarchically into clans. Pfam defines a clan as a collection of families that have arisen from a single evolutionary origin. Although domain families in the same clan are considered to be homologous, they are so evolutionarily diverse that the family members cannot be identified with a single HMM. Of the 808 domain events, 52 events  involved two domains in the same Pfam clan and were excluded from the subsequent analyses. The distribution of the remaining 756 events is provided in It has been proposed that novel combinations of preexisting domains had a major role in the evolution of protein networks and more complex cellular activities . In agreAnother example  is the NAn example of a true AD event is observed in all NHP orthologs of the human protein CMAH_HUMAN (Q9Y471), an inactive cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Gc) hydroxylase . The humIn this section, we investigate the 3406 domain events (1243 MD and 2163 AD) that were retained as true events in the fungi dataset. As for primates, we first checked for MD/AD in the same Pfam clan. Of the 3406 domain events, 567  involved two domains in the same Pfam clan and were excluded from the subsequent analyses. The distribution of the remaining 2,852 events is provided in . Most hemiascomycetes can grow on galactose as a sole carbon source, and in S. cerevisiae seven genes (the GAL genes) function exclusively in this pathway. Although orthologs of the GAL genes are present in many yeast genomes, they have been lost independently in several lineages  all share the GDP-mannose 4,6 dehydratase domain. However, only Schizosaccharomyces pombe exhibits both domains, while 4 orthologs correspond to partially uncharacterized genome regions and 12 have confirmed losses of the Aldose 1-epimerase domain. Cryptococcus neoformans is missing the DNA binding domain (PF00172), but the corresponding genome sequence is partially uncharacterized. A. oligospora has the dimerization domain (PF03902) after correction of the gene prediction. The loss of the dimerization domain is confirmed in the other identified orthologs.Other examples of true NSF MD events include the galactose metabolic processlineages . This spY. lipolytica ortholog of LDH1_YEAST (P38139), lipid droplet hydrolase 1, that plays a role in maintaining lipid homeostasis in S. cerevisiae  with Y. lipolytica and A. oligospora, but these two orthologs also contain a C-terminal dual specificity phosphatase, catalytic domain (PF00782).An example of a NSF AD is observed in the revisiae . YarrowiS. pombe and the two Microsporidia). The MaoC like domain (PF01575), with possible hydratase activity, is lost in the two Chytridiomycota, and the Basidiomycota W. ichthyophaga and Coprinopsis cinerea. In C. cinerea, the MaoC like domain is found in a separate downstream gene, suggesting a possible fusion/fission event.More complex MD/AD events are found in orthologs of the reference protein FOX2_YEAST (Q02207), a peroxisomal hydratase-dehydrogenase-epimerase trifunctional protein, with 17 identified NSF orthologs (no orthologs were found for Ustilago maydis), clearly demonstrating the necessity for reliable quality control and error detection.Analysis of domain architecture changes is important for fundamental studies of evolution and applications in the use of non-human primates in medical studies, for example , or in tAfter error filtering, true domain events are observed with a frequency of 0.5% for NHP orthologs and 7.7% for NSF orthologs. This difference highlights the different nature of the two datasets used in this work. The datasets were chosen to represent the main challenges for domain annotation pipelines. The first dataset includes closely related NHP species (sharing >89% protein sequence identity with human) with a common ancestor that appeared recently around 50–55 Mya . True doThe fact-checking method used here confirms that errors or inconsistencies are widespread in the protein database entries corresponding to the two datasets. For both NHP and NSF, the most frequent source of errors was heterogeneous domain annotations between orthologs. InterPro combines predictive models from several different databases, including Pfam into a single resource. The entire InterPro database is regularly reviewed for accuracy, and matches for sequences from UniprotKB are updated or removed where necessary . These eGene prediction failures were the second most frequent source of errors, particularly for NHP proteins. The relative difference between NHP and NSF is probably explained by the fact that the problems of gene prediction are less severe in the case of fungi that generally have less intron-rich genomes than primates. Furthermore, in primates, some potential domain events also reflect the presence of alternative splicing isoforms, and the fact that the UniprotKB database may define different canonical isoforms for the orthologs of different species.Finally, 22% of domain events for NHP and 7% for NSF, were mapped to incomplete or uncharacterized genome sequences. In this case, the protocol could not distinguish between true and false events. Nevertheless, genome sequence quality should improve in the near future with the application of long read sequencing technologies, for example.Three important caveats should be kept in mind. First, we extracted ortholog information from the OrthoInspector database, which identifies pairwise orthologs based on protein sequence comparisons. This implies that orthologs with very different domain architectures may be missed by the algorithm, and thus our list of domain events may be incomplete. In contrast, we assumed that the identified ortholog pairs are accurate, since OrthoInspector achieved a precision >99% in independent benchmarking tests . AlthougSecond, our more relaxed definition of the ‘domain content’ of proteins considers only the set of domains present in a protein irrespective of domain order or domain duplications. This definition focuses on the important evolutionary events involving loss or gain of unique domains and has been used previously to study evolution of multidomain proteins . NeverthS. cerevisiae). This does not necessarily reflect the underlying evolutionary mechanisms and cannot distinguish between a domain being recruited in one protein or lost in the other. Therefore, our study does not provide further evidence concerning the debate about the relative rates of domain losses and gains in a given clade. Resolution of the evolutionary mechanisms underlying the domain content differences observed in this work would require detailed phylogenetic analyses and reconstruction of ancestral states.Third, it should be noted that our protocol uses pairwise sequence comparisons and MD/AD events are defined with respect to the reference protein (human or Based on the true events, we performed some preliminary analyses to investigate the functional relevance of our findings. After filtering of errors, similar rates of MD and AD are observed in the closely related NHP, while AD are more frequent than MD in the NSF that have adapted to more specific ecosystems. The latter is in line with previous findings that emergence of novel domains is foremost associated with environmental adaptations . The mosIn the future, we plan to extend the functional analyses to investigate the potential enrichment of the domain gain/loss events observed here in specific cellular pathways or processes. We will also apply the fact-checking approach to other domain events, such as domain shuffling or domain duplications, observed in primate and fungi proteins. An important application will be the comparison of the evolution of domain architectures between orthologs and paralogs. For example, are domain architectures more similar between orthologs than paralogs at the same degree of evolutionary separation? While orthologs are generally expected to experience stronger evolutionary pressure to maintain the same function than paralogs, it would be interesting to test whether functional conservation is associated with a higher conservation of domain architectures, i.e., if domain architecture is an important vehicle of protein function.


