Protein-shelled viruses have been thought as “tin cans” that merely carry the genomic cargo from cell to cell. However, through the years, it has become clear that viruses such as rhinoviruses and caliciviruses are active and dynamic structures waiting for the right environmental cues to deliver their genomic payload to the host cell. In the case of human rhinoviruses, the capsid has empty cavities that decrease the energy required to cause conformational changes, resulting in the capsids “breathing”, waiting for the moment when the receptor binds for it to release its genome. Most strikingly, the buried N-termini of VP1 and VP4 are transiently exposed during this process. A more recent example of a “living” protein capsid is mouse norovirus (MNV). This family of viruses have a large protruding (P) domain that is loosely attached to the shell via a single-polypeptide tether. Small molecules found in the gut, such as bile salts, cause the P domains to rotate and collapse onto the shell surface. Concomitantly, bile alters the conformation of the P domain itself from one that binds antibodies to one that recognizes receptors. In this way, MNV appears to use capsid flexibility to present one face to the immune system and a completely different one to attack the host tissue. Therefore, it appears that even protein-shelled viruses have developed an impressive array of tricks to dodge our immune system and efficiently attack the host. Non-enveloped viruses have been thought to have protein shells that simply move the viral genome from cell to cell. The following review will discuss human rhinovirus (HRV) and mouse norovirus (MNV) which intricately respond to environmental cues. With HRV14 , the capsids undergo a “breathing” process, where the buried N-termini are transiently extruded . It is lPicornaviruses are among the largest of animal virus families and include polio-, rhino-, foot-and-mouth disease, Coxsackie, and hepatitis A viruses. The rhinoviruses—of which, there are more than 100 serotypes—are major causative agents of the common cold in humans . The virA hydrophobic cavity lies directly beneath the canyon floor into which some non-polar antiviral compounds (WIN) bind . Upon biAnalysis of HRV14 trypsin digestion products using mass spectrometry provided evidence that the drug-binding cavity plays a role in capsid dynamics [It is possible that the digestion seen in these experiments was due to a mixture of intact and damaged HRV14 particles. To control for this, WIN compounds were added to the digestion mix since they were known to stabilize the HRV capsid. WIN compounds completely blocked cleavage at all sites but the drug did not affect the intrinsic activity of trypsin itself . This stFurther evidence of the stabilizing effects of the WIN compounds on HRV14 came from experiments where the virus was subjected to high hydrostatic pressure, low temperature, and urea in the absence and presence of an antiviral drug [WIN compounds have also been shown to affect the receptor-virus interactions of the major but not the minor serotypes of HRV . On thisIt was suggested that certain host cell molecules, “pocket factors,” bind to the WIN drug-binding cavity beneath the canyon floor and provide transient stability to some of the picornaviruses . There wTo test whether putative “pocket factors” might play a role in the viral life cycle, HRV14 was mutated in  and around S1223G) the drug-binding pocket [3G the drThis simplified model is supported by studies demonstrating that drug-resistant mutations can arise distal to the drug-binding region. While some of the mutations that confer resistance in HRV14 make direct contact with the bound drug, many of the other residues point away from the bound WIN compounds and most lie on strands that remain relatively unchanged by drug binding. Therefore, these latter mutations may facilitate the canyon-associated conformational changes in the presence of drug rather than simply affecting receptor or drug binding.HRV16 is a better example of distal, drug-resistant mutations. Resistant mutations V1210A, M1103T, and T1208A lie midway up the south canyon wall. These mutations destabilize virions, and hence, virions can only assemble in the presence of drug. This is an important finding in that it demonstrates that crucial residues are not only confined to the very bottom of the canyon and