Using a high-throughput mass spectrometry (MS)-based assay in combination with scanning alanine mutagenesis, residues contributing to the KR-binding epitope of the ACP domain were identified. Application of carbene footprinting revealed the ACP-binding site on the KR domain surface, and molecular docking simulations driven by experimental data allowed production of an accurate model of the complex. Interactions between ACP and KR domains from trans-AT PKSs were found to be specific for their cognate partner, indicating highly optimised interaction interfaces driven by evolutionary processes. Using detailed knowledge of the ACP:KR interaction epitope, an ACP domain was engineered to interact with a non-cognate KR domain partner. The results provide novel, high resolution insights into the ACP:KR interface and offer valuable rules for future engineering efforts of biosynthetic assembly lines.The biosynthesis of polyketides by type I modular polyketide synthases (PKS) relies on co-ordinated interactions between acyl carrier protein (ACP) domains and catalytic domains within the megasynthase. Despite the importance of these interactions, and their implications for biosynthetic engineering efforts, they remain poorly understood. Here, we report the molecular details of the interaction interface between an ACP domain and a ketoreductase (KR) domain from a  trans-AT polyketide synthase is elucidated in molecular detail, providing unique insights into recognition and specificity of the interface.The interaction epitope between a cognate KR–ACP domain pairing from a  These multi-domain enzymes conform to a paradigm of covalent substrate attachment for exceptional processivity, and as a result, are often likened to molecular assembly lines. A co-enzyme A (CoA)-derived phosphopantetheine (Ppant) moiety, post-translationally attached to the acyl carrier protein (ACP) domains by a 4′-phosphopantetheinyl transferase (PPTase), serves to tether biosynthetic intermediates to the ACP domain via a thioester linkage.4Polyketides constitute a valuable family of natural products, many of which find application in both medicine and agriculture.5–7 Evolution has given rise to two discrete classes of modular PKS: cis-AT and trans-AT. Whilst the cis-AT PKSs have AT domains integrated into each module; the trans-AT (or AT-less) PKSs employ a standalone AT domain to supply malonyl extender units to all ACP domains in the assembly line.8–10 In addition to lacking an integrated AT domain, the trans-AT PKSs have other characteristic features, these include modules that are split across subunits, non-elongating KS domains and an assortment of other trans-acting catalytic domains.11 The archetypal trans-AT PKS responsible for the biosynthesis of bacillaene highlights some of these aberrant features  domain loads an malonyl-derived extender unit onto the ACP domain, allowing the ketosynthase (KS) domain to catalyse a Claisen-like condensation with the upstream polyketide chain yielding a β-keto thioester intermediate. Optional processing domains in the module affords additional diversity at the α- and β-carbon positions of the resulting β-keto thioester. These include C-methyltransferase (MT) domains, which methylate the α-carbon, in addition to ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains that generate hydroxyl, olefinic and fully saturated intermediates, respectively.features .cis- and trans-AT PKSs.12,13 Within a module, the ACP domain must shuttle biosynthetic intermediates between catalytic domains in an ordered manner, engaging in specific protein–protein interactions with each domain.14 Although the natural tethering of PKS domains means that the effective local concentration of the ACP domain is high,15 as a consequence, the interactions with catalytic domains are often low affinity, making them difficult to characterise experimentally. This has resulted in the intra-molecular interaction network between ACP domains and catalytic domains remaining largely unexplored, with the majority of our current knowledge limited to cis-AT PKS systems. Of particular note are a set of cryo-EM structures from an entire cis-AT module, which capture the various substrate-bound forms of the ACP domain interfacing with the KS, AT and KR domains.16,17 Although this provided remarkable insights into the positioning of the ACP domain during the catalytic cycle, precise residue-level information regarding each interface is obscured by the resolution of the technique at the time. The development of mechanism-based crosslinking has allowed certain ACP domain-mediated interactions to be covalently ‘trapped’, permitting crystallisation of these complexes.18 To date, this approach has yielded high-resolution structures for various FAS and PKS systems,19–22 however, structures of crosslinked ACP-catalytic domain complexes from modular PKSs have proved more challenging. It should be noted, that although crosslinkers have been developed to target most catalytic domains, probes for trapping complexes with the KR and ER domain are yet to be established.Intra-molecular interactions between the ACP domain and catalytic domains play a pivotal role in ensuring biosynthetic fidelity in both pro-S hydride of NADPH to yield a β-hydroxy thioester product.23 The stereochemical outcome of this reaction is controlled by the nature of the KR domain: ‘type A’ (KRA) and ‘type B’ (KRB) domains generate (3S) and (3R)-configured 3-hydroxy thioester products, respectively, information that can be obtained by inspection of the primary sequence , and a C-terminal catalytic subdomain (KRC) which binds NADPH; both of which exhibit a Rossmann-like fold  and KR (PksJ KR2) domains from module 3 of the bacillaene s-AT PKS . Our datEscherichia coli as N-terminal pHis6 fusion proteins, purified to near-homogeneity using immobilized metal-ion affinity chromatography (IMAC), and analysed by intact protein mass spectrometry (MS) to confirm their identity -3-hydroxy-pentanoyl thioester product, yielding the expected stereochemistry for the A-type family of KR domains.31 Building on this, an MS-based assay was devised to monitor conversion of 3-keto-butyryl-PksJ ACP4 to (3S)-3-hydroxy-butyryl-PksJ ACP4 catalysed by the PksJ KR2 domain, thereby forming an experimental platform to probe interactions between the domains.In the first instance, excised PksJ ACP4 and PksJ KR2 domains were overproduced in enzyme A . Conversenzyme A . PreviouS)-3-hydroxy-butyryl-PksJ ACP4, we elected to monitor ejected phosphopantetheinyl ions from the intact PksJ ACP4 protein.32 This allowed an unambiguous mass shift associated with ketoreduction to be identified, yielding ions at m/z = 345.15 and 347.16 corresponding to the 3-keto-butyryl and (3S)-3-hydroxy-butyryl species, respectively (S)-3-hydroxy-butyryl-PksJ ACP4 in the presence of NADPH after a 10 min incubation period. Time-resolved analysis of the reaction showed near-complete conversion of 3-keto-butyryl-PksJ ACP4 to (3S)-3-hydroxy-butyryl-PksJ ACP4 after 5 min and was used as the time-point at which to quench the reaction, in order to observe changes in reaction efficiency as a result of mutations  associated with PksJ KR2-catalysed conversion of 3-keto-butyryl-PksJ ACP4 to . Following analysis of the model, a total of 50 surface-exposed residues were then individually mutated to Ala, overproduced, and purified to homogeneity in their apo-form. It is worth noting that five of these mutants  could not be expressed in a soluble form and were therefore excluded from the study. The remaining 45 X → Ala mutants resulted in ∼95% surface coverage of the solvent exposed non-Gly/Ala residues from the core 4α-helix bundle of the PksJ ACP4 domain -3-hydroxy-butyryl species, and then expressed as a percentage of the WT PksJ ACP4:KR2 reaction (The suite of 3-keto-butyryl-loaded PksJ ACP4 Ala-mutants were then subjected to the MS-based ketoreduction assay to assess the impact of each residue on the PksJ ACP4:KR2 interaction interface. The extent of ketoreduction was measured as a ratio of the Ppant ejection ions corresponding to the 3-keto-butyryl and (3reaction . The majreaction . Circulareaction . This prII), with I47 and I48 directly adjacent to S46. The Q51, R55 and R58 residues are located at sequential turns running down HII, indicating the importance of the residues displayed on the surface of this helix. Interestingly, residues E25 and R27 are located on the loop region connecting HI and HII, and therefore distant at the sequence level, but are close in three-dimensional space to the critical residues on HII, contributing to the well-defined binding epitope  and conformational changes  to be identified.We next sought to establish residues on the PksJ KR2 domain which contribute to complex formation with the PksJ ACP4 domain. To achieve this, we employed the recently developed carbene footprinting methodology; a structural mass spectrometry technique which exploits covalent labelling of solvent-exposed residues on a protein surface using a reactive carbene species, formed by E. coli as an N-terminal pHis6 fusion protein and purified to near-homogeneity  , suggesting a conformational change that increases solvent exposure in this region, possibly to accommodate part of the 24-residue linker (Carbene footprinting experiments were carried out on solutions containing PksJ KR2 domain and  S75KTK) .31 This e linker . Given te linker , appearse linker . This suS subdomain situated at the open side of the substrate binding channel or (ii) at the KRC subdomain close to the NADPH co-factor binding site. Furthermore, guided by our experimental data, analysis in ChimeraX virtual reality40 and preliminary docking using high ambiguity driven biomolecular docking (HADDOCK)41 suggested that both sites had the potential to be viable solutions for the PksJ KR2:ACP4 complex. We therefore opted to mutate surface exposed residues at each site, and monitor the effect using our MS-based ketoreduction assay to identify the site of PksJ ACP4 complex formation. Using information from carbene footprinting and preliminary docking simulations, four residues at each site were individually mutated to alanine: E378, E467, E469 and K541 from the site adjacent to the KRS subdomain; and R228, M327, D328 and E421 from the site located near the NADPH binding site on the KRC subdomain. It should be noted that although residues M327, D328 and E378 are located in peptides not