An equiatomic CrCoNi medium-entropy alloy was subjected to high-energy shot peening (HESP) to fabricate a gradient nanostructure (GNS) in this work. The microstructures of the GNS samples at different depths within the deformed layer were thoroughly investigated. The microstructure exhibited a transformation from unstressed coarse grains to deformed coarse grains, followed by the formation of ultrafine grains, and ultimately reaching a final nanocrystalline structure with a uniform size of approximately 50 nm. Detailed structural analysis indicated that the deformation process was primarily influenced by the interaction between dislocations and deformation twins, which was attributed to the low stacking fault energy (SFE) of the alloy. The nanocrystalline mechanism was divided into three stages. In the coarse-grained deformation stage, the dislocation band divided twin/matrix lamellae into sub-segments, and the cross twin divided coarse grains into ultrafine grains simultaneously. In the ultrafine grain deformation stage, dislocations were arranged around the deformation twins in order to break the twins to form incoherent boundaries, destroying the coherent relationship between the twin and matrix. Finally, in the nanocrystalline deformation stage, the nanocrystalline structure was further divided into smaller segments to accommodate additional strains through the interaction between dislocations and twins. The phe1120]HCP ,43. Rece5Mo0.05C , Fe20Mn2Co20Cr20 , and NiCCo20Cr20 ,46 MEAs.Co20Cr20 . In addiThe gradient nanostructure layer was prepared on the surface of CrCoNi MEA after HESP. The high density of dislocations and twins always exists in the deformed grains, new boundaries are constantly formed in the deformation process, and segmented microstructures appear on smaller scales. The deformation mechanism, the interactions between twins and dislocations, and the grain refinement mechanism can be summarized in the process of microstructure evolution.The current study discovered that as the stress level increases, the evolution of dislocations in the grains progresses from low-density dislocation lines to high-density dislocation entanglement and eventually to much higher-density dislocation bands. However, after the formation of a nanocrystalline structure, the dislocation density in the grains gradually decreases. Dislocation activity always plays a crucial role during the deformation process. Firstly, the slip of Shockley partial dislocation can generate stacking faults and deformation twins, which is a typical phenomenon in FCC metals with low SFE during severe plastic deformation . SecondlThe evolution of deformation twins in the deformation process of CrCoNi MEA varies with the strain level. The deformation twins begin to form at 150 μm depth in large quantities and maintain a high-density structure at each deformation stage. Several studies have found that when a dislocation slips across the twin boundary, mobile dislocation, pinned dislocation, and stacking faults occur at the twin boundary, complicating the twin boundary structure ,50. At t2) [2) [The nanocrystallization formation mechanism of CrCoNi MEA induced by HESP is schematically illustrated in 2)  and Ni (2) [2) . As the At the stage of ultrafine grain deformation, the dislocation–twin interactions dominate the deformation process. (1) A gradient nanostructure was formed on the surface of the CrCoNi MEA, consisting of distinct zones: a nanocrystalline zone, an ultrafine grain zone, a coarse-grained deformation layer, and a strain-free coarse-grained zone. The nanocrystalline layer had a thickness of approximately 20 μm, with the grain size refined to approximately 50 nm.(2) The morphology of dislocations in the grains evolved from low-density dislocation lines to high-density dislocation entanglements and dislocation bands, progressing from the core to the surface. Dislocation activity resulted in the generation of stacking faults, deformation twins, and HCP structures. The deformation twins penetrated the grains, contributing to grain refinement, and the spacing between twin lamellae decreased with the increasing strain level.(3) The nanocrystallization formation mechanism could be summarized as follows: the dislocation–twin/matrix and twin–twin interactions separate coarse grains into ultrafine grains. With further deformation, the severe interaction between the dislocation and twin breaks the coherent twin area, and highly misoriented HAGB is generated by sub-grain rotation. At a higher stress level, the stepped deformation twins refine the nanocrystalline structure to a smaller scale.


However, due to the brittle nature of SiO2, such micromirrors may be incapable to survive even slight collisions, which greatly limits their application range. To improve the robustness of electrothermal micromirrors, a polymer material is incorporated and partially replaces SiO2 as the electrothermal isolation and anchor material. In particular, photosensitive polyimide (PSPI) is used, which also simplifies the fabrication process. Here, PSPI-based electrothermal micromirrors have been designed, fabricated, and tested. The PSPI-type micromirrors achieved an optical scan angle of ±19.6° and a vertical displacement of 370 μm at only 4 Vdc. With a mirror aperture size of 1 mm × 1 mm, the PSPI-type micromirrors survived over 200 g accelerations from either vertical or lateral directions in impact experiments. In the drop test, the PSPI-type micromirrors survived falls to a hard floor from heights up to 21 cm. In the standard frequency sweeping vibration test, the PSPI-type micromirrors survived 21 g and 29 g acceleration in the vertical and lateral vibrations, respectively. In all these tests, the PSPI-type micromirrors demonstrated at least 4 times better robustness than SiO2-type micromirrors fabricated in the same batch.Electrothermal bimorph-based scanning micromirrors typically employ standard silicon dioxide (SiO Several types of MEMS micromirrors exist, including electromagnetic, electrostatic, and electrothermal micromirrors10. Compared with electromagnetic and electrostatic micromirrors, electrothermal micromirrors use thermal bimorph actuators and offer the advantages of large displacement, large nonresonant angular scan range, high fill factor, and low driving voltage12. Thus, they have been applied in optical endoscopic imaging13 and Fourier transform near-infrared spectrometers14. The most commonly used bimorph actuator materials for electrothermal micromirrors are Al and SiO2 due to their wide availability and large differences in their coefficients of thermal expansion (CTE). SiO2, due to low thermal conductivity, is also used to provide thermal isolation at anchors to reduce power consumption15. However, electrothermal micromirrors with SiO2 anchors are susceptible to impact failure due to the brittle nature of SiO2. The typical fracture strength of thin-film SiO2 is only 0.77 MPa m1/2 (ref. 16), which is 1/40 that of aluminum17. As a result, such electrothermal micromirrors are easily damaged by accidental collisions or drops. For instance, some electrothermal micromirrors may not even survive a drop from a height of a few centimeters18. Experimental analyses have shown that device failure is mainly caused by the fracture of SiO2 anchors at the ends of bimorph actuators19. This susceptibility to impact failure largely limits the application range of electrothermal micromirrors, especially in hand-held optical imaging probes, which often experience accidental drops or collisions.Microelectromechanical system (MEMS) scanning micromirrors have recently attracted broad interest due to their potential applications in LiDAR18, but the unidirectional operation, nonstationary center of rotation, and large initial tilt angle of this 1D micromirror made the device packaging and optical design more complex and difficult20. Furthermore, the PI etching required a hard mask material, and the fabrication process was not applicable to make electrothermal micromirrors with more sophisticated bimorph actuators, such as triple-bimorph lateral-shift-free (LSF) actuators. Zhang et al. proposed an LSF 2D micromirror that used Cu/W bimorphs to replace Al/SiO2 bimorphs to improve its robustness21. However, it proved challenging to passivate Cu layers due to how Cu/W bimorphs must operate under high temperatures. In addition, fragile SiO2 was used for thermal isolation at anchor regions, such that the breakage issue was not obviated.Some methods have been proposed to improve the impact resistance of electrothermal micromirrors. For instance, Pal et al. reported a simple bimorph-based one-dimensional (1D) electrothermal micromirror that