that the canyon region is very sensitive to mutations. Similarly, drug-resistant mutations are found in three regions of poliovirus: the drug-binding pocket itself, the north wall of the canyon (~17 Å away from the bound drug), and at the viral capsid inner surface. A number of these latter mutations are clustered around the VP4–capsid interface. Again, these results suggest that the neutralizing effects of the drug are not immediately localized to the bottom of the canyon.We proposed that the drug-binding cavity evolved to maintain flexibility of this region . Empty cAntibodies are a major defense against picornavirus infection and several mechanisms of antibody-mediated neutralization have been proposed. Large changes in pI were observed when antibodies bound to virions ,29,30,31The hypothesis that neutralizing antibodies act by inducing conformational changes was directly tested by the determination of several antibody/virus complexes. Neutralizing monoclonal antibodies against HRV14 are divided into three groups: strong, intermediate and weak ,36. All The structures of three different Fab/HRV14  complexes and of one mAb/HRV14 (mAb17) complex have been determined ,38,39,40H1CL) of 2-fold related Fabs in close proximity that could facilitate bidentate binding across icosahedral 2-fold axes. In contrast, Fab1 binds almost vertically to the virion surface with a “twist” that made it seem unlikely that these antibodies could bind bivalently. These studies, at resolutions of 20–30Å, consistently demonstrated that antibodies do not induce large conformational changes in the virion upon binding. In the case of the mAb17/HRV14 complex, it was thought that bivalent binding itself might induce conformational changes in the capsid. Bivalent binding was subsequently visualized in the structure of the mAb17/HRV14 complex [Fab17 and Fab12 both bind to the Nim-IA site at a somewhat tangential orientation that placed the constant domains  yielded some interesting results on antibody recognition and capsid breathing . C5 bindFab fragments of C5 were added to HRV14 at 33 or 4 °C and the structures of the complexes were determined with cryo-EM. In all cases, Fab fragments were observed to bind to the capsid. The major difference was that there were more empty particles at 33 °C in the presence of the Fab than at 4 °C or in the absence of Fab at 33 °C. The treatment at 33 °C resulted in ~30%–60% of the particles releasing their genome, while only a small fraction of the virus was empty when treated at 4 °C. The major changes in the capsid upon release of the genome was expansion of the entire capsid and separation of the capsid at the icosahedral 2-fold axes. Interestingly, this separation of the VP2 α-helices at the icosahedral axes was discussed as a possible location of the N-termini extrusion in the previous mass spectrometry study [While it was suggested that the antibodies induced viral uncoating, it seems likely that the process is more complicated than that. From the studies reviewed above , the HRVThese results are a clear demonstration of the changes in the HRV capsid upon release of the genome. However, it still is unclear if antibodies induce conformational changes upon binding and if this is a significant part of neutralization. Firstly, it is a bit of a misnomer to suggest that antibodies induce conformational changes per se. Even in this study, the model is suggesting that the higher temperatures are driving the changes in the intersubunit interactions and an antibody might be selecting for the altered conformation. However, even here, there is no obvious structural mechanism for either C5 recognition of the eclipsed particle or antibody induced conformational changes. In addition, if the main mechanism of C5 neutralization is the destabilization of the capsid, then one might expect some of the escape mutants would have stabilizing effects and lie distal to the epitope. Instead, all escape mutants to HRV14 monoclonal antibodies all lie on the capsid surface and are important antibody contact residues. Distal, stabilizing mutations are clearly possible as reviewed above in the case of escape mutations to the antiviral WIN compounds. Interestingly, distal antibody escape mutations are possible in viruses and discussed below in the review of mouse norovirus. Finally, it is unlikely that the common mechanism of antibody