detected in carbene footprinting experiments , a significant decrease in activity was observed for all mutants at the KRC subdomain site (6–66% of WT PksJ KR2)  next to the KRksJ KR2) . Based o5KTK) and the homology model of apo-PksJ ACP4 as input structures. The cluster of solutions with the highest score showed excellent agreement with the experimental data, which positioned the PksJ ACP4 Ppant attachment site (S46) at the opening of the NADPH-binding channel, ∼22 Å from the 4-pro-S hydride of NADPH. A Ppant arm was then manually modelled into the structure using Chimera X virtual reality and the complex was energy minimised, followed by a 200 ns classical molecular dynamics simulation in AMBER. The model shown in pro-S hydride, allowing sufficient space for the 3-ketoacyl biosynthetic intermediate to enter the active site and position the C3 keto group proximal to the 4-pro-S hydride of NADPH and the catalytic tyrosine (Y386) residue. The rest of the polyketide intermediate likely sits in the largely hydrophobic substrate binding channel, running away from the NADPH co-factor  both sit in a different phylogenetic clade to that of PksJ ACP4, reflective of the MU from which it originates (DH–KRB––KS + ER)  and ‘clade h’ (e.g. PksJ ACP3a/b) are distinct from each other, with sequence logos of these clades highlighting the differences in conserved residues  were cloned and overproduced in E. coli followed by intact protein MS analysis to confirm their identity   . We postand N73) . This suE. coli yielded insoluble protein, as did a chimeric construct replacing HII of the PksJ ACP3a domain with that of the PksJ ACP4 domain. The mutagenesis data described previously indicated that Q51 plays critical role in the interaction epitope (Although the PksJ ACP3a domain was unable to interact productively with the PksJ KR2 domain, residues required for the binding epitope on the ACP domain had been elucidated from our mutagenesis data and docked model. Using this knowledge, a set of mutations and chimeric constructs were produced to engineer the PksJ ACP3a domain towards a productive interaction with the PksJ KR2 domain. In the first instance, a hepta-mutant construct of was produced, where all seven residues of the PksJ KR2-binding epitope were introduced into the equivalent positions on the PksJ ACP3a domain. Overexpression of the hepta-mutant construct in  epitope  and is h epitope . Althoug epitope . The E25 epitope . These f epitope . Althoug epitope , introduI–HII from the PksJ ACP4 domain, and HIII–HIV from the PksJ ACP3a domain yielded soluble recombinant protein, which encompassed all seven residues of the PksJ KR2 domain interaction epitope. Furthermore, the PksJ ACP4 [HI–HII]/ACP3a [HIII–HIV] chimera was able to restore ketoreduction activity to ∼60% of WT PksJ ACP4 domain levels (Surprisingly, a chimeric construct comprised of Hn levels . Despitetrans-AT PKS, allowing production of a docked model driven by residue-level experimental data. The KR-binding epitope of the ACP domain encompasses key residues situated on HI and HII, and combined with previous observations,17 the ACP domain docks at the same KRC subdomain site in both cis- and trans-AT systems. However, unlike cis-AT PKSs,29,30 we have shown that ACP domains from trans-AT PKSs are highly specific for the KR domain within their module/migratory unit; a result of the distinct evolutionary origins of cis- and trans-AT PKSs. Exchange of the HI–HII region within an ACP domain promoted communication with a non-cognate KR domain, highlighting the minimum requirements for interaction with the KR domain and the potential for manipulation of the ACP domain. Taken together, these results provide important domain-level compatibility rules for engineering efforts on trans-AT PKS pathways, and serves to highlight the requirement for a fundamental understanding of the carrier protein interaction network to ensure these endeavours are successful.In summary, we have elucidated molecular details of the interaction interface between an ACP domain and a KR domain from a 10.17632/8shpf4mrs6.1.The 200 ns MD trajectory stripped of water and ions with frames every 1 ns is available for download from Mendeley Data DOI: M. J. conceived and designed the study. M. P. and M. J. generated expression constructs, overproduced and purified proteins, constructed mutant plasmids and performed biochemical assays. M. J. performed and analysed the carbene footprinting experiments. A. G., and J. R. L. conducted docking studies and molecular dynamics simulations. M. J. and M. P. wrote the manuscript with input from all authors.There are no conflicts to declare.SC-012-D1SC03478B-s001


This study was conducted to determine the effect of composite flour (CF) constituents and different extrusion temperatures on the production of analog rice and community perceptions about the produced rice. CF was produced by mixing modified cassava flour (Mocaf) and snakehead fish flour (SF) in the following ratios: 100:0; 97:3; 94:6; 91:9; and 88:12. Analog rice was then extruded from CF at the following temperatures: 50, 70, and 90°C. The analog rice was tested for physical properties (bulk density and cooking time), chemical properties (nutrient content), and organoleptic properties. The results showed that CF and extrusion temperature affect the nutrient content of analog rice. The best analog rice formulation constituted of CF with Mocaf:SF ratio of 91:9 and extrusion temperature of 70°C, and contained 14.34% water, 0.85% ash, 71.83% carbohydrate, 11.24% protein, 1.12%, calcium 1,113 ppm, and 2.43% dietary fiber. This study included 42 respondents aged 20–23 years, including 20 males and 22 females. All the respondents showed acceptance for the analog rice, suggesting that it could substitute ordinary rice as a staple food. This study was conducted to determine the effect of composite flour constituents and different extrusion temperatures on the production of analog rice and community perceptions on the produced rice. Composite flour (CF) was produced by mixing modified cassava flour (Mocaf) and snakehead fish flour (SF) in the following ratios: 100:0; 97:3; 94 6; 91:9; and 88:12. Analog rice was then extruded from the CF at the following temperatures: 50, 70, and 90°C. The best analog rice formulation constituted of composite flour with Mocaf–SF ratio of 91:9 and extrusion temperature of 70°C The nutritional composition of white rice is 40%–80% calories, 78.9% carbohydrate, 6.8% protein, and 0.8% iron  and different extrusion temperatures. The study also aimed at determining respondents' perception of the best analog rice and its nutritional content.22.1The materials used in this study were modified cassava flour/Mocaf from Omah Tani, Gunung Pati, Semarang, Central Java; snakehead fish meat from a fish market in Semarang, Indonesia; corn starch (CS); and additional ingredients . Other chemicals used in this work were of analytical grade and used directly without pretreatment.2.2SF was made using a modified procedure by Widodo et al. . Cleaned2.3CF was prepared by mixing Mocaf and SF flour in the following ratios(%w)  with a basis weight of 280 g; then, 120 g CS flour was added to each mixed flour.2.4CF dough was obtained by mixing each sample of CF flour with 400 ml water and 4 g glycerol monostearate. The dough was wrapped and compacted in a cloth, then steamed for 30 min with a temperature of approximately 80°C. The dough that had been preconditioned was placed in the extruder. The extruder used in this study is a single screw extruder equipped with a heating controller assembled from CV Teguh Jaya Teknik Ungaran, Semarang, Indonesia. The extruder operating temperatures were set at  for constituents of composite flour variable and  for extrusion temperature variable. After extrusion, the wet analog rice grains produced were dried at room temperature for 24 hr , did not like (2), neutral (3), liked (4), and very much liked (5). Forty‐two people were selected for the hedonic test  at the Department of Chemical Engineering, Universitas Diponegoro. The parameters assessed include aroma, color, taste, and texture.2.7Data processing for proximate analyses, calcium analysis, and hedonic rating was performed using one‐way analysis of variance, and the Duncan post hoc test was used to separate means.33.1The result of the analysis is represented by Tables 3.2Table There were statistical differences in calcium content among the CF formulations; the CF formulation with Mocaf:SF ratio of 91:9 had the highest calcium concentration 1,113.0 ppm), whereas the CF formulation with Mocaf only had the lowest (662.5 ppm) as shown in Figure .0 ppm, wThere were statistical differences in protein content among the CF formulations  was found in sample 12 (97:3), whereas the lowest crude fiber content (2.43%) was found in sample 9 (91:9)  was determined based on the best proximate analysis of various compositions. Respondents were assessed for their analog rice preference using a 1–5 scale ranging from “very much did not like” to “very much like.” The results of organoleptic tests on analog rice are presented in Table Based on Table 4In conclusion, based on the proximate analysis on various compositions of CF, sample 9 (with a ratio of Mocaf to CF of 91:9 and extrusion temperature of 70°C) is the analog rice with the best formulation. The highest calcium and carbohydrate levels in this sample of analog rice could make it a staple food.The authors declare that they do not have any conflict of interest.Siswo Sumardiono: Conceptualization (lead); Formal analysis ; Funding acquisition (lead); Investigation ; Methodology (lead); Project administration (lead); Resources ; Software (supporting); Supervision (lead); Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Budiyono: Conceptualization ; Formal analysis ; Investigation ; Methodology . Heny Kusumayanti: Data curation ; Funding acquisition (supporting); Investigation (supporting); Project administration ; Resources . Novian Indra Agung Prakoso: Data curation ; Investigation ; Validation ; Writing‐original draft . Fawzia Puti Paundrianagari: Data curation ; Investigation ; Validation ; Writing‐original draft . Heri Cahyono: Formal analysis ; Software (lead); Writing‐review & editing .Ethical Review: This study was approved by the Institutional Review Board of Universitas Diponegoro.Informed Consent: Written informed consent was obtained from all study participants.