used polyimide (PI) as a stress buffer2 bimorph LSF 2D electrothermal micromirrors with flexible multimorph beams enabled by the incorporation of photosensitive polyimide (PSPI). In this new process, PSPI partially replaces SiO2 as the electrothermal isolation material and as the anchor material, making this new type of electrothermal micromirrors much more robust to impact and more power efficient. These new features can extend the applications of electrothermal mirrors to a much wider range, including portable or hand-held devices. In the following sections, the design, fabrication, and characterization of the micromirror are described.Here, a new fabrication process is presented for robust Al/SiO2-based LSF actuators  process at a temperature of 300°C, followed by a wet etching process to form part of the multimorph patterns. In Fig. 2 layer is deposited. In Fig. 2 layer is deposited to form electrical isolation on the multimorphs, followed by a reactive ion etching (RIE) process to form vias. In Fig. 2 layer is removed by RIE to define the shape of the multimorph actuators and dicing grooves.The micromirrors are fabricated on an SOI wafer using a combined surface and bulk micromachining process. The fabrication process is illustrated in Fig. 22, and then the wafer is gradually cooled down to room temperature.The fabrication process of the flexible connect anchor is shown in Fig. 2 is used to remove the residual silicon underneath the multimorphs and PSPI beams  of silicon on the handle layer and 1 μm buried oxide removal by RIE are carried out sequentially to facilitate device release Fig. . Finallyams Fig. . BecauseFigure 2 as the connection anchor material. These are referred to as SiO2-type micromirrors. Except for the connection anchor structure, the SiO2-type micromirrors have exactly the same structural dimensions as the PSPI-type MEMS micromirrors. SEM images of the SiO2-type micromirror are shown in Fig. We also fabricate electrothermal micromirrors with SiOWhen the same driving signal is applied on all four actuators, the micromirror works in the piston mode. One pair of opposing actuators can be activated to perform one-axis rotational scanning, while the other pair can generate orthogonal axis rotational scanning. Thus, the micromirror can work in the piston scan or two-axis angular scan mode.Joule heating is generated by the Pt resistors. The measured resistance of the heaters in the four actuators of the PSPI-type micromirror is 520 ± 20 Ω. The temperature coefficient of resistivity (TCR) of Pt is measured using a temperature-controlled oven, and the measured value of the TCR is 0.0022/K Figure . The mea2-type. These angular scan ranges can satisfy many application requirements, including endoscopic OCT imaging. The applied voltage can exceed 4 V DC, but nonlinearity and plastic deformation may occur at this condition19. Likewise, 4 V DC meets the human body safety requirement. Thus, in this study, we limited the applied voltage up to 4 V DC.The optical scan angle is measured using a position-sensitive detector (PSD). When a laser beam points at a working micromirror, the PSD records the position of the laser spot reflected from the mirror plate, and then the scan angle can be deduced. Figure τ = 31.8 ms based on 27.To measure the frequency response, a sinusoidal voltage varying between 0 and 2 V is applied to one of the actuators of the micromirror. The test results are shown in Fig. 21. In contrast, the response times of the SiO2-type micromirror are slightly smaller than those of the PSPI-type micromirror, as shown in Fig. 2-type micromirror because PSPI has a smaller thermal conductivity than SiO2  for ball 2, g is the acceleration due to gravity (m/s2), M (g) and R (m) are the mass and radius of each ball, respectively, and σ and E are Poisson’s ratio and Young’s modulus of the material of the balls. These balls are made of stainless steel, from which the material properties are taken29. For lateral impact, the mirror plate is parallel to the radial direction of the ball, which is shown in Fig. 2-type mirrors are damaged when the drop height exceeds 2 cm, corresponding to a maximum survival acceleration of only 54 g. In contrast, the PSPI-type micromirror withstands impact from the ball at a drop height of more than 19 cm, corresponding to a maximum survival acceleration of 209 g. For vertical impact, the mirror plate is perpendicular to the radial direction of the ball, which is shown in Fig. 2-type and the PSPI-type micromirrors are 6 cm and 24 cm, respectively, and the corresponding maximum survival accelerations are 104 g and 240 g, respectively.As illustrated in Fig. 2-type micromirror. Additionally, the impact resistances of both micromirrors in the vertical direction are better than those in the lateral direction. This is because the mirror plate is free to move in the vertical direction while constrained in the lateral direction.In the experiments involving both impact directions, the PSPI-type micromirror shows vastly improved robustness compared to the SiO2-type micromirrors cannot even survive drops from a height of 5 cm. In contrast, nearly all of the PSPI-type micromirrors still work normally after free falling from a height of 21 cm. Detailed experimental data about the PSPI-type micromirror resistances and resonant frequencies after different drop heights are given in Table The impact tests given by Newton’s cradle may only simulate a single impact event, whereas micromirrors in real-world use may suffer from many complex impact loads. Thus, drop tests presented in Fig. 2-type micromirror, the device is damaged after free falling just when the height exceeds 5 cm. In contrast, the PSPI-type micromirror can withstand fall heights greater than 15 cm. Compared to the SiO2-type micromirror, the PSPI-type micromirror may offer greater potential in practical applications due to its improved robustness.Electrothermal micromirrors can be widely used in OCT endoscopic imaging. OCT probes may inevitably suffer from accidental falling in actual operation, so the robustness of the micromirror is very important for the service life of OCT probes. An experiment was established to imitate the free-falling motion of the two types of micromirrors that are encapsulated in an OCT probe. A customized transparent and hard plastic shell is used for packaging to observe the micromirror after the OCT probe free falls. Figure 30, the vibration frequency is set to sweep from 20 to 2000 Hz and then back to 20 Hz, which is increased on a logarithmic scale, and the duration is set to 12 min. The acceleration amplitude is increased by 1 g after each frequency sweep until the device is damaged. During the whole process of vibration, the health status of the micromirror can be directly observed with the naked eye.In practical applications, environmental vibrations also bring challenges to the normal operation and service life of the micromirror. A vibration test system , as shown in Fig. 2-type micromirrors fail after the acceleration exceeds 3 g, while the PSPI-type micromirrors withstand more than 21 g. In addition, similar results are observed for horizontal vibrations, as are shown in Fig. 2-type micromirrors still cannot survive after the acceleration exceeds 5 g, while the PSPI-type micromirrors can withstand at least 29 g acceleration. Compared with the SiO2-type micromirrors, the PSPI-type micromirrors exhibit much improved vibration resistance in both vertical and horizontal directions, and their safe acceleration range is increased by at least 4–5 times. These experimental results show that the micromirrors are easier to damage when vibrating in the vertical direction, which is opposite from the cases of the impact test and drop test. More study is needed to fully understand this phenomenon.Figure 2-type micromirrors. Representative photos of the damaged SiO2-type and PSPI-type devices are shown in Fig. 2-type micromirrors, the anchor structures are composed of only one layer of 1-μm-thick SiO2, such that they are easily damaged due to the brittle nature of SiO2. The breakage occurs either at the joint to the mirror plate  is applied to replace SiOSupplementary Information