neutralization is induction of conformational changes. Indeed, all antibodies to the NIm-IA site in HRV14 have been shown to stabilize the capsid against pH denaturation rather than causing uncoating .As previously reviewed ,43, in aNoroviruses are the major cause of epidemic gastroenteritis in humans and, as such, are important pathogens , causinEfforts to make effective norovirus vaccines have been hampered by our lack of understanding of the structural mechanisms of viral escape. In addition, noroviruses are constantly evolving and generate new strains every 2–4 years ,47,48 thCaliciviruses are T = 3 icosahedral particles with 180 copies of the major capsid protein VP1; ~58 kDa), that is divided into the N-terminus (N), the shell (S) and C-terminal protruding (P) domains ,54,55,56 kDa, thaCalicivirus capsid flexibility likely plays important roles in receptor binding and escape from immune surveillance. There are at least two aspects of capsid flexibility; the entire P domain freely moves about the capsid surface and the conformation of the P domain itself is highly flexible and sensitive to antibody escape mutations and receptor binding.The cryo-EM structure of MNV was determined to a cryo-EM resolution of ~8Å ,61. The Since this unusual “floating P domain” conformation (“extended state”) was so different than the crystal structure of NV (“contracted state”), the structure of rabbit hemorrhagic disease virus (RHDV) VLP was also determined . SimilarThe flexibility of the entire P domain may also play a role in receptor interactions. The Virgin lab found that the cell receptor for MNV was proteinaceous and was not dependent upon carbohydrates for binding . Using tUsing these soluble forms of CD300lf and the P domains, they also directly measured the interaction affinity using surface plasmon resonance . The monTo determine the atomic details of CD300lf binding to the receptor, the atomic structure of the soluble P domain/CD300lf complex was determined ,68 folloThere were several major changes in the MNV capsid upon the addition of GCDCA . The P dThis flexibility of the P domain may play an important role in receptor interaction in MNV by creating more space for the receptor to bind ,66. In FThe P domain mobility could also affect antibody recognition. In the expanded state, the flexible tether between the shell and the P domains could be sensitive to proteases. This could release soluble P domains to act as a “smoke screen” for the immune system and present antigenic sites not normally exposed on the viral surface. Similarly, the marked flexibility of the P domains in the expanded state might expose these antigenic sites without having to be cleaved from the shell. In either case, if any of these normally buried sites are immunodominant, then the immune response might focus on producing antibodies that bind poorly or not at all to the contracted capsid. This hypothesis is supported by the findings that the immune response to MNV included antibodies to the buried shell domain  and fromFrom the results above, the P domain is like a balloon floating above the viral shell, attached only by a thin tether. The P domain is structurally isolated from the rest of the capsid and, therefore, it seems highly unlikely that antibodies or receptors can bind to the P domain and transmit conformational changes to the shell. However, there is growing evidence that the P domain itself is highly flexible and that this motility is necessary for viral function and antibody escape.In the initial crystal structure of the MNV P domain , the outTo understand how noroviruses escape antibody neutralization, we determined the cryo-EM structure of MNV complexed with Fab fragments from a neutralizing A6.2 . It was Using the A6.2/MNV-1 docking model ,61 as a An even more extreme case of P domain plasticity was observed with a second neutralizing antibody, 2D3 . CompareTo understand the difference between these two antibodies, the cryo-EM structure of the 2D3 bound to MNV was determined  Figure . Like A6To understand how the 2D3 escape mutants, that are distal to antibody contact and buried beneath the surface, can block 2D3 binding, dynamic simulations were performed on the P domain where the V339I mutation was modeled into both the open and closed conformations . The resTogether, these results strongly suggest that the P domain is in a dynamic