Trypanosoma brucei. Centromere localization of these proteins is mediated by the unique centromere localization domain.Kinetoplastids are evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. Marcianò et al. show that KKT2 and KKT3 play important roles in kinetochore assembly in  Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain , which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite  The kinetochore is the macromolecular protein complex that drives chromosome segregation during mitosis and meiosis in eukaryotes. Its fundamental functions are to bind DNA and spindle microtubules . In mostTrypanosoma brucei  cells that kinetochore localization of KKT2 and KKT3 was not affected by KKT4 depletion  peptide (GFP-NLS) in . brucei . We prev. brucei . The pre. brucei .672–1030 copurified with several kinetochore proteins, with KKIP1 being the top hit . A corollary is that, in WT cells, the constitutively localized KKT2 protein recruits these transient kinetochore proteins onto kinetochores using KKT2672–1030 and DPB domains. It is noteworthy that KKT14 was not detected in the immunoprecipitates of any KKT2 fragments we tested, despite our findings that KKT2 was one of the most abundant proteins in the immunoprecipitates of KKT14 . KKT3594–811 copurified with limited amounts of KKT7, KKT1, and KKT2  phasing . Although the CL domain and the C1 domain share some structural similarity, their superposition revealed fundamental differences . The sequence alignment of the BsKKT2 C2H2 zinc finger shows that residues at the positions −1 and 3 are highly conserved in trypanosomatids, while those at positions 2 and 6 are not , PkKKT2b (XU18_0308), and PkKKT2c (XU18_4564), because they tend to have higher sequence similarity to KKT2 than KKT3, especially in the kinase domain: 34.4% identical between PkKKT2a and TbKKT2 compared with 24.9% between PkKKT2a and TbKKT3; and 34.5% identical between PkKKT2b and TbKKT2 compared with 25.8% between PkKKT2b and TbKKT3. Interestingly, similarities among these Perkinsela proteins are higher than those between them and KKT2 or KKT3 in other kinetoplastids , with the exception of some differences being localized to the loop insertion and the absence of a C2H2 domain in PkKKT2a551–679 , it is likely that the unique CL domain structure is conserved among kinetoplastids.We determined the crystal structure of  phasing , which c phasing . The CL-a551–679 , consista551–679 . Taken tPerkinsela KKT2a lacks a C2H2 zinc finger, our sequence analysis of the KKT2 central domain revealed a putative C2H2 zinc finger not only in trypanosomatids and bodonids, but also in one of Prokinetoplastina’s KKT2-like proteins, PhM_4_m.86555  of ∼500 nM on three DNA probes that have different GC contents  and found that it has a similar DNA-binding affinity compared with WT PkKKT2a551–679  and found that it has similar DNA-binding activity as WT TbKKT2562-677 . We found that mutations in zinc-coordinating residues of the CL domain  all abolished kinetochore localization  suggested that TbKKT3 likely consists of two domains that correspond to the CL domain and the C2H2 zinc finger present in KKT2  did not affect kinetochore localization.The central domain of ll cycle . The seq in KKT2 . Consist protein . We also protein  was esse protein . In contTbKKT3 with a C-terminally YFP-tagged construct that has either WT or mutant versions of the central domain and performed RNAi against the 3′UTR of TbKKT3 to knock down the untagged allele of TbKKT3  were unable to localize at kinetochores, while the TbKKT3 C2H2 zinc finger mutant (C707A and C710A) localized normally , we previously showed that KKT4 is important for the kinetochore localization of KKT20, but not many other proteins, including KKT2 and KKT3 . Importantly, mutational analyses of TbKKT2 revealed that the CL domain is important for the localization of the central domain, while the C2H2 zinc finger is not. Furthermore, although full-length TbKKT2 CL mutants localized at kinetochores (likely due to interactions with other kinetochore proteins via other domains of TbKKT2), they were not fully functional. Taken together, these data have established that the CL domain is essential for the function of TbKKT2, which is consistent with the presence of CL, but not C2H2 zinc finger, in Perkinsela KKT2a. Given that the CL domain mutants of TbKKT2 and PkKKT2a still have DNA-binding activity, it is unlikely that centromere localization of the KKT2 central domain relies solely on its DNA-binding activity.By ectopically expressing fragments of KKT2 and KKT3 in TbKKT3 was also essential for the kinetochore localization and function, while the equivalent domain of C2H2 zinc finger was not, showing that the functional importance of CL is conserved in KKT3. It will be important to obtain KKT3 central domain structures to reveal structural similarity or difference between KKT2 and KKT3. It is noteworthy that all identified KKT2/3 homologues in deep-branching Prokinetoplastina have greater similarity to KKT2 than KKT3. We speculate that ancestral kinetoplastids had only a KKT2-like protein(s) that performed all necessary functions, and that KKT3 in trypanosomatids and bodonids represents a product of gene duplication that became specialized in certain functions, such as more efficient centromere localization by its central domain compared with KKT2.It remains unclear whether the structure of the central domain is conserved between KKT2 and KKT3. Nonetheless, our functional studies showed that the equivalent domain of CL in T. brucei has four histone variants: H2AZ, H2BV, H3V, and H4V; however, none of them is specifically enriched at centromeres , BAG95 synthetic DNA fragment was digested with HindIII/BamHI and subcloned into the HindIII/BamHI sites of pBA310. pBA2052 (KKT2/3 double-hairpin RNAi construct targeting KKT2 5′UTR from −342 bp to start codon as well as KKT3 3′UTR from stop codon to +370 bp) was made with BAG129 as above. Similarly, pBA861  was made with BAG24, pBA864 (KKT6 hairpin RNAi targeting 12–499 bp) with BAG27, pBA869  with BAG32, pBA1316 (KKT8 3′UTR hairpin RNAi targeting from +31 to +446 bp) with BAG79, pBA1845 (KKT22 hairpin RNAi targeting 466 bp to stop codon) with BAG105, pBA1997  with BAG115, and pBA2021  with BAG127. To make pBA1091 (KKIP1 RNAi), 297–856 bp of KKIP1 coding sequence was amplified with primers BA1541/BA1543 and cloned into p2T7-177 using BamHI/HindIII sites . To makePlasmids linearized by NotI were transfected into trypanosomes by electroporation into an endogenous locus (pEnT5-Y derivatives and pBA148/pBA192/pBA892 derivatives) or 177-bp repeats on minichromosomes (pBA310 derivatives and p2T7-177 derivatives). Concentrations of drugs used were as follows: 5 µg/ml phleomycin, 25 µg/ml hygromycin, 10 µg/ml blasticidin, 30 µg/ml G418, and 1 µg/ml puromycin. To obtain endogenously tagged clonal strains, transfected cells were selected by the addition of appropriate drugs and cloned by dispensing dilutions into 96-well plates. Clones that express mutant versions of KKT2 or KKT3 from the endogenous locus were screened by Sanger sequencing of genomic DNA. Expression of GFP-NLS fusion proteins (pBA310 derivatives) was induced by the addition of doxycycline (10 ng/ml). RNAi was induced by the addition of doxycycline (1 µg/ml).To analyze fluorescently tagged proteins, cells were washed once with PBS, settled onto glass slides, and fixed with 4% paraformaldehyde in PBS for 5 min . Cells w6 cells/ml and expression of GFP-NLS fusion proteins was induced with 10 ng/ml doxycycline for 24 h. Cells were pelleted by centrifugation , washed once with PBS, and extracted in PEME  with 1% NP-40 and protease inhibitors  and phosphatase inhibitors  for 5 min at RT, followed by centrifugation . Samples were kept on ice from this point on. The pelleted fractions that contain kinetochore proteins were resuspended in modified buffer H  containing protease inhibitors and phosphatase inhibitors. Samples were sonicated to solubilize kinetochore proteins . 12 µg of mouse monoclonal anti-GFP antibodies  that had been preconjugated with 60 µl slurry of Protein-G magnetic beads  with dimethyl pimelimidate  with carbamidomethyl cysteine as a fixed modification. Up to two missed cleavages were allowed. Oxidization (Met), phosphorylation , and acetylation (Lys) were searched as variable modifications. Mass tolerances for mass spectrometry and tandem mass spectrometry peak identifications were 20 ppm and 0.02 D, respectively. Proteins identified with at least two peptides were considered significant and listed in To identify interaction partners of KKT2 or KKT3 fragments fused with GFP-NLS, we performed immunoprecipitation using anti-GFP antibodies and identified copurifying proteins by mass spectrometry . Typicallimidate  were inchttps://www.sanger.ac.uk/), UniProt , the central domain of B. saltans KKT2 (TriTrypDB accession no. BSAL_50690) was amplified from BAG50 , the central domain of Perkinsela CCAP 1560/4 KKT2a (UniProt accession no. XU18_4017) was amplified from BAG48 , designed zinc finger domain was amplified from BAG136 (a synthetic DNA codon optimized for expression in E. coli) with primers BA3077/BA3078 and cloned into RSFDuet-1 using BamHI/EcoRI sites with an NEBuilder HiFi DNA Assembly Cloning Kit. To make pBA283 (His6-TbKKT2562–677), the central domain of T. brucei KKT2 (TriTrypDB accession no. Tb927.11.10520) was amplified from genomic DNA with primers BA670/BA574 and cloned into pNIC28-Bsa4 using ligation-independent cloning. To make pBA1178 , the central domain of T. brucei KKT2 was amplified from pBA493  with primers BA1563/BA1567 and cloned into RSFDuet-1 using BamHI/EcoRI sites with an NEBuilder HiFi DNA Assembly Cloning Kit. Recombinant proteins were expressed in BL21(DE3) E. coli cells at 20°C using auto induction media . Cells were harvested by centrifugation and resuspended in 50 ml per liter of culture of lysis buffer phosphine (TCEP), 10 mM imidazole, and 1.2 mM PMSF). Proteins were extracted by mechanical cell disruption using a French press , and the resulting lysate was centrifuged at 48,384 g for 30 min at 4°C. Clarified lysate was incubated with 5 ml TALON beads (Takara), washed with 150 ml lysis buffer, and eluted in 22 ml of elution buffer  in a gravity column, followed by tobacco etch virus treatment for the removal of the His6 tag. Salt concentration of the sample was subsequently reduced to 50 mM NaCl using buffer A  and the sample was loaded onto a 5-ml HiTrap Heparin HP affinity column  preequilibrated with 5% buffer B  on an ÄKTA pure 25 system. Protein was eluted by using a gradient from 0.05 to 1 M NaCl, and protein-containing fractions were combined, concentrated with an Amicon stirred cell using an ultrafiltration disc with 10-kD cutoff (Merck), and then loaded onto a HiPrep Superdex 75 16/60 size exclusion chromatography column  preequilibrated with 25 mM Hepes (pH 7.5), 150 mM NaCl, and 1 mM TCEP. Fractions containing the protein of interest were pooled together, concentrated with an Amicon stirred cell using an ultrafiltration disc with 10-kD cutoff, and stored at −80°C. Designed zinc finger was buffered exchanged into 50 mM Tris (pH 7.5), 1 mM ZnCl2, 50 mM NaCl, and 1 mM TCEP prior to storage. Protein concentration was measured by Bradford assay.Briefly, 500 ml of cells were grown at 37°C in 2.5-liter flasks at 300 rpm until ODBsKKT2572–668 and PkKKT2a551–679 crystals were optimized at 4°C in sitting drop vapor diffusion experiments in 48-well plates using drops of overall volume 400 nl, mixing protein, and mother liquor in a 3:1 protein:mother liquor ratio. BsKKT2 central domain crystals grew from the protein at 26 mg/ml and mother liquor 40% PEG 400, 0.2 mM (NH4)2SO4, and 100 mM Tris-HCl (pH 8). The 40% PEG400 in the mother liquor served as the cryoprotectant when flash-cooling the crystals by plunging into liquid nitrogen. PkKKT2a551–679 crystals grew from the protein at 13 mg/ml and mother liquor 19% 2-methyl-2,4-pentanediol (MPD), 50 mM Hepes, pH 7.5, and 10 mM MgCl2. The crystals were briefly transferred into a cryoprotecting solution of 30% MPD, 50 mM Hepes, pH 7.5, and 10 mM MgCl2 before flash cooling.Both BsKKT2 central domain crystal were collected at the I04 beamline at the Diamond Light Source at the Zinc K-edge wavelength (λ = 1.28297 Å). A set of 1,441 images were processed in space group I222 using the Xia2 pipeline  and processed using the autoPROC pipeline  with accession nos. 6TLY  and 6TLX (Perkinsela KKT2a).All images were made with Pymol (Schrödinger) and CCP4mg . TopologDNA oligonucleotides were purchased from Thermo Fisher Scientific . To makedK.All experiments were performed in binding buffer  using 1 nM fluorescein amidite–labeled dsDNA sequences purchased from IDT . Prior tPkKKT2 and TbKKT2 central domains. 558–679, KKT2672–1030, KKT21024–1260, KKT21024–1260 (W1048A), KKT3594–811, and KKT3846–1058 by mass spectrometry. BsKKT2 CL domain structure. BsKKT2 C2H2 zinc finger structure. Review HistoryClick here for additional data file.Table S1558–679, KKT2672–1030, KKT21024–1260, KKT21024–1260 (W1048A), KKT3594–811, and KKT3846–1058 by mass spectrometry.lists the proteins identified in the immunoprecipitates of YFP-tagged KKT2Click here for additional data file.Table S2BsKKT2 CL domain structure.lists the DALI search hits for the Click here for additional data file.Table S3BsKKT2 C2H2 zinc finger structure.lists the DALI search hits for the Click here for additional data file.Table S4lists the trypanosome cell lines, plasmids, primers, synthetic DNA, and DNA probes used in this study.Click here for additional data file.