Treponema species (spp.). Clinical inspection of the feet is the common way to diagnose the disease, but is laborious. There are no simple diagnostic tools, which makes it difficult to monitor DD prevalence in a herd. Therefore, we developed, validated and implemented a Treponema antibody detecting ELISA for use in bulk tank milk. A weak relation between clinical scores of claws with the results of ELISA in individual milk samples was found. When using bulk milk, a clear increase in the ELISA response was observed when mean clinical scores of the claws in the herd were higher. Using the results of the Treponema ELISA in bulk milk, herds with a low or high number of cattle with DD could be distinguished. This ELISA is now being used in a claw health monitoring program for dairy cattle in the Netherlands. This helps farmers to obtain insight into the level of DD in their herd and is helpful to improve claw health in dairy cattle.Digital dermatitis (DD) is a painful infection of the skin at the coronary band of the claws, a major cause of lameness in dairy cattle and associated with several Treponema spp. Clinical inspection of the feet is the best way to diagnose DD, but this is laborious and stressful for cattle. A simple diagnostic tool was developed to monitor DD prevalence at the herd level. An antibody ELISA based on antigens from four different Treponema spp. has been developed and validated in two field studies. In one study, bulk milk and individual milk samples of seven dairy herds, of which clinical claw scores were obtained, were tested. In the second study, bulk milk was tested from 110 herds of which clinical scores were obtained. A weak correlation between clinical scores of cows and the ELISA results in individual milk samples was observed. The ELISA response in bulk milk was higher in herds with higher mean clinical scores. Using the ELISA results in bulk milk, herds with a low or high proportion of cattle with DD lesions could be distinguished. This ELISA is useful to obtain insight into the DD status at the herd level, and is nowadays being used in a claw health monitoring program for dairy cattle in the Netherlands.Digital dermatitis (DD) is a painful inflammation at the coronary band of the claws, a major cause of lameness in cattle and associated with infections with several  Treponema [T. medium/T. vincentii-like, T. phagedenis-like, and T. pedis bacteria according to the 16S rDNA and flaB2 gene homology [Bovine digital dermatitis (DD) is a painful, infectious claw disorder that causes ulcerative lesions mainly at the coronary band of the hind legs of dairy cows and lameness ,2. The creponema . Althoughomology ,14,15,16Dichelobacter nodosus and Fusobacterium necrophorum [Other bacteria found were rophorum   last estimated by the claw trimmer at the moment of regular preventive trimming. We attempted to create an extensive database of dairy cattle that includes herds with varying DD prevalence in order to include enough variability and power for the analysis. The database was based on a total of 110 farms, visited between August 2017 and July 2018, and young animals (<21 months) were excluded. Seven herds, determined by the claw trimmer to have few, moderate and severe DD, were visited by a GD employee (trained by MH before the start of this study), three times, with 6 months between each visit, at the moment of regular preventive claw trimming. Inter-observer agreement for the M-score between the observers was tested with photographs by using Cohen’s kappa coefficient (k)  . All obsIn seven herds, at that moment of regular claw trimming, all claws were checked 3× with 6 month intervals by trained employees, scored and noted according to the M-score system for the presence and severity of DD ,18, and n = 897 cows). In herds (n = 110), from which we used data (M-scores) of one regular claw trimming, bulk milk samples on the day of claw trimming and for one year, up to 5 additional bulk milk samples from these herds were collected with approximately 10 week intervals.The dataset for laboratorial investigation comprised individual milk samples of lactating dairy cows of 7 herds, collected 3 times, 4 weeks after previous trimmings  in coating buffer and incubated overnight at 4 °C. Then, 100 µL/well blocking solution was added and plates were incubated for 1 h at room temperature. Subsequently, the contents of the plates were discarded, and plates were dried for four hours at 37 °C, vacuum sealed and stored at 4–8 °C. Plates were incubated with 100 µL per well of either sera diluted 1:100 diluted in assay buffer, non-diluted individual milk samples or non-diluted bulk milk samples for 1 h at 37 °C after washing five times with washing buffer (PBS + 0.05% Tween 20) using a Biotek automatic washing station. Then, 100 µL per well diluted horseradish peroxidase (HRP)-conjugated Protein G  in assay buffer was added and the plates were incubated for 1 h at 37 °C. Subsequently, the plates were washed again. Then, 100 µL/well TMB  was added, and the plates were incubated for 30 min at room temperature. The reaction was stopped by adding 50 µL/well 0.5 M sulfuric acid . The optical densities (OD) were measured at 450 nm using an ELISA microplate reader . S/P values were calculated by the formula: S/P = (OD Sample–OD Negative control)/(OD Positive control–OD Negative control).High-binding microtiter plates  were coated with a mixture of p < 0.05) only including 1 covariate (X), assuming a linear association, the coefficient of determination (r2) was determined between X and Y. X was either the individual or the bulk milk S/P ratio and Y could be the individual S/P ratio; the M2-score was the prevalence or the average M-score depending on the association that was investigated.Using Stata version 17.0 , tables The mean number of cows trimmed in the first visits to the seven herds that were scored 3 times was 98 (SD: 14.1) and 63% of these cows had an M-score ≠ 0. The percentage of cows with an active lesion (M2) in the 110 herds varied from 2% to49% at the herd level. The number of records collected per farm varied between 94 and 262 . From these records, 38.5% of animals were diagnosed with one of the DD stages other than M0 in one or both hind legs. The leg prevalence for each of the DD stages is shown in The results of the agreement (Cohen’s kappa coefficient) for the M-score of the students who performed the M-scores in the herds varied between 0.44 and 0.71. Six out of seven students were in the category ‘moderate’ and one was in the category ‘substantial’. The overall kappa value was 0.51 (moderate).n = 20, r2 = 0.82, p < 0.001) between the average S/P ratio of the individual samples and the S/P ratio of the bulk milk samples  M-score 2 counted as 3, an M1, M3, or M4.1 score as 2, an M4-score as 1, and an M0-score as 0. The total M-score of a cow was the total score of both hind legs . There was a general increase in the ELISA S/P ratio with the higher alternative total M-score see also .Treponema spp. in the bulk milk, two cut-off values were determined based on the 25 percentile and 75 percentile of the S/P ratio obtained for all the bulk milk samples from the seven herds. The lower cut-off was 0.87, while the higher cut-off was 1.24.To distinguish between herds with high and low levels of antibodies against These ELISA cut-offs were evaluated in the 110 herds of the second part of this study. Herds with a prevalence of M2 lesions higher than 20% were considered to have a high rate of clinical lesions, while herds with a prevalence of M2 lesions lower than 5% were considered to have no or few lesions. The herds with an M2 prevalence between 5% and 20% were herds with intermediate clinical signs. One of the herds had a high ELISA S/P ratio in bulk milk, but a low M2 prevalence, while 11 herds were found with a high M2 prevalence and a low ELISA S/P ratio .From 2019, the Treponema ELISA was added to the Royal GD claw health program, using quarterly bulk milk testing. Since 2020, the S/P ratios are reported and the result is categorized into ‘little or no antibodies’, ‘many antibodies’ or ‘very many antibodies’. In 2022, 1558 herds participated in the Treponema bulk milk program. Per quarter, over the period 2020–2022, the percentage of farms with ‘little or no antibodies’ was 37.2% . Promising results were found when the ELISA S/P ratios were compared with the number of legs affected and the severity of DD in those herds.The purpose of the present study was to develop and evaluate an ELISA for detection of digital dermatitis (DD)-related antibodies against Treponema spp. related to DD [r2= 0.82), which was also observed in another study [r2 = 0.51) was found between the ELISA results and the severity of the lesions in the individual animals, a good correlation between the clinical presentation of the different M-stages on the hind legs and the BTM ELISA S/P ratios. However, the ELISA is not intended for use as an individual diagnostic tool. Additionally, a good correlation was seen between the average M-score or the prevalence of the M2-lesion and the BTM ELISA S/P ratios in these herds (r2 = 0.23). During the clinical observations, no attention was paid to other disorders