structural equilibrium where the crystallographically observed open and closed states represent two possibilities of a montage of conformations. The site directed escape mutations to A6.2 and the naturally occurring escape mutations to 2D3 suggest that the virus can block antibody binding by shifting this equilibrium towards the closed state. If true, then it necessarily follows that the closed state is the viable and infectious form of the virus. However, it is not at all clear why the 2D3 contacts on the P domain are so immutable and suggests that this region has an important role.The details of the MNV–receptor interactions have been elucidated with the atomic structure of the P domain/CD300lf complex ,68 and tThe conformation of the A’B’ and E’F’ loops in the receptor complex is nearly identical to that of the closed conformation. The major difference is that the C’D’ loop is “turned up” compared to the P domain alone. This affords enough space for the bile salt, GCDCA (tan spheres), to bind. It may be that the structural equilibrium in solution is shifted by GCDCA binding to the closed form, which may be preferred by CD300lf. In this way, the bile salts may enhance receptor binding, indirectly, by altering the dynamics and structural equilibrium of the P domain. What is particularly interesting is that the V339I escape mutant (black spheres) lies immediately adjacent to the bile salt binding site. As with the bile salts, we had proposed that the V339I mutation shifted the equilibrium towards the closed conformation that does not favor antibody binding ,70,71. IThe conformation of the P domain in solution is more than likely a montage of structures including the observed open and closed configurations. This structural equilibrium can be disrupted by environmental cues and antibody escape mutants. In the open conformation, the A’B’ and E’F’ loops are splayed apart, making room for antibodies  to bind. The closed conformation does not expose the hydrophobic region between the two loops and the E’F’ loop clashes with the CDR3 loop, therefore, preventing antibody binding. All of the escape mutations to 2D3 are distal to the antibody binding contact and dynamic simulation studies with the V339I escape mutation suggest that it causes long-range structural disruption in the P domain dimer and may be pushing the structure from the open (that binds antibody) to the closed (that does not bind antibody) conformation . SimilarFrom these two cases, viruses with simple protein shells are more dynamic and flexible than might be expected. In the case of the rhinoviruses, the capsids appear to be breathing where the capsid may be opening and closing around the 2-fold axes and exposing the myristoylated N-termini of VP4. The receptor, ICAM, may prefer one of the conformations sampled during this breathing process and begin the uncoating process. As suggested by the effects of the WIN compounds and mutagenesis studies, the empty drug-binding pocket minimizes the energy required for these conformational changes. While it is attractive to suggest that antibody binding might similarly drive the capsid towards uncoating, the fact is that the most potent antibodies do not cause conformational changes. Even in the one study reviewed here that suggests such conformational changes might occur, the antibody does not appear to prefer one conformation over the other. In the end, the immune system simply recognizes epitopes presented to it and does not select for antibodies that induce conformational changes. While it is attractive to consider vaccines that recognize viruses in a transition state, trying to force the B cells to recognize a conformation that is not prevalent in solution will likely yield a poor adaptive immune response.While the rhinoviruses appear to breathe in preparation for receptor interactions and uncoating, MNV has several modes of flexibility for quite different reasons. MNV has a highly mobile P domain that can move around the capsid surface that likely facilitates binding to the target cell. This flexibility of the P domain might also be involved in avoiding the immune system by presenting antigenic sites exposed in one state but not the other. This gross movement of the P domain is clearly sensitive to environmental conditions and appears to improve virus binding to the receptor. Similarly, there is evidence that the structure of the P domain itself responds to environmental cues. In physiological phosphate-buffered saline, the C’D’, E’F’, and A’B’ loops adopt an “open” conformation that appears to be recognized by antibodies. As the virus enters the gut, solutes such as bile salts move these loops to the “closed” conformation that is no longer recognized by antibodies but is favored by receptors. So, unlike HRV14, the plastic nature of the capsid allows the virus to present one “face” to the immune system and a very different one for cell attachment. Nevertheless, it is clear that neither of these viruses are simply inanimate “tin cans”.