Due to climate change sorghum might gain widespread in the Western countries, as the grain is adapted to hot climate. Additionally sorghum contains a notable amount of health-promoting nutrients. However, Western countries do not have a long history of sorghum consumption, and thus little experience in processing it. Milling systems in these areas were mostly developed for wheat or rye milling. In the present work, the effectiveness of sorghum milling when using a stone and a roller milling system  was investigated as well as its impact on the chemical and physical properties of the obtained flour fractions and whole-grain flours. Results showed that both milling systems could be successfully adapted to producing chemically and physically distinct flour and bran fractions from the small sorghum kernels. Fractions with increased bran material that contained higher amounts of ash, protein, fat, total dietary fiber, and total phenolic content but less starch, showed enhanced water absorption indices and water solubility indices. Interestingly, no significant difference was found in the ash and fat content of the different fractions obtained from stone milling. Overall, the study provided information on the production and composition of distinct flour fractions, which offer a wider range of future food applications. Sorghum is the fifth most produced cereal worldwide and it is mostly harvested in Africa, followed by America and Asia . TraditiWhen using cereals for food processing, flour production is often the first step. As flour properties and composition greatly affect end-product quality, milling, and fractionation processes play a crucial role. Knowledge on sorghum processing has been mostly gained in Africa and Asia. Some studies have described grinding of sorghum (whole-grain) flour using lab scale mills or local milling systems, e.g., hammer mills ,6. MoreoIn Austria, different varieties of sorghum are cultivated in very small quantities. The harvested grain is used for animal feed and bioethanol production, but not for human nutrition. Therefore, these sorghum varieties have neither been investigated for their food processability nor been chemically analyzed before. In order to assess the potential of these Austrian grown sorghum varieties, the aim of this study was (1) to produce sorghum whole-grain flours, as well as flour fractions; and (2) to characterize all the obtained flours and fractions for their chemical and physical properties. Milling and fractionation of sorghum was carried out using two pilot scale milling systems (stone mill and roller mill). For comparison purposes, an industrial scale whole-grain flour, milled by a roller milling system was included. Chemical and physical analyses were performed to evaluate on the other hand, whether the (wheat) pilot scale stone mill and/or the roller milling system can be used for sorghum milling and fractionation. For roller milling, all single fractions were collected separately, in order to follow the performance and effectiveness of this rather complex milling system see . The stuArmorik), where grown on an experimental farm in Hörsching, Austria and harvested in 2019. They were obtained from Caj. Strobl Naturmühle GmbH  and were used for the milling trials  was obtained.DFS-PSM : Several pre-trials were performed to select the appropriate equipment and settings for stone milling. Based on these results (data not shown) a pilot scale dry flake squeezer  and a pilot scale stone mill  produced a WSF, which was separated by a sifter  into three flour fractions with particle sizes >400 µm, 400–180 µm, and <180 µm. The purpose of squeezing the kernels prior to stone milling is to increase the flour yield. As a first step, the sorghum kernels were squeezed in the dry flake squeezer at 1000 kg/h, then they were ground in the pilot scale stone mill (PSM), set with the smallest possible gap between the stones, 507 revolutions per minute and an hourly rate of 100 kg/h. The gap between the stones was estimated 0.2–0.5 mm, which could not be quantified more accurately. One stone of the mill veered and the other one backed. For fractionation, the whole-grain sorghum flour was first sieved with a 400 µm sieve at 2000 kg/h. The <400 µm fraction was further sieved through a 180 µm sieve, thus obtaining two further fractions (400–180 µm and <180 µm). DFS-PSM was performed from 100 kg of sorghum grain.PRM : The second milling trial applied a pilot scale roller milling plant made of several roller mills  and plansifters  after each passage. Milling was performed with an hourly rate of 150 kg/h. The configuration and settings of this pilot scale roller milling plant were established according to pre-trials on Bühler MLU-202 , which is a lab scale roller milling system (results not presented here). The process of  roller milling/sifting is based on various breaking and reduction steps see . UsuallyAll produced WSFs, as well as all defined flour fractions were analyzed, as described below. From the 14 flour fractions obtained in the second milling system five fractions were selected, representing different sections of the sorghum kernel: bran fine (outer layer), B1, C1 (white endosperm flour), C4, C5/C6/B4/B5 (dark endosperm flour), as well as a reconstituted WSF. The selected fractions were chosen in order to follow if fractions from different parts of the kernel were characterized by different chemical or physical properties.Color analyses were carried out as described in Bender et al. . A DigiEg. Supernatants were separated from the sediment and the liquid phase was collected. After adding 10 mL ethyl acetate to both, the solid and the liquid phases, the mixtures were shaken for 5 min and afterwards centrifuged for 2 min at 3092× g. For these analyses ethyl acetate was chosen as extraction aid in order to extract the liberated phenolics, as well as the previously unesterified phenolics, from the aqueous extract. Ethyl-acetate is a suitable solvent for the phenolics and, in contrast to more polar solvents like methanol or ethanol, can be separated from the aqueous phase via centrifugation. The supernatants (ethyl acetate phases) of the solid and the liquid phases were collected together in a shaded round bottom flask. The extraction with ethyl acetate was repeated twice. Samples were concentrated using a rotavapor  and dissolved in 4 mL of 50% methanol. For TPC determination 100 µL of the extracts were mixed into 0.5 mL of freshly prepared 10-fold diluted Folin-Ciocalteu reagent. After 2 min waiting time 0.8 mL sodium carbonate (75 g/L) was added. The mixtures were incubated for 5 min at 50 °C. The absorbances were read at 755 nm against a blank. The results were expressed as mg ferulic acid equivalent (FAE) per 100 g dry substance. All chemical analyses were carried out in triplicate.Moisture content was determined by ICC standard method Nr. 110/1 . Ash cong for 10 min and residues were weighed for WAI calculation. Supernatants were collected in aluminum dishes and evaporated for 4 h in a drying cabinet at 103 °C. Dried solids were used for WSI calculation. WAI was expressed as absorbed water in g/g dry sample, WSI was expressed as percentage of dry solids/2.5 g dry sample. Measurements were carried out in triplicate.The water absorption index (WAI) and the water solubility index (WSI) were measured based on the method from Anderson et al. . BrieflyStatistical data analyses were carried out using Statgraphics Centurion 18, Version 18.1.13 . Significant differences were evaluated by performing an ANOVA and Fisher’s least significant test with a significance level of α = 0.05. Results were expressed as mean ± standard deviation and significant differences between results were expressed by different superscript letters.Milling yields obtained from the two investigated milling systems are visualized in The most abundant flour fraction in DFS-PSM, was the flour with a particle size of <180 µm, 43.9%, followed by almost equal parts of the remaining two fractions >400 µm, 27.5%, and 400–180 µm, 28.6% see .As described in the methodology, all PRM passages were collected separately. As for the PRM milling system, one-fifth of the flour fractions ended in bran material (bran coarse and bran fine) and further 8% in the bran-rich animal feed flour. As expected, the flour fraction B1 was extracted to a very low extent, as it is the first endosperm flour obtained after grain breaking. Higher yields were obtained during the reduction milling system (C1–C5). The flour fraction C1 showed a milling yield of 11%. However, the highest amount within the flour fractions C and B was extracted after the C5/C6 passage, and after the B4/B5 passage, respectively. Total endosperm flour accumulated to 72%, while the remaining 28% were bran and animal feed. Only five flour fractions were selected from this milling system for subsequent chemical and physical analyses and are highlighted in Overall, both milling trials were successful to produce not only whole-grain flour, but also different flour fractions. Although the sorghum kernel differ greatly from wheat or rye kernels in size and shape, both the stone mill and the roller milling system could be used and adapted for production of sorghum milling fractions. Endosperm flour was successfully extracted in both cases, as could visually be observed, and will be described by the color and chemical data in the subsequent sections, Well configurated industrial scale mills with even higher number of passages are able to extract up to 80% of endosperm flour in wheat milling.In In the DFS-PSM milling fractions lightness  increased with decreasing particle sizes. They were also less red and yellow, which indicated a lower content of bran in the finer fractions.In the PRM milling fractions, the bran fraction had the highest a*-values (red) and b*-values (yellow), as expected. These value trends are further in line with