such as udder cleft dermatitis, teat necrosis and hock lesions. This type of lesions have also been associated with the presence of Treponema spp., might have influenced the ELISA results [Our indirect ELISA used a mixture of crude antigens from ed to DD ,13. In ter study . Additio results ,35, and Treponema spp. in the pathogenesis of clinical DD, as extensively reported earlier [Our findings in individual animals are in line with the results of Gomez et al. , althoug earlier ,36,37.T. phagedenis was performed to evaluate the discrimination between infected and non-infected cows and a BTM test was used to quantitatively evaluate the level of antibodies in BTM and the sum of serological responses of the individual cows [T. phagedenis antibody ELISA was applied on BTM samples to accurately estimate the DD within-herd prevalence. They concluded that the BTM antibody ELISA showed great promise at the herd level for screening purposes during DD management programs [In a study in Sweden, an ELISA for detection of antibodies against ual cows . In a Frprograms . Finallyprograms .Treponema spp. in the areas where the studies were performed, infection pressure, single or repeated infection, management and housing. The observed variation might be partly associated with the age of the animals, due to the higher S/P ratio observed in older cows that probably have had repeated infections. Other sources of variation might be due to infections with other Treponema spp., change in infection pressure per herd or in time, management and housing. In older studies, cross-reacting antibodies to Treponema pallidum and Treponema phagedenis due to Borrelia burgdorferii infections have been described in humans [The results of the first phase of our study were used to determine cut-offs for the ELISA using BTM samples. That was based on the P25 percentile and the P75 percentile of the BTM ELISA results, respectively. In that way, BTM samples with high levels of antibodies (>P75) could be differentiated significantly from BTM samples with low levels of antibodies (<P25). The ELISA cut-offs for BTM samples were validated in the second phase of the present study, where BTM samples were taken in 110 herds four weeks after trimming, upon which M-scores of all animals were also registered. It was found that herds with high levels of BTM antibodies (>P75) in general were also the herds with a high prevalence (>20%) of M2 lesions, while herds with a low level of BTM antibodies (<P25) had less than 10% M2 lesions. The observed variation might be caused by the age (parity) of the animals in the herd, different n humans ,40. It iAnother factor causing discrepancy between the bulk milk results and the DD observation might be correlated to other lesions in cattle related to Treponema infections, such as non-healing claw disorders, udder cleft dermatitis and skin lesions ,41,42. As in the study of Gomez et al. , in our Treponema spp. in the dairy herd. Laboratory support for individual diagnosis is less useful where, at claw trimming, the clinical presentation of different M-stages is so obvious that this is sufficient to start treatment.In most countries, regularly claw trimming is performed two or three times per lactation. Where especially chronic DD stages do not result in lameness, but are infectious for susceptible animals , it is fWe conclude that the Treponema antibody ELISA is useful for monitoring on the herd level, but not for individual diagnosis.In the Netherlands, the number of dairy herds that took part in our Treponema monitoring program steadily increased to 1630 in May 2023.Treponema spp. is valuable to monitor the DD prevalence at the herd level and may support dairy farmers in applying the correct curative and preventive measures [To summarize, periodically, BTM testing for antibodies against measures .A Treponema bulk milk ELISA was developed to obtain insight into the DD status at the herd level, and the ELISA is suitable to be used in a claw health monitoring program for dairy cattle in the Netherlands.


The Arrhenius constitutive equation of the alloy was established based on the true stress–strain curves to describe the rheology behaviors during the deformation of the alloy. The processing maps under the strain of 0.2–0.8 were constructed, which indicates the efficiency of power dissipation and instability of the deformed alloy. It was found that the instability domains are more likely to occur in the regions of low deformation temperature and high strain rate, corresponding to the high Zener–Hollomon (Z) parameter. The microstructure evolution of the studied alloy with different Z parameters was characterized. Then, the dynamic recrystallization (DRX) behavior was studied by electron backscatter diffraction, and the misorientation angle of deformed specimens was analyzed. The effect of different deformation temperatures and strain rates on the microstructure of the alloy and the behavior of dislocations and precipitations were investigated by transmission electron microscopy. The results demonstrate that continuous dynamic recrystallization (CDRX) and geomatic dynamic recrystallization (GDRX) mainly occur at the deformation conditions of a low Z value, and discontinuous dynamic recrystallization (DDRX) is likely to occur with increasing Z values.The 2198 Al–Li alloy has unique superiority in mechanical performance and has been extensively used in the aerospace field. In this study, the hot deformation behavior of the 2198 Al–Li alloy was investigated on a Gleeble-1500 thermomechanical simulator with a strain rate of 0.01–10 s With the increasing demand for lightweight structure materials, the Al–Li aluminum alloy has been widely used in the aerospace manufacturing industry due to its excellent mechanical properties. As one of the newly developed alloys, the 2198 Al–Li alloy shows superiority for use in aerospace structural materials, such as low density, good formability, high strength, and weight reduction effect ,4. The hIt has been extensively accepted that the flow behavior and microstructure are relevant to several factors, including deformation temperature, strain rate, and total strain ,8. ThereIn addition, extensive research has shown that dynamic recovery (DRV) and dynamic recrystallization (DRX) play a crucial role in the microstructure evolution during the hot deformation of the alloy ,20,21,22Z parameters were observed by comparing the variation of misorientation angles and distribution of dislocations, where the dynamic softening mechanism in the compression process was analyzed. In addition, the effect of deformation temperature and strain rate was also investigated by characterizing the microstructure.In the present work, the hot deformation behaviors of the as-homogenized 2198 Al–Li alloy were investigated. The constitutive equation and processing maps were established based on the stress–strain curves to describe the effect of deformation temperature and strain rate on rheology behaviors and determine the optimal hot-working process. Deformation microstructure characteristics with different The chemical composition of the studied alloy in this paper is shown in −1. As the schematic diagram of the hot compression shows in Hot compression tests were executed on a Gleeble-1500 thermomechanical simulator  at temperatures of 330, 360, 390, 420, 450, 480, and 510 °C and strain rates of 0.01, 0.1, 1, and 10 s−1) due to the lower critical shear stress, the flow stress drops rapidly after reaching the peak stress [−1 and a deformation temperature of 510 °C, as shown in Uniaxial isothermal hot compression is a dynamic process of work hardening and microstructure softening of the alloy. In the initial stage of hot deformation, the flow stress increases rapidly due to the increased dislocation density caused by deformation. As deformation proceeds, the thermal effect on work hardening occurs when the flow stress reaches the peak stress. When the deformation temperatures are lower than 510 °C, the dynamic softening driven by deformation energy eliminates stress concentration and decreases true stress, and then the flow stress gradually reaches the steady deformation stage. The increase in the strain rate accelerates the increment of dislocations in the alloy, which results in an increase in dislocation density and flow stress as well. The increase in deformation temperature accelerates the dislocation movement and annihilation rate, which leads to a decrease in dislocation density and flow stress. Because that dynamic softening cannot work effectively at a relatively low strain rate (0.01 sk stress . In addi−1); T is the deformation temperature; Q represents the thermal strain activation