The BCL6 BTB domain has been adapted to form a promiscuous assembly block as a basis for affinity-capture crystallography. The production of diffraction-quality protein crystals is challenging and often requires bespoke, time-consuming and expensive strategies. A system has been developed in which the BCL6 BTB domain acts as a crystallization chaperone and promiscuous assembly block that may form the basis for affinity-capture crystallography. The protein of interest is expressed with a C-terminal tag that interacts with the BTB domain, and co-crystallization leads to its incorporation within a BTB-domain lattice. This strategy was used to solve the structure of the SH3 domain of human nebulin, a structure previously solved by NMR, at 1.56 Å resolution. This approach is simple and effective, requiring only routine protein complexation and crystallization screening, and should be applicable to a range of proteins. The main bottleneck in this technique is the production of well-ordered crystals that produce high-quality diffraction. Strategies to aid crystallization include the use of chaperones  domain from the transcriptional repressor BCL6 to serve as a promiscuous assembly block that may assist the crystallization of a guest protein. The 129-residue BCL6 BTB domain forms a symmetrical strand-exchanged homodimer that contains two lateral grooves that bind corepressors with high affinity (Ahmad 2.2.1.1GITTIKEMGRSIHEIPR17 (NCoR1BBD1) and 1RERIAAASSDLYLRPGS17 (NCoR1BBD2). The peptides were dissolved in appropriate buffers for NMR or crystallography and the pH was adjusted to experimental conditions prior to use.Peptides were synthesized by GL Biochem Shanghai Ltd. The sequences were 2.2.et al., 2003BBD2-BCL6BTB and NCoR1BBD2(link)-BCL6BTB chimeric proteins were made by fusing sequences encoding the residues SSDLYLRPG and SSDLYLRPGGG, respectively, onto the N-terminus of the BCL6 BTB domain using PCR. DNA sequences encoding the nebulinSH3-NCoR1BBD1 fusion protein were synthesized by GeneArt (Thermo Fisher Scientific) and encoded the nebulin SH3 domain  fused to the NCoR1 BBD1 via a glycine linker (GGGGITTIKEMGRSIHEIPR); this was then cloned into the modified pET-28a vector as above. All constructs were verified by DNA sequencing (Genewiz).The human BCL6 BTB domain  was cloned into a modified pET-28a vector using ligation-independent cloning ; the vector encoded an N-terminal hexahistidine-MBP tag. The point mutations C8Q, C67R and C84N were introduced by PCR using Phusion High-Fidelity DNA polymerase (Thermo Scientific); these mutations enhance protein solubility and have previously been used in crystallographic studies (Ahmad 2.3.Escherichia coli BL21 (DE3) pLysS cells. The cultures were grown in 2YT medium at 310 K to an optical density (600 nm) of 0.6, cooled to 291 K and the recombinant proteins were induced by growth for a further 18 h in the presence of 500 µM isopropyl β-d-1-thiogalactopyranoside. The cells were harvested by centrifugation at 11 000g and resuspended in 20 mM Na2HPO4, 500 mM NaCl, 40 mM imidazole, 3 mM β-mercaptoethanol pH 7.4. Bovine deoxyribonuclease 1 (Sigma) and Protease Inhibitor Cocktail VII (Merck) were added and the cells were lysed at 207 MPa using a cell disruptor (Constant Systems). The lysate was clarified by centrifugation at 39 000g for 45 min and the proteins were purified via immobilized metal-affinity chromatography using a 5 ml HisTrap HP column . The N-terminal His-MBP tag was removed by cleavage with HR3C protease, and the protein was further purified by size-exclusion chromatography on a Superdex 75 HiLoad 26/60 column . The NCoR1BBD2(link)-BCL6BTB/nebulinSH3-NCoR1BBD1 complex was obtained following size-exclusion chromatography of the mixed