previous findings . HoweverThe results of the chemical analyses are displayed in The dry matter content of all analyzed sorghum flours ranged between 88.73 annd 91.72%. Among the sorghum flour fractions obtained from DFS-PSM, no significant differences in ash and fat content were observed. This was also seen in a previous study, which stated no differences in ash, fat, and protein in sieving fractions of whole-grain wheat . The floBased on protein, starch, TDF, and total polyphenol content, it seemed that nutritional fractionation within the DFS-PSM sieving took place; although, ash and fat contents did not differ significantly among different fractions. Both, fat and ash, are the predominant nutrients in the germ ,26. An eAs described in type W480 according to the Austrian codex Alimentarius [The C1 passage flour fraction showed similar results to the B1 passage fraction flour. On the basis of ash, protein, starch, fat, TDF, and TPC values, it can be concluded that the C1 fraction flour corresponds to a rather pure endosperm flour. In wheat milling, the flour obtained after the C1 passage milling corresponds to a very white endosperm flour, which is categorized as entarius . The redAs mentioned previously, milling has a great effect on the resulting flour properties. The effect of the two applied milling systems on flour quality can be followed when comparing the whole-grain sorghum flours produced in this study. For comparison, a commercially available whole-grain sorghum flour was included . Again, it has to be considered that PRM WSF was reconstituted. Ash was the only component which did not differ significantly between the three whole-grain sorghum flours. The highest protein value was observed for PRM WSF, followed by DFS-PSM and IRM. An explanation for the higher protein content in PRM could be an improper reconstitution of WSF based on the fraction yields. Endosperm flour yields may have been underestimated, as an undefined amount of flour residue inevitably remained in the mill. So far, lower protein contents in tartary buckwheat flours obtained from a roller mill compared to a stone mills have been reported , but no Generally, rather low and insignificant differences were observed between the stone milled and roller milled flours. Pagani et al.  also didResults of water absorption index (WAI) and water soluble index (WSI) are given in Flours obtained from DFS-PSM decreased in WAI with decreasing particle size. Al-Rabadi et al. (2012) evaluated comparable results on sorghum flour for similar particle sizes. However, the authors reported no consistent WAI trend in flours of different particle sizes . Cadden Water solubility indices (WSI) of DFS-PSM flour fractions were lower with decreasing particle size. Looking at the PRM flour fractions, the bran had the highest WSI. Flour fractions from B and C fractionation showed similar WSI ranging from 2.42 to 3.7 1%. Comparing the whole-grain flours, WSI from PRM milling had a significant higher WSI than from PSM or IRM milling. This might also be related to the higher ratio of bran in the reconstituted whole-grain flour of PRM.Considering the chemical data in combination with the physical properties, it can be stated that the obtained sorghum flour fractions offer different food uses, both, either in terms of intended nutritional enrichment, and in terms of specific processing needs. Some bakery products require very defined physical or rheological conditions of flours, e.g., fine bakery products or wafers. Replacing wheat flour with any other flour therefore has major effects on the end-product quality and requires thorough adaptation of recipe and processing parameters. Detailed knowledge on flour properties is one of most important factors to be considered in this respect. The data obtained in this study for sorghum flour fractions provide a step forward to future use of sorghum and incorporation into Western type bakery products.Results of this research indicated that stone and roller milling, both typically applied for wheat milling, were effective for milling sorghum and could successfully produce defined flour fractions, as well as whole-grain flours.With regard to stone milling, the chemical and physical properties of the obtained flour fractions were clearly distinct, demonstrating that fractionation was not only based on their particle sizes , but also on their chemical profile.In the roller milling system, flour fractions were not separated by sieving only, but by a typical sieving/sifting combination after each roller mill passage. Although the size and shape of sorghum kernels differed to wheat, roller milling settings could be adapted to sorghum milling. Chemical and physical properties of defined roller milled flour fractions indicated that sorghum grains were separated into different anatomical parts. Considering the chemical data, it can be concluded that the B1 and C1 flour fractions were highly concentrated from endosperm parts. The C4 and C5/C6/B4/B5 fractions can be defined as dark endosperm flours, which was confirmed by a lower starch content, but a higher ash, protein, fat, and TDF content compared to B1 and C1 flour fractions. As the bran fraction was shown to be largely composed of outer kernel layers and germ, it was confirmed, that it could successfully be separated from the endosperm. The WSF from the roller milling system was significantly higher in protein and TPC, but lower in starch compared to the WSF from stone milling. This was likely the result of an improper reconstitution of WSF based on the fraction yields.Overall, the results of the chemical and physical analyses of the flour fractions, obtained from stone or roller milling systems, seem to confirm that sorghum milling can be carried out on typical wheat milling systems without detailed or complex technical adaptations. Both milling systems were successful in producing flour fractions with distinct chemical and physical properties. The two milling systems differed in their ability to produce distinct fractions, with roller milling being superior to stone milling. Depending on the intended food use, the availability of defined flour fractions can be advantageous for selected food applications ,9 as somThis study provided information on defined sorghum flours and flour fractions that might facilitate the future use of sorghum in the Western diet.


Oxyuropoda—reported in 1908 from the Late Devonian floodplains of Ireland, and left with unresolved systematic affinities despite a century of attempts at identification. Known from a single specimen preserved in two dimensions, we analysed its anatomy using digital microscopy and multispectral macroimaging to enhance the contrast of morphological structures. The new anatomical characters and completeness of Oxyuropoda, together with a phylogenetic analysis with representatives of all major Eumalacostraca groups, indicate that Oxyuropoda is a crown peracarid, part of a clade including amphipods and isopods. As such, Oxyuropoda is the oldest known species Peracarida, and provides evidence that derived peracarids had an incursion into freshwater and terrestrial environments as early as the Famennian, more than 360 Ma.Peracarida (e.g. woodlice and side-swimmers) are, together with their sister-group Eucarida (e.g. krill and decapods), the most speciose group of modern crustaceans, suggested to have appeared as early as the Ordovician. While eucarids' incursion onto land consists of mainly freshwater and littoral grounds, some peracarids have evolved fully terrestrial ground-crawling ecologies, inhabiting even our gardens in temperate regions (e.g. pillbugs and sowbugs). Their fossil record extends back to the Carboniferous and consists mainly of marine occurrences. Here, we provide a complete re-analysis of a fossil arthropod— In peLigia, a coastal terrestrial oniscoid woodlouse, earned it its name Oxyuropoda ligioides Carpenter and Swain, 1908. In addition to its round and short head fused to larger thoracic segments, it possesses a tapering and short pleon, with curved and pointed lateral pleurae on the abdominal segments, typical of isopods. However, its anatomy reveals six thoracic segments instead of the usual seven in isopods. Six such segments exist in other peracarids but these are very different in shape [A crustacean-looking arthropod, found in the early twentieth century from the Famennian (Late Devonian) of Ireland , has lonin shape . From itin shape  the fossin shape ,24–29, pin shape , arachnoin shape –33 or otin shape –35. Here. 2 (a)Oxyuropoda ligioides consists of a single specimen  housed in the palaeontological collection of the National Museum of Ireland, Dublin—Natural History, and recovered from Kiltorcan Hill, County Kilkenny, Ireland. Besides O. ligioides, Kiltorcan Hill outcrops have yielded a range of continental freshwater-type organisms, including lycophytes, progymnosperms, green algae Bythotrephis, freshwater bivalves, placoderms [Oxyuropoda from what can be recognized as the Classic or Old Quarry of Kiltorcan [New Quarry [Roadstone Quarry [acoderms ,36 and facoderms –39. Carpacoderms  reportediltorcan ,36,40. Te Quarry ,36,37) y (b)f/2.8 Macro lens was used to photograph the specimen under artificial lighting from multiple directions. Polarizing filters at the lens and the light source created crossed polarization that reduced reflections and increased contrast. Digital microscopy images were collected using a Keyence VHX-7000 digital microscope equipped with a VH-ZOOT Macro lens (0×–50× magnification), connected to a VXH-7020 high performance 3.19-megapixel CMOS camera. High-resolution three-dimensional images were collected through an automatic stitching process. Three-dimensional images presented herein in natural  or false warm–cold  colours were produced through an automatic vertical stacking process that creates three-dimensional surface profiles.A Canon EOS 800D digital SLR camera fitted with an EF-S 60 mm  (c)b) red—illumination 435 nm/detection 435 ± 20 nm (reflection), green—illum. 