energy of hot deformation (kJ/mol), which reflects the degree of difficulty of atomic rearrangement during the deformation, and R is the molar gas constant (R = 8.314 J/(mol·K)).The constitutive equation of the metallic material describes the relationship among flow stress, deformation temperature, and strain rate. The corresponding flow stress of the alloy under different deformation conditions can be predicted by the constitutive equation. The Arrhenius constitutive model was applied to establish the correlation for characterization of the flow stress behavior of the 2198 alloys, which can be expressed as Equation (1):At a low-stress level  and (3) are substituted into Equation (1),and logarithms are taken on both sides of the equation.The deformation activation energy of the material is different because of the different microstructural evolution during the hot deformation, as well as the material constants . AssuminB), which is the ratio of the volume of the specimen after deformation divided by the initial volume (the diameter is measured at the midlength of the specimen), can evaluate the effect of friction on flow stress. When B ≥ 1, it indicates that friction has a non-negligible effect on flow stress and must be corrected. The dimensions of specimens after deformation were measured, and the barreling coefficient B was calculated for the specimen under each deformation condition, and the values are between 1 and 1.1, indicating that the effect of friction on the flow stress of the alloy 2198 is negligible and friction correction is not necessary.−1 and the deformation temperature is 360 °C, the flow stress curve of the specimen fluctuates abnormally, and therefore the stress needs to be corrected [Otherwise, when the strain rate is 10 sorrected . The equTaking logarithm on both sides of Equation (8), the rearranged equation can be expressed as:T, and taking a strain of 0.3 as an example, the relationship between its measured stresses and temperature is the relationship between The fixed strain rate n1 = 6.4209 and β = 0.0996 and then β/n1 = 0.0155 MPa−1 are obtained.The stresses at strains from 0.00 to 0.80 with an interval of 0.05 are corrected and the dot line plotted as stress–strain curves shown in The natural logarithm is taken on both sides of Equation (5) and can give the equation expressed as:Q can be obtained by calculating the partial differential of Equation (8):The expression of deformation activation energy n and Q for the present alloy, the functional relations between n value is 4.4600. According to the mean slope of Q is estimated as 184.46 kJ·mol.To calculate the Z) parameter. The deformation temperature T and strain rate Z parameter related to flow stress The relationship between flow stress and hot deformation conditions can be expressed by the Zener–Holloman  is transformed into Equation (15) after logarithms are taken from both sides.In order to obtain the exact value of constant Q is substituted into Equation (13), and the Z parameter under various deformation conditions can be calculated by the Zener–Hollomon method, as listed in A can be determined according to the slope and the intercept of the fitting line. It also can be seen from Z and 13 s−1.The deformation activation energy The hot processing maps are composed of a power dissipation diagram and an instability diagram ,33, whicP, in which the energy consumed by plastic deformation is G, and the dissipated energy of microstructure evolution is J. The relationship among P, G, and J is presented as follows [In the dynamic material model, it is assumed that during the hot compression process, the entire power of the alloy is  follows :(17)P=σεP, in which G is the energy depleted by plastic deformation, while J is the energy depleted by microstructure evolution.In the dynamic material model, it is assumed that during the hot-working process, the overall power per unit volume of the alloy is J to the power dissipation rate dG, which is expressed as Equation(18).The strain rate sensitivity index m is the ratio of the dissipation coefficient dG and the dissipator co-content J are regarded as the energy generated and consumed by plastic deformation, respectively. From the stress–strain curve, the consumption rate of dislocations is always less than or equal to the generation rate during thermal deformation. Thus, the energy loss by the microstructure evolution can be expressed as Equation(19).The dissipator content J is equal to ideal G, and at this time, m is 1, and J obtains the maximum value maxJ:For the ideal linear dissipater, J < G, and the efficiency of the power dissipation η can be expressed by the ratio of J to maxJ. The dimensionless index η is the power dissipation efficiency and can be expressed by Equation (21).For the nonlinear dissipater, η corresponding to different strain rates and deformation temperatures can be obtained, and the isoline of power dissipation efficiency η of T is drawn on the power dissipation efficiency diagram. Narayana et al. [According to the DMM theory, the power dissipation efficiency a et al.  proposedWhen the −1. The shaded area and the unshaded area in the processing maps represent the instability and the safety regions, respectively, and the number on the contours indicates the power dissipation coefficient η.η decreases from high temperature to low temperature and from low strain rate to high strain rate, representing the decrease in the dynamic consumption of energy in the alloy. When the strain is lower than 0.4, the maximum of the power dissipation factor η is 0.26 due to the inadequate plastic deformation, and the unsafe region with a small area only appears in the corner of the processing maps and is concentrated in which the power dissipation factor η is less than 0.13, as shown in η gradually increases to 0.38. As the deformation proceeds, the density of dislocations and the deformation energy in the alloy increase, resulting in the occurrence of recrystallization and other microstructure behaviors [η.ehaviors . At a st−1 approximately, as region B shown in Based on the preliminary analysis of the information in the processing maps, the relatively good processing performance corresponds to the optimal parameters of the deformation process of the 2198 Al–Li alloy, which are 390–480 °C and 0.01–0.1 sZ parameters was characterized [To study the microstructure evolution of the deformed 2198 alloy during the hot compression process, the microstructure of specimens with different cterized . InverseFurthermore, the deformation conditions in Z values, the grain boundaries of deformed grains are relatively clear and smooth with uniform distribution, while at high Z values, the distribution of deformed grains is more concentrated and uneven, where the deformed microstructure shows severe flow localization phenomenon caused by shear band concentration. The adiabatic shear band will easily form during the deformation with the increase in local temperature and then promote the flow localization. Under the deformation conditions with a high Z value, the deformation time is too short to make the heat dissipate in time, which is more likely to cause a local temperature concentration, resulting in flow localization. Moreover, with the further increase in the Z value, the phenomenon of flow localization becomes more obvious. In addition, the uneven and insufficient dynamic softening is also the reason for the formation of flow localization, where the thickness of banded deformed grains is smaller and broken obviously. It indicates that the deformation is severer in the area of flow localization and leads to an increase in the deformation resistance of the alloy. Therefore, the more serious flow localization at a high Z value is also the reason for higher flow stress.At low Z is presented in Z. With the decrease in lnZ, which represents the decrease in deformation rate or the increase in deformation temperature, the fraction of LAGBs tends to decrease gradually. The LAGB fraction reflects the dynamic softening ability of the alloy, and the LAGBs indicate that most of the dislocations disappear through climbing or rearrangement, resulting in the decrease in the deformation energy and resistance. However, because the location of the deformation condition of 390 °C/1 s−1 is at the unsafe region in the processing map, the fraction of LAGBs does not conform to the law of change [Z values, based on increasing deformation temperature or decreasing strain rate, the frequency of recrystallization increases, which corresponds to the increase in HAGB fractions. Contrary to the low lnZ, a lot of LAGBs are formed inside the deformed grains, and the subgrains have complete profiles with a