660 nm/det. 650 ± 30 nm (refl.), blue—illum. 435 nm/det. 571 ± 36 nm (luminescence); (ii) (figure 1c) red—illum. 385 nm/det. 935 ± 85 nm (lum.), green—illum. 435 nm/det. 571 ± 36 nm (lum.), blue—illum. 435 nm/det. 435 ± 20 nm (refl.).Building on the concept of UV photography  and visifigure 1 (d)Oxyuropoda using a morphological dataset for malacostracan crustaceans modified from [Oxyuropoda, following the approach of [https://morphobank.org/permalink/?P3989). We included in a second analysis †Tealliocaris, a Late Devonian–Carboniferous eumalacostracan with debated peracarid or decapod affinities [. We analysed the dataset using Bayesian inference as implemented in MrBayes v. 3.2.6 [7 generations each. We used the default settings of four chains (one cold and three heated) per independent run. The relative burnin fraction was set to 25% and the chains were sampled every 200 generations. The temperature parameter was set to 0.01 as determined by preliminary runs to achieve chain mixing values in the optimal range (0.4–0.8). The convergence of independent runs was assessed through the average standard deviation of split frequencies ASDSF = 0.01 and potential scale reduction factors . We userameters  to deterrameters , Isopodarameters ,11,13 anrameters .Figure . 3O. ligioides specimen resolved both new anatomical features, never observed by optical microscopy, and the artefacts leading to previous erroneous interpretations of the fossil (a–h). Elongated elements have earlier been questioned to correspond either to antennae/uropod parts or to plant remains that look very similar to the animal appendages and are very abundant in the specimen [b). A three-dimensional surface rendering using digital microscopy accentuated a different extent of deformation between the right and left pleurae, with the left ones having bent more vertically than the right ones . Parallel to the left pleurae, the specimen exhibits a marked groove that we interpret as a break associated with the more pronounced compression of the left side of the thorax (b). This localized bending implies that the body of Oxyuropoda was generally flattened dorsoventrally, but had the extremities of its pleurae directed ventrally rather than laterally. Elements of the cephalon previously suggested to be the eyes are here revealed to be topographical artefacts , which we cannot exclude to reflect more ventrally located organs, but which we rather interpret as local, and asymmetric, bulges reflecting superposition of appendages. The first segments of the antennae—with large peduncles —and the outlines of the buccal appendages are observed for the first time . These are comparable to the derived appendages of modern malacostraca, being elongated in shape . The mandible structure shows a strong incisor, crenelated at its very tip end and bearing four teeth, as well as an elongated palp directed forward . The number of these appendages suggests that there are three somites forming the cephalon part of the cephalothorax ; the presence/absence of a carapace above these segments cannot be established. The thorax comprises six thoracomeres  increasing in size from anterior to posterior , each crossed by two transverse suture ridges and likely bearing a pair of pereopods the bases of which are locally visible . Some pleurae exhibit longitudinal to oblique grooves, evoking the coxal articulations found in Isopoda thoracomeres. Close examination of these structures reveals they correspond to be taphonomic cracks, meaning there is no anatomical evidence for coxal plate articulations in Oxyuropoda . The pleon is composed of six pleomeres , with the last one bearing the telson and a pair of uropods posteriorly oriented and styliform in shape . See electronic supplementary material, text ST1 for systematic palaeontology and full description, including measurements.The advanced imaging of the only known e fossil a–h. Elonspecimen ,32. Somee thorax b. This leduncles e,f—and tollowing ) and genOxyuropoda displays only seven directly observable cephalothoracic somites out of the nine typical of malacostracans, requiring further digging into other diagnostic characters such as its uropodal features [Oxyuropoda. The basipodite is straight without extensions, and the uropod is without carinae  and uropods are malacostracan features ,8,65–68,features . In eumaation in , ridge cation in .Oxyuropoda among a strongly supported late diverged clade of Peracarida (posterior probability 83)—previously found in [figure 2a; electronic supplementary material, figure SF2a). The internal phylogenetic relationships of these main peracarid clades, with the exception of a few more-often recovered patterns (e.g. Mysida grouped with Lophogastrida), are so far not resolved [Oxyuropoda maintains (i) the sister-relationship of Isopoda and Amphipoda recovered in [Tealliocaris, whose affinities remain unresolved in our analysis, is found outside of modern peracarid clades . With its Late Devonian age, Oxyuropoda is the oldest known crown-group Peracarida, outdating Anthracocaris scotica Peach, 1882 from the Visean (Early Carboniferous) of the Calciferous Sandstone of the UK [figure 2a). Recovering Oxyuropoda  as a crown peracarid may help polarize the direction of change of cephalic integration of anterior thoracomeres among Peracarida, yet this remains tentative until intrarelationships within crown Peracarida are better resolved.Bayesian inference phylogenetic analysis places found in ,7,69—tharesolved –67,69–74vered in ,74, as wvered in –74, follf the UK , which hf the UK ). No stef the UK . Based of the UK ,11,64, af the UK , suggestOxyruropoda gives insight into the timing of colonization of non-marine environments by peracarids. Oxyuropoda plots either as the direct sister taxon, or within a sister clade (clade ACTI for Spelaeogriphacea) of groups with partial or strict freshwater representatives, the record of which even extends to their fossil representatives  (a). Being likely part of an independent lineage of peracarids, Oxyuropoda provides further evidence that derived Peracarida were present in continental settings as early as the Fammenian, implying the fast colonization of continental waters (and land?) during the evolution of clade ACTI and likely of Spelaeogriphacea. Overall, our results indicate that besides Branchiopoda [figure 2a,b; electronic supplementary material, figure SF3).Finally, the affinity of iphacea) a. Being chiopoda ,76–80, a


R155H, p97R191Q, p97A232E, p97D592N, as well as p97E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains  by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97R155H and p97R191Q), (2) the interprotomer interactions (p97A232E), or (3) the coupling between D1 and D2 nucleotide domains . We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function.The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97 Co. CoA) ineriments . All threriments , 4.FigurDue to the essential role played by p97 in cellular processes, it is no surprise that a number of multisystem diseases are associated with mutations and dysfunctions in p97. Diseases arising from protein degradation defects  such as Prior structural information on p97 mutants largely comes from X-ray crystallographic studies of N-D1 truncated p97 constructs , NMR invOur experiments with these mutants were designed to explore the extent to which the mutations influence the conformational landscape. While p97 is amenable to structural analysis in both substrate-free forms and in the presence of bound protein substrates and cofactors, we chose to study substrate-free complexes since our goal is to unravel the intrinsic effects of the mutations on quaternary structure. Previous studies have shown that the primary effect of MSP-1 mutations is in the transition between conformations (I-III) rather than directly on the hydrolytic domains  or indeeWe also report cryo-EM structures of full-length wild-type p97 complexed to CB-5083 in the presence of ADP and ATPγS and describe how the binding of the inhibitor influences the overall quaternary arrangement of the N, D1, and D2 domains. Taken together, our results provide a detailed understanding of the structural mechanisms underlying the effects of the disease mutations and an explanation of the resistance of the E470D mutation to inhibition by CB-5083.C and S1A) (WT are observed in the presence of bound ADP but not ATPγS (D) or in the presence of bound ATPγS but not ADP (E). These effects are described in greater detail below.All five mutants displayed relative ATPase activities similar to previously reported biochemical studies C and S1Aand S1A) , 16, 19.ot ATPγS D or in t not ADP E. These R191Q, p97R155H, and p97A232E mutants, the primary variation in conformational outcome as compared with p97WT is observed when the D1 and D2 domains are both occupied by ADP (D). In these mutants, the ADP-bound conformation of the D1 domain displays many of the structural signatures observed only upon ATPγS binding to p97WT, suggesting that these mutations shift the conformational equilibrium toward promoting ATP binding. This effect is most readily visible in p97R191Q, with p97R155H and p97A232E displaying similar effects in the D1 domain, but of a smaller magnitude .In the p97d by ADP D. In theB). Mutation to histidine may disrupt interactions resulting in greater flexibility of the N-domain, consistent with disorder in this domain, observed as a decrease in local resolution in the N-domain cryo-EM density. This finding is consistent with previously published NMR studies where the N-domain flexibility, i.e., exchange between “up” and “down” conformations in the ADP-bound state, is inversely proportional to the charged/hydrogen bonding nature of the residues at position 155 . R191 may stabilize the “down” position of the N-domain by maintaining an extended loop conformation in the linker. A unique aspect of the mutation-induced structural changes in the ADP-bound state of p97R191Q is a dramatic ordering of the linker connecting the N and D1 domains into an α-helical structure between residues 190–199 (B and S1A). This structural ordering is observed in p97WT only when ATPγS is bound to the D1 domain, which arises from changes propagated at the other side of the linker proximal to the D1 nucleotide-binding site. The dramatic shift from extended linker to an α-helix upon loss of charge by mutation from arginine to glutamine appears to confirm the vital role R191 plays in stabilizing the “down” position in the N-domain of p97WT. Further inspection of the structure of p97R191Q in the ADP-bound state shows a shift in the local conformation of the loop region between K425 and L445 of the D1 domain that is located at the junction between the N and D1 domains (E and S1B). The change in the structure of this loop is such that it is close to the conformation observed for this region in p97WT upon the occupancy of the D1 nucleotide-binding site with ATPγS.R191 is located at the start of the linker connecting the N and D1 domains A and 2,  190–199 B and S1A domains E and S1BB) separating the putative hydrogen bonding residues T127 and T436 of the N and D1 domains of the adjacent monomer, respectively (C). This disruption likely accounts for the increased flexibility of the N-domain and also results in a shift similar to that of p97R191Q in the loop region between K425 and L445 of the D1 domain adjacent to the E232 residue (B and S1D). Of note, a T127A mutation has been reported in an individual with FTD  WT, all twith p97 . Indeed,ofactors .E470D and p97D592N mutants, minimal structural differences are observed as compared with p97WT when the D1 and D2 domains are occupied by ADP . These results imply that the mutations decouple ATPγS binding in the D1 domain from inducing the structural rearrangement of N domain from the “down” to “up” conformation.In contrast to the mutants discussed above, in the p97d by ADP . Howeverof p97WT  and S1E.C), indicating that interactions distal to the D1-D2 interface can also influence the coupling between these domains. As observed during the activity assays, the p97E470D and p97D592N mutants differ in terms of their effects on ATPase activity (C). This suggests that p97E470D stimulates increased ATPase activity in the D2 domain, possibly by removing the requirement for ATP binding to the D1 domain for D2 hydrolysis, whereas this intra-protomer D1-D2 communication across the linker is maintained in the p97D592N mutant.Although we would expect the E470D mutation to have minimal effects because of the conservative substitution, residue 470 is located at a critical juncture between the D1 and D2 domains. This major effect highlights the structural role of this side chain in maintaining the network of interactions by which the structural changes in the D2 domain are communicated to the D1 domain. D592 is located in the pore loop region of the hexamer at the inter-protomer interface C, indicaactivity C. This s50 of ∼10 nM . Notably, the vast majority of p97 particles bound to CB-5083 were in the dodecameric state, regardless of D1 nucleotide occupancy . However, unlike these mutants, CB-5083-bound p97WT is locked in conformation I and retains the same tertiary structure in the D2 domain as that seen for ADP-bound p97.CB-5083 is a potent inhibitor of p97 function, with an ICf ∼10 nM . The binf 3.75 Å . In ordeing site A and S2Bccupancy  and S16;or ATPγS B. HoweveC). The same step is blocked by the phenyl indole-based allosteric inhibitor UPCDC-30245, which binds p97 distal to the nucleotide binding site, but prevents the transition to conformation II because of a steric block of the conformational change in the D2 domain by the bound inhibitor  phosphate moiety is involved in H-bonding interactions with R766 and R635 in the neighboring protomer. These additional interactions explain why ATPγS binding initiates the sequence of quaternary structural changes via interactions at the inter-protomer interface. Although CB-5083 binding site overlaps with the nucleotide-binding site of the D2 domain and shares interactions with some of the residues that interact with ADP/ATP, the key inter-protomer interaction between R766 and the γ-phosphate of ATP is missing in the CB-5083-bound structure, preventing the generation of the type of quaternary conformational changes seen upon ATPγS binding to p97WT.A detailed inspection of the interactions in the nucleotide-binding pocket provides an explanation for the effects observed with CB-5083 binding. As shown in Figure 4R155H and p97R191Q or through inter-protomer steric effects in p97A232E. We expect that the primary effect of the N-D1 mutants on p97 is a disruption in cofactor recruitment in-vivo, due to the shift in the conformational equilibrium toward the N domain “up” state. This is supported by previous biochemical and NMR analysis , and it is possible that the requirement for nucleotide binding in the D1 domain for ATP hydrolysis in the D2 domain in WT is abolished by the E470D mutation, allowing the D2 domain to cycle through ATP, independent of D1 nucleotide state. Previously, it was shown that the D1–D2 linker residue, L464, is key to transmitting D2 nucleotide-dependent motion to the D1 domain via an inter-protomer pathway  in regulating D2 ATP hydrolysis.Analysis of the p97er ATPγS  or CB-50activity  ; c.572G>A, 573T>A (R191Q); c.695C>A (A232E); c.1410G>C (E470D); c.1774G>A (D592N) (see E. coli BL21 (DE3) (Thermo Fisher) cells expressing plasmid-encoded mutant p97 were suspended in 20 ml of lysis buffer; 50 mM Tris-HCL pH 8, 300 mM NaCl, 0.5 mM β-Mercaptoethanol (BME) (Fisher Scientific), and 1× Protease Inhibitor Cocktail tablet (Thermo Fisher) and lysed. Following centrifugation, imidazole was added to the resulting supernatant to a final concentration of 20 mM and loaded onto a Ni-NTA column (Thermo Fisher) and eluted with 300 mM imidazole. The eluted protein was flash frozen in liquid nitrogen and stored in aliquots at –80 °C without glycerol.Full-length recombinant His6x-tagged human p97 mutants were purified with some adaptations as described in . BrieflyProtein concentrations were first normalized using Pierce BCA Protein Assay kit (Thermo Fisher), a standard series was prepared with the BSA provided (2 mg/ml–25 μg/μl). Twenty-five microliter of each of the standards and unknown sample were added to a 96-well plate in triplicate, 200 μl of working reagent was added to each well, and prepared according to manufacturer's guidelines. The plate was covered and incubated at 37 °C for 30 min, the plate was cooled to room temperature (RT), and the absorbance at 562 nm was measured for each well.2, 0.1 mg/ml BSA, and incubated at 37 °C for 30 min. After incubation, the solutions were equilibrated to RT for 5 min and 5 μl of ADP-Glo Reagent was added to stop the reaction and consume the remaining ATP. The solution was incubated for 40 min at RT. Ten microliter of Kinase Detection Reagent was added and incubated for 30 min. The sample's luminescence was recorded using Varioskan LUX Multimode Microplate reader (Thermo Fisher). Each sample was averaged across the three measurements and plotted as mean ± standard deviation.The ATPase activity was examined using the ADP-Glo Kinase Assay (Promega), as previously described in . The prog for 10 min, and the supernatant was diluted to a concentration of 2 mg/ml in protein storage buffer with 0.5 μM octyl glucoside, 1 mM TCEP (Fisher Scientific), and 1 mM ADP (Sigma Aldrich) or 1 mM ATPγS (Sigma Aldrich) or 1 mM CB-5083  depending on the experiment and incubated on ice before vitrification. Protein vitrification was achieved using the Leica GP2 plunge freezer on Quantifoil  holey carbon grids (Electron Microscopy Sciences). Firstly, coated grids were glow discharged at 25 mA for 15 s and placed on a pair of tweezers in the blotting chamber. Chamber temperature was set to 20 °C with 100% humidity. Three microliter of prepared protein solution was loaded on the grid. The grid was blotted for 6 s, followed by plunge-freezing in liquid ethane.In total, 10 mg/ml frozen aliquots of the mutant solution was spun down at 12,000−/Ǻ2 on either a 200 kV Glacios or 300 kV Krios (Thermo Fisher Scientific) transmission electron microscope fitted with a K3 (Gatan), Falcon3 or Falcon4 (Thermo Fisher Scientific) direct electron detector camera. Please refer to Frozen grids were stored in cryo grid boxes in liquid nitrogen until imaging. Digital micrographs of frozen hydrated protein particles were typically recorded at 190,000× magnification with defocus range between –1 and –2.5 μm with a total dose of 50 eIn general, all data processing was performed in cryoSPARC v.2.15 or v.3.0.1  unless sWT cryo-EM structures in the ADP (PDB ID: 5FTK) or ATPγS (PDB ID: 5FTN) bound states reported previously (The p97 PDB ID: FTK or ATeviously , the hexeviously . The reseviously .7RL6, 7RL7, 7RL9, 7RLA, 7RLB, 7RLC, 7RLD, 7RLF, 7RLG, 7RLH, 7RLI, and 7RLJ respectively, for R155H-ADP, R155H-ATPγS, R191Q-ADP, R191Q-ATPγS, A232E-ADP, A232E-ATPγS, E470D-ADP, E470D-ATPγS, D592N-ADP, D592N-ATPγS mutant p97, and for p97 bound to CB-5083-ADP and CB-5083-ATPγS. All other data are available from the corresponding authors upon request.The density maps and refined atomic models have been deposited in the Electron Microscopy Data Bank with accession numbers EMD-24518, 24519, 24522, 24523, 24524, 24525, 24526, 24528, 24529, 24530, 24531, and 24532 and in the Protein Data Bank with matching accession numbers of PDB-This article contains S. S. is Founder and CEO of Gandeeva Therapeutics Inc, a drug discovery company based in Vancouver. All other authors declare that they have no conflicts of interest with the contents of this article.