smaller size compared with the DRX grains [The distribution of the boundary misorientation angle of the 2198 alloy with different lnf change . Becausef change . Due to Z. The DRX effect is limited under the deformation conditions with high lnZ, but when the lnZ value is high, the region where the deformed microstructure has obvious flow localization is also recrystallized along the grain boundary, as revealed in The microstructures of the deformed 2198 alloy were composed of deformed grains, newly formed equiaxed grains, and subgrains. According to the fine-equiaxed grains along the serrated grain boundaries shown in Z are 23.73 and 26.03, as shown in The DRX mechanisms differ in the interior of grains or at the grain junctions, and they may also be inconsistent near grain boundaries. It is necessary to distinguish the type of recrystallization mechanisms. There are mainly three dynamic recrystallization mechanisms: DDRX, CDRX, and GDRX. DDRX has a typical process of nucleation and growth of recrystallized grains and is also related to the gradual accumulation of dislocations ,41. CDRXZ, and the recrystallization fraction decreases, which indicates that the nucleation and growth of dynamic recrystallization decrease correspondingly. Under the deformation conditions with a low lnZ value, the CDRX and GDRX mechanisms work in the deformed microstructure. The migration ability of the dislocations and grain boundaries is sensitive to deformation temperature. At high deformation temperatures, the migration speed of dislocations is faster, and HAGBs are more likely to bow out and migrate to form discontinuous dynamic recrystallization nuclei, which also grow faster. Under the deformation conditions with a high lnZ value, there is a certain number of DDRX grains in the alloy. By controlling DRV and DRX during hot working, the substructure of the alloy is strengthened, and the mechanical properties of the material are improved. Moreover, hot working includes forging, extrusion, and rolling, and the strain rates of them are generally in the range of 0.05–5 s−1 in industrial production. Based on the further analysis supported by microstructure, the optimal lnZ value of the 2198 Al–Li alloy is 23.73–31.16, which corresponds to the parameters of the deformation process around 450–480 °C approximately.In summary, the average grain size of dynamic recrystallization decreases with the increase in lnTEM was used to study the influence of different deformation temperatures and strain rates on dislocation behavior and submicrostructure characteristics. −1, the subgrain form with a low density of intragranular dislocations owing to the climbing and cross-slipping of dislocations, as shown in −1, the dispersed distribution of dislocations can be observed inside the deformed grains because the time for the migration of strain-induced dislocations is shortened [−1, there is a slight decrease in the overall density of dispersed dislocation inside the grains and an increase in the dislocation wall due to the slow rearrangement of dislocations, as shown in −1 and 390 °C/1 s−1, precipitation zones can be observed in the interior of grains and at grain boundaries clearly, hindering grain boundary migration and inhibiting the formation of DRX. The secondary phases may also be the reason that DRX does not occur easily under the deformation conditions of 2198 Al−Li alloys with a lower deformation temperature and higher strain rate. It is hard to observe the existence of secondary phases at a temperature of 510 °C.hortened , and the(1)The flow stress of the 2198 Al–Li alloy during the hot compression test increases with the increase in strain in the initial stage of deformation, and then gradually tends to be stable due to the increasing effect of microstructure softening. In general, the characteristic stress is inversely proportional to the deformation temperature and proportional to the strain rate.(2)The Arrhenius constitutive equation of flow behavior for the 2198 Al–Li alloy was determined as (3)Z values, corresponding to the conversion of the misorientation angle of grain boundaries from LAGBs into HAGBs, while the DDRX mechanism mainly occurs at moderate and high Z values.The CDRX and GDRX mechanisms mainly occur at low (4)Z value, constraining the forming of DRX grains by hindering the migration of grain boundaries.At low deformation temperatures and high strain rates, flow localization occurs at the triple junctions of deformed grains. The density of secondary phases increases with the increase in the In this work, the hot deformation behavior of the 2198 alloy and microstructure evolution were investigated based on the flow stress curves obtained through hot compression tests at various deformation temperatures and strain rates. The main results can be summarized as follows:


Enterococcus faecalis and Enterococcus faecium bacteriophages were analysed for gene shuffling in the lytic cassettes of bacteriophages infecting. It was found that enterococcal bacteriophages could be classified into well-defined groups based on the size of their genomes and each size group had its own conserved gene composition of lytic cassettes. Enterococcal bacteriophages use a relatively broad spectrum of holins and endolysins with variable cell-wall binding (CWB) and catalytic domains, and most of them utilise a lytic cassette with more than two genes. Enterococcal bacteriophages most commonly use endolysins with amidase catalytic domains and the CWB domain SH3_5. Some bacteriophages possess in their lytic cassette a holin-like gene with the XhlA domain protein, characteristic of hemolysin. Regardless of the shuffling of genes encoding holins and endolysins in lytic modules, a novel example of CWB domain shuffling within enterococcal endolysins was identified.The genomes of The online version contains supplementary material available at 10.1007/s13205-023-03775-w. Enterococcus faecalis and Enterococcus faecium, are known to cause secondary infections in hospital environments. With the emergence of multidrug-resistant strains over the past two decades, they have become an important nosocomial pathogen causing urinary tract infections, bacteremia, and endocarditis  species, have become one of the major nosocomial pathogens worldwide  domain and the N—terminal catalytic domain. The CWB domain keeps endolysin tightly bound to the bacterial cell wall, while the catalytic domain cleaves specific bonds in peptidoglycan Donovan . The CWBEnterococcus spp. bacteriophages were obtained from the GenBank database , Holin_BhlA (pfam: PF10960), Phage_holin_1 (pfam: PF04531), Phage_holin_Dp1 (pfam: PF16938), Phage_holin_2_2 (pfam: PF10746), Phage_holin_5_2 (pfam: PF16079), XhlA (pfam: PF10779), and Holin_SPP1 (pfam: PF04688).Endolysins of enterococcal bacteriophages were found to possess 7 different catalytic domains already known to protein databases: Amidase_2 (pfam: PF01510), endopeptidase domain like (CATH Superfamily 3.90.1720.10), CHAP (pfam: PF05257), Glyco_Hydro_25 (pfam: PF01183), Amidase_5 (pfam: PF05382), Peptidase_M23 (pfam: PF01551), and Glucosaminidase (pfam: PF01832); and 4 types of CWB domains: ZoocinA_TRD (pfam: PF16775), SH3_3 (pfam: PF08239), SH3_5 (pfam: PF08460), and LysM (pfam: PF01476).Based on sequence comparisons, there were 21 different combinations of proteins and their domains in lytic cassettes observed in enterococcal bacteriophages, and the distribution and composition of individual types of lytic cassettes were non-random regarding the size groups of bacteriophages. All types of lytic cassettes, together with corresponding numbers of bacteriophages possessing particular cassettes, are shown in Fig. The bacteriophages with the smallest genomes  had three types of lytic cassettes. The first type had holin with a Phage_holin_4_1 domain, followed by endolysin with an Amidase_2 domain, a protein with an unknown function, DUF3310, and an endopeptidase domain-containing protein. Based on multiple sequence alignments, Amidase_2 endolysin from phage MDA1 (MW623430.1) was used as the type endolysin for this lytic cassette. Secondary structure prediction showed that the Amidase_2 domain is connected with an unknown CWB domain via a proline-rich interdomain linker. The second type of lytic cassette in Group 1 contained holin with the HolinBhlA domain, endolysin with the Amidase_2 domain, and a CHAP  domain-containing protein, which functions mainly in peptidoglycan hydrolysis. The predicted structure of Amidase_2 endolysin from this type of lytic cassette showed a similar proline-rich interdomain linker connecting another unknown CWB domain. Only one bacteriophage had the third type of lytic cassette consisting of protein