Comparing scientific impact among countries is fundamental for the development of effective strategies for science financing and assessment . Such coUsually, the number of scientific papers and the number of citations of a country are the main variables used for these comparisons. These metrics are frequently combined in a single indicator, such as the Citations Per Publication (CPP)  or the wFor instance, Belgium was the country with the highest research impact in Medicine in 2019 (Scimago data), with a CPP value of 1.38 , whereas its CPP value for 2016 is 17.73  and for 2013, 30.91 (22 times higher than in 2019). This limitation leads to the question: How can one avoid this caveat and meaningfully compare research impact among countries in a year-per-year basis?In this essay we present two simple methods to standardize CPP values along time, thus allowing for comparisons among countries in a time-independent fashion. Therefore, two metrics are presented: The Relative CPP and the Impact-Relative Rank Score (IRRS). Both of these procedures are based on the principle of normalizing the metric according to maximum-minimum values in the common set of observational units (countries) used for comparison.First, consider the CPP definition for a given country and year as the number of citations received by the country’s publications in that year, divided by the number of those publications . Next, iIn general, the inclusion of countries with a modest number of publications per year  leads to atypical CPP values, distorting a metric and thus reducing its efficacy. In order to minimize this effect, a cut-off value is commonly used for country selection, preferably resulting in the inclusion of more than 95% of all publications in a base year. Therefore, below, the approach is illustrated for the field of Medicine, with a cut-off of 2,000 papers/year .At first, the ranking of the 52 selected countries according to their CPPs in Medicine was obtained from the Scimago platform . In thisAs mentioned, taking into account the CPP value of the benchmark country, it is possible, then, to calculate the impact of other countries relatively to it in each year. For example, medical United Kingdom papers had CPP = 1.07 in 2019 and CPP = 4.71 in 2018. Therefore, according to the Relative CPP procedure described above, the relative impact of the United Kingdom in those years was 77.5 and 79.3% respectively - the United Kingdom Relative CPPs.A).B. As expected, most country trends in the yearly-based IRSS scores are similar to those based on the Relative CPPs. The IRRS scores of Belgium, Sweden and the United Kingdom are clearly stable, while Chile and Hungary presented a rise in their values. The United States and Russia experienced a decrease in the period. Brazil, on the other hand, shows a reasonably stable IRSS, while, as mentioned, its Relative CPPs presented a modest rise.The IRRS in Medicine for the same set of countries is shown in Panel C) showed a high correlation between these metrics . Panel D shows that Relative CPPs and IRSS values are also highly correlated in the Agricultural and Biological Sciences, 2018, for Russia, Brazil, Chile, Italy, the United States, Hungary, the United Kingdom, and Switzerland . The country with the highest impact in this area in 2018  was Switzerland, with CPP = 4.92. Finally, The Linear Coefficient of Determination between the Relative CPPs and the IRRS values for Medicine  is more sensitive to small changes, while the Relative CPP allows one to more globally visualize the extent of the difference between a country and the top ranker in the field.The main limitation of the proposed indicators is the mentioned common problem among cross-country impact performance metrics: Relative performance evaluation can change contingent on a specific set of countries analyzed, and the inclusion of small-impact countries can greatly distort an analysis.In summary, we presented here two metrics for cross-country comparison of scientific performance: The Relative CPP and the IRRS, and we hope that these will allow for a more informative discussion of government policies and their impact over the world scientific output.RA, LB, DM, and MH contributed to the study design, data analysis, and manuscript drafting.Part of this research was funded by a Ministry of Education/CAPES grant (Code: 001), to whom we thank.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We advanceUlva’s genetic tractability and highlight its value as a model organism by characterizing its APAF1_C/WD40 domain-encoding gene, which belongs to a family that bears homology to R genes.Ulva mutabilis  is native to coastal ecosystems worldwide and attracts both high socio-economic and scientific interest. To further understand the genetic mechanisms that guide its biology, we present a protocol, based on adapter ligation-mediated PCR, for retrieving flanking sequences in U. mutabilis vector-insertion mutants. In the created insertional library, we identified a null mutant with an insertion in an apoptotic protease activating factor 1 helical domain (APAF1_C)/WD40 repeat domain-encoding gene. Protein domain architecture analysis combined with phylogenetic analysis revealed that this gene is a member of a subfamily that arose early in the evolution of green plants (Viridiplantae) through the acquisition of a gene that also encoded N-terminal nucleotide-binding adaptor shared by APAF-1, certain R-gene products and CED-4 (NB-ARC) and winged helix-like (WH-like) DNA-binding domains. Although phenotypic analysis revealed no mutant phenotype, gene expression levels in control plants correlated to the presence of bacterial symbionts, which U. mutabilis requires for proper morphogenesis. In addition, our analysis led to the discovery of a putative Ulva nucleotide-binding site and leucine-rich repeat (NBS-LRR) Resistance protein (R-protein), and we discuss how the emergence of these R proteins in green plants may be linked to the evolution of the APAF1_C/WD40 protein subfamily.The multicellular chlorophyte alga The online version contains supplementary material available at 10.1007/s00425-022-03851-0. Chlorophyte and streptophyte algae  are diverse oxygenic photosynthetic eukaryotes that obtained their chloroplasts through an ancient endosymbiotic event with a cyanobacterium McFadden . Our undGreen algae are important to the global carbon cycle and local ecosystems as primary producers. Although some of them may also occupy terrestrial, subaerial niches, streptophyte algae are regarded as strict freshwater lineages, whereas chlorophyte algae have adapted to oceanic and coastal marine environments, as well as freshwater  , U. mutabilis morphogenesis also depends on the activity of Roseovarius sp. MS2, which releases compounds that promote cell division and blade formation. Only under the synergistic effect of the compounds produced by both Roseovarius sp. and Maribacter sp., U. mutabilis will develop properly   at 18 °C. For phenotyping of transgenic plants, axenic cultures were prepared according to Califano and Wichard  was used for genomic DNA extraction using the GenElute Plant Genomic DNA Miniprep Kit (Merck). Genotyping PCR was performed using Prime-STAR GXL DNA Polymerase (Takara) in the presence of 5% DMSO using a primer pair specific for the resistance cassette and as a control for the gene UM008_0183. Oligomers are listed in Suppl. Table S1.Polyethylene glycol-mediated transformation was performed according to the protocol described in Oertel et al.  and subsU. mutabilis genome sequence . Oligomers are listed in Suppl. Table S1.To create a template suited for PCR, genomic DNA (gDNA) containing the pPIBT7 insert was digested with a restriction enzyme and adapters were ligated to the overhanging ends of the gDNA. A short strand and a long strand make up the adapter. When the two strands of the adapter are hybridized, the short strand has a 5′ overhang, which is complementary to the restriction half-site ‘sticky end’ of the gDNA and will anneal to it; subsequently, the fragments are fused by ligation . First, total RNA was extracted using the Spectrum plant total RNA kit, including the optional on-column DNase I digestion (Merck). Then, (nested) 5′RACE was used to determine the 5′UTR region, and subsequently, a forward primer in the 5′UTR was designed for 3′RACE of the full-length transcript, which was cloned in pJET1.2 (Thermo Fisher) and sequenced. Normalized relative expression of UM033_0004 and UM005_0337 was determined using real-time quantitative PCR (qPCR) using the reference genes UM008_0183 (Ubiquitin) and UM010_0003 (PP2A 65 kDa regulatory subunit A). For qPCR, 1 µg of total RNA was reverse transcribed using the SuperScript IV VILO Master Mix Reagents (Thermo Fisher). Subsequent qPCR was done using PowerUp SYBR green chemistry (Thermo Fisher) on a CFX96 Real-Time PCR Detection System (Bio-Rad). The Cq values and melting curves were analyzed with CFX Maestro qPCR Analysis Software that implements the Pfaffl method (Pfaffl The cDNAs of wild-type d Pfaffl . Amplifihttp://smart.embl-heidelberg.de) and Interproscan . Chlorophyte homologs of UM033_0004 were identified by performing a blastp query  were selected and then filtered for the presence of the APAF1 helical domain  or its corresponding gene3D signature (G3DSA:1.25.40.370). To identify homologs from more distantly related taxa, hidden Markov model (HMM) searches were performed , the prasinodermophyte Prasinoderma coloniale, the glaucophyte Cyanophora paradoxa and Rhodophyta. Complete species lists and data sources are listed in Suppl. Table S2. Also, an additional HMM search against all the streptophyte algae included in the One Thousand Plant Transcriptomes Initiative project was performed . To root the phylogenetic tree, we also aligned the WD40 repeat domain of the fungal protein HET-E, which contains an N-terminal NACHT-domain ATPase. An initial protein alignment was created against the custom APAF1_C HMM profile, whereas the C-terminal part, containing the WD40 repeats, was aligned using MUSCLE  with the lowest LE Edgar . SequencLE Edgar , using tU. mutabilis, we first created a collection of insertional mutants via polyethene glycol-mediated vector transformation. After selection and genotyping, an adapter-ligation PCR approach  and in a membrane-bound protein-encoding gene (UM005_0337) that were also identified in the initial BLAST search  revealed the effect of the intronic insertions on the accumulation of the gene transcripts. Using primer pairs that anneal downstream or on opposite sides of the insertions, no transcript of the WD40 repeat-encoding gene could be detected Fig. c, and trUM033_0004, we further investigated its potential function using protein domain analysis. Genome annotation and the SMART protein domain architecture tool indicated it contained 13–15 WD40 repeats. InterProScan additionally identified the presence of the APAF1_C domain located N-terminally to the WD40 repeat domain  , we identified two proteins that only contained the APAF1_C helical domain. In contrast, two out of four Tetraselmis striata (Chlorodendrophyceae) homologs possessed both an additional N-terminal NB-ARC and a domain resembling the WH-like DNA-binding domain superfamily. In one out of three investigated Chlorella species, the N-terminal WH-like domain was detected, too, and in one case, accessory C-terminal transcription factor 25 (TCF25) Ribosome quality control complex subunit 1 in yeast) and the actin-binding domain Ysc84 domains were also present. Among the other major groups of Viridiplantae, searches using the HMM profile did not detect the APAF1_C domain in the investigated Embryophyte, Prasinodermophyte or Glaucophyte proteomes However, it was present in the Mesostigmatophyceae (Streptophyta) and Rhodophyta sequences. The retrieved Mesostigma viride CCAC 1140 and the rhodophyte proteins displayed N-terminal NB-ARC and WH-like domains, resembling the domain architecture observed in two of the T. striata proteins. However, the M. viride CCAC 1140 protein had an additional C-terminal ankyrin domain repeat, whereas the M. viride NIES-296 sequence only contained the WH-like and APAF1_C domains. A similar HMM search against the streptophyte algal proteomes included in the One Thousand Plant Transcriptomes Initiative did not deliver any further homologs. The HMM hits with the lowest e-value from non-archaeplastidal eukaryotes belonged to basal groups of metazoans, whereas the most significant bacterial hits were cyanobacterial. Interestingly, in these proteins, the APAF1_C/WD40 module was linked to NB-ARC and WH-like domains as in T. striata and M. viride CCAC 1140, and in the metazoan homologs, it was additionally linked to an N-terminal caspase activation and recruitment (CARD) domain, which is typical for APAF1-like proteins  and an NB-ARC domain-containing protein (UM003_0200.1) with C-terminal LRR-domains . Therefore, the latter protein is likely a representative of the NBS-LRR protein family that is involved in immunity  and UM005_0337 (membrane-bound protein-encoding) on the phenotype of the mutant plants, we propagated the primary transformants and grew new generations of gametophytes from unfertilized gametes. Microscopic inspection of germlings 12 days after gamete release did not show apparent phenotypic alterations compared with untransformed control plants  are missing, as previously observed  split from the other streptophytes. The latter event also explains the absence of this gene family in the investigated land plants. The resulting scattered distribution indicates that the cooption of the genes was highly selective rather than that they played a role common to green algae.Identifying the Ulva, suggests that this gene family acquired a restricted or specialized role in these lineages during evolution that solely depends on the APAF1_C/WD40 domain repeat. Because the WD40 repeats fold into beta-propellers that promote protein–protein interactions ]  and two Botryococcus braunii accessions (class Trebouxiophyceae)  therefore considerably strengthens Shao et al.’s conclusion (2019) that a protein with an NBS-LRR architecture was already present in the common ancestor of the entire green lineage. However, the exact origin and molecular mechanisms that led to the emergence of this ancestral plant R-protein are still under debate. It has been suggested that NBS-LRR proteins evolved through an NB-ARC/WD40 domain-containing intermediate Supplementary file2 (XLS 2656 KB)Supplementary file3 (DOCX 228 KB)Below is the link to the electronic supplementary material.