with domains Phage_lysozyme2 and Peptidase_M23, holin with domain Phage_holin_4_1, and a second lytic protein with the Glyco_hydro_25 catalytic domain.Group 2, consisting of 73 bacteriophages with genome sizes ranging from 20 to 50 kbp, was the most diverse in lytic cassette types. Most of the lytic cassettes had an endolysin with an N-terminal Amidase_2 domain, followed by a C-terminal SH3_5 or ZoocinA CWB domain, and a holin with a Phage_holin_1 domain. In some of these lytic cassettes, no known CWB domain was identified using InterProScan, but multiple sequence alignment showed that multiple different Amidase_2 sequences contained various C-terminal sequences, probably representing unknown CWB domains. Bacteriophages MSF2 (MK982307.1) and AUEF3 (KJ127304.1) were used for secondary structure prediction of endolysins with SH3_5 and ZoocinA domains. Surprisingly, the predicted structure of endolysin from MSF2 revealed another possible CWB domain between Amidase_2 and SH3_5. The second most common type of lytic cassette comprises endolysin with N-terminal Glyco_hydro_25 catalytic domain, followed by two C-terminal LysM domains, and holin with Phage_holin_Dp1, except for two bacteriophages with Phage_holin_1. All lytic cassettes either with Amidase_2 or Glyco_hydro_25 domains had holin-like protein with XhlA hemolysin domain. Several bacteriophages had unique lytic cassettes  was the only one with a holin-like protein with an XhlA domain, a holin with a Phage_holin_1 domain, an endolysin with an N-terminal Amidase_2 domain, and an unknown C-terminal CWB domain. Although this phage belongs to size Group 4, multiple sequence alignment of Amidase_2 endolysins clearly showed that its endolysin is almost identical to endolysins from phages Aramis (LR990833.1) and dArtagnan (LR991625.1) from Group 2. The rest of the bacteriophages had holin with the Phage_holin_5_2 domain and endolysin with N-terminal Amidase_2 domain and C-terminal SH3_5 CWB domain. Multiple sequence alignment showed that bacteriophage nattely (MT119360.1) had the same sequence of the Amidase_2 domain as other phages in this group but a slightly different sequence of the putative CWB domain. However, the predicted secondary structure showed a surprisingly similar domain composition with the Amidase_2 catalytic domain, a putative unknown CWB domain, and the SH3_5 CWB domain. Bacteriophage 9183 (MT939241.1) had a unique amino acid sequence of endolysin with an Amidase_2 domain and two CWB domains, SH3_5 and SH3_3. The secondary structure of this endolysin resembled that of MSF2 (Group 2), nattely phage, and other phages from Group 4, but their amino acid sequences were different.Group 5 was formed by 41 bacteriophages encoding three types of lytic cassettes. All three types had endolysin with an N-terminal Amidase_2 domain, an aggregation-promoting factor containing a LysM domain, and holin with a Holin_SPP1 domain, except for one bacteriophage with a Phage_holin_4_1 domain similar to the holins of Group 1. All bacteriophages from this group had a similar composition of lytic cassettes, but sequence analysis of endolysins revealed that there were two different types of endolysins with the Amidase_2 domain. The first was a protein with a shorter sequence of 289 amino acid residues. The second type had the same N-terminal amidase domain but consisted of 416 amino acid residues. Multiple sequence alignment and secondary structure prediction showed that there were CWB domains not previously identified by protein databases. Surprisingly, bacteriophages with longer endolysin contained another protein in their lytic cassette with an Amidase_5 catalytic domain and an SH3_5 CWB domain.In summary, the most common catalytic domain was Amidase_2, followed by the CHAP domain, the Amidase_5 domain, and the Glyco_hydro_25 domain. Considering only known domains from the Pfam database, the most common combination of catalytic and CWB domains was found to be Amidase_2 + SH3_5, followed by Amidase_2 + ZoocinA, Amidase_5 + SH3_5, and Glyco_hydro_25 + LysM Table . CombinaMany bacteriophages from Group 2 with either Amidase_2 domain endolysins or Glyco_hydro_25 domain endolysins had in their lytic cassettes the holins Phage_holin_1 and Phage_holin_Dp1. However, GenBank records for another holin-like gene were found in the majority of enterococcal bacteriophage genomes from Group 2 with those specific lytic cassettes. Analysis using InterproScan revealed that this holin-like protein contained an XhlA hemolysin domain. This gene was always present in combination with Phage_holin_1 and Phage_holin_Dp1. However, in many genome annotations, this gene was misidentified as "tail fiber protein" or simply denoted as "hypothetical protein". Close localization of these holin-like protein genes to genes encoding holins might suggest that this gene plays an important role in bacteriophage lytic activity as well.Gene shuffling in lytic cassettes of Group 2 and Group 5 bacteriophages In most lytic cassettes, holins were always paired with specific endolysins. Holins were also highly specific for each group. Surprisingly, some holins as well as endolysins were found in groups in which they were not expected. Some genes encoding holins were shuffled among bacteriophages with similar but not identical compositions of lytic cassettes. Gene shuffling was identified in lytic cassettes of bacteriophages from Group 2 and Group 5.Two types of lytic cassettes were detected in Group 2 bacteriophages, consisting of identical endolysins with Glyco_hydro_25 catalytic domains and LysM CWB domains. These bacteriophages mostly encoded Phage_holin_Dp1, which we considered specific for this holin-endolysin pair. However, two of those bacteriophages were found to use Phage_holin_1 instead. Both lytic cassettes contained an XhlA holin-like protein. Fig. A.Fig. 3AA similar situation could be seen in Group 5 bacteriophages, which contained endolysin with an Amidase_2 domain and a separate protein containing a LysM CWB domain. All bacteriophages in Group 5 used HolinSPP1 except one, which had Phage_holin_4_1 in its lytic cassette Fig. B.We also discovered that there were two types of Amidase_2 endolysins in Group 5. The endolysin with the longer amino acid sequence was present in lytic cassettes with another endolysin consisting of Amidase_5 and SH3_5 domains. Both types of lytic cassettes, the ones with longer Amidase_2 and Amidase_5 endolysins and the ones with shorter Amidase_2, had HolinSPP1. Multiple sequence alignments showed that the Amidase_2 domain regions of these endolysins were almost identical in sequence but differed in their putative CWB domain regions. This suggests that both gene and domain shuffling might have occurred in the evolution of the largest enterococcal bacteriophages , vB_EfaS-DELF1 (LC513943.1), vB_GEC_Ef_S_9 (MW672041.1), and Aramis (LR990833.1) were used. Endolysins from bacteriophages 9184, vB_EfaS-DELF1, and vB_GEC_Ef_S_9 had the same amino acid sequence of the Amidase_2 domain with differences in their putative CWB domain regions. Putative CWB domains were denoted as UCWB (unknown CWB) domains. Secondary structure predictions of representative endolysins showed that those UCWB domain regions contained large CWB domains as opposed to SH3_5-containing endolysins. Since the Amidase_2 domain and interdomain linkers had high sequence similarity, domain shuffling may have happened between UCWB domains in these endolysins. Surprisingly, endolysin from bacteriophage Aramis had a different Amidase_2 domain but the same UCWB as endolysin from bacteriophage 9184. That suggests possible shuffling not only between CWB domains but between catalytic domains as well. The putative interdomain linker of this endolysin slightly differed from the consensus sequence. The same Amidase_2 and UCWB domains were found in Group 4 bacteriophage 9181. A similar situation probably happened with bacteriophages vB_EfaP_Zip (MK360025.1) and vB_EfaP_IME199 (KT945995.1) from Group 1. Their endolysin contained the same UCWB domain as previously mentioned bacteriophages 9184 and Aramis but a completely different Amidase_2 domain . Most Amidase_2 domain-containing endolysins had unknown CWB domains (32.75% of lytic cassettes). Domain SH3_5 was found to be typical in the Amidase_2 endolysins of enterococcal bacteriophages. The common lytic cassettes of Enterococcus spp. phages were different from those of other Lactobacillales phages. The combination of Amidase_2 + SH3_5 domains is typical for Enterococcus phages and prophages. Lactobacillus prophages lytic cassettes have the majority of endolysins with domains Glyco_hydro_25 + LysM Below is the link to the electronic supplementary material.