individual purified proteins, with nebulinSH3-NCoR1BBD1 in a 1.5:1 molar excess.Proteins were expressed in 2.4.M protein in 20 mM Na2HPO4 pH 6.8, 300 mM NaCl, 3 mM β-mercaptoethanol.NMR spectra were collected on a 750 MHz Bruker Avance spectrometer equipped with a cryoprobe. Data were collected at 298 K using 400 µ2.5.BTB, NCoR1BBD2-BCL6BTB and NCoR1BBD2(link)-BCL6BTB) were concentrated to 300 µM (monomer) in 20 mM HEPES pH 7.5, 300 mM NaCl, 3 mM β-mercaptoethanol, and all peptides were dissolved in the same buffer. For all crystallizations, standard sparse-matrix screening was carried out using 400 nl drops with a 1:1 protein:precipitant ratio at 298 K using a Mosquito Liquid Handling Robot (TTP Labtech). Initial hits for crystals of the BCL6BTB/NCoR1BBD2 complex were obtained in the conditions depicted in Supplementary Table S1.All variations of the BCL6 protein  PEG 3350, with the NCoR1BBD1 peptide in a 4:1 molar excess. Crystals of the BCL6 BTB domain in complex with the low-affinity NCoR1BBD2 peptide were obtained in 13.4%(v/v) PEG 400, 0.335 M K2HPO4/NaH2PO4 pH 7.5, with the NCoR1BBD2 peptide in an 8:1 molar excess. Crystals of the NCoR1BBD2-BCL6BTB chimera were obtained in 2.2 M NaCl, 10% MPD, 0.1 M imidazole pH 6.5, 0.1 M CaCl2. Crystals of the NCoR1BBD2-BCL6BTB chimera in complex with the high-affinity NCoR1BBD1 peptide and of the NCoR1BBD2(link)-BCL6BTB chimera in complex with the high-affinity NCoR1BBD1 peptide were obtained in 2 M NaCl, 5%(w/v) PEG 4000, 0.1 M Tris–HCl pH 8.5 and in 1.34 M ammonium sulfate, 0.67%(v/v) MPD, 0.1 M HEPES/sodium hydroxide pH 7.5, respectively, each with the NCoR1BBD1 peptide in a 4:1 molar excess. The NCoR1BBD2(link)-BCL6BTB chimera in complex with nebulinSH3-NCoR1BBD1 was purified by size-exclusion chromatography and concentrated to 7 mg ml−1 (determined using an assumption of 1:1 stoichiometry and combined extinction coefficients). Crystals were obtained in 0.66 M ammonium sulfate, 3.3%(v/v) glycerol, 0.05 M magnesium sulfate, 0.1 M imidazole–HCl pH 6.5. Prior to data collection, all crystals were vitrified in mother liquor containing 25% ethylene glycol and were rapidly cooled using liquid nitrogen.For structure determination, crystals of the BCL6 BTB domain in complex with the high-affinity NCoR1AIMLESS -BCL6BTB chimera in complex with the NCoR1BBD1 corepressor peptide showed signs of pseudotranslational NCS. Examination of the data suggested that the crystal may belong to space group P41212, with a Patterson off-origin peak of 83.7% at fractional coordinates 0.5, 0.5, 0.5 and a distance of 151.4 Å from the origin. Analysis of the acentric reflections also showed that 〈I2〉/〈I〉2 = 3.035, 〈F〉2/〈F2〉 = 0.665 and 〈|E2 − 1|〉 = 0.990; for the centric reflections 〈I2〉/〈I〉2 = 3.904, 〈F〉2/〈F2〉 = 0.585 and 〈|E2 − 1|〉 = 1.153. In space group P41212, six BTB monomers were present in the asymmetric unit, and were placed using a combination of Phaser and MOLREP -BCL6BTB/NCoR1BBD1, 6zbu; NCoR1BBD2(link)-BCL6BTB/nebulinSH3-NCoR1BBD1, 6y17.The structures were deposited in the PDB with the following accession codes: BCL63.3.1.Supplementary Fig. S1). We refer to this NCoR1 sequence as BBD2; it is distinct from the NCoR1 sequence BBD1 which binds to the lateral groove of the BTB domain with high affinity . The structure of the BCL6BTB/NCoR1BBD2 complex was solved to 1.39 Å resolution .In the crystals of the BCL63.2.BBD1  and Table 1BBD2 in our previous structures. We reasoned that the high-affinity NCoR1BBD1 peptide would displace the NCoR1BBD2 residues from the BTB-domain β1 strand of the NCoR1BBD2-BCL6BTB chimera, whilst still leaving the NCoR1BBD2 sequence free to potentially interact with the HP of an adjacent BTB domain; this might result in the effective polymerization of the BTB homodimer , Supplementary Fig. a)S3S3Supplementary Fig. S3 to 2215.2 Å2 (NCoR1BBD2-BCL6BTB/NCoR1BBD1).We next sought to adapt the BTB domain to form an assembly that might act as a crystallographic scaffold for hosting a guest protein of interest. By solving the crystal structure, we showed that the classic high-affinity NCoR1 BTB-binding sequence, NCoR1BBD2-BCL6BTB/NCoR1BBD1 complex were highly porous, with a solvent content of 82.30% (VM = 6.95 Å3 Da−1). The N-termini of the NCoR1BBD1 peptides were exposed in solvent channels of ∼100 Å in diameter as estimated from projections across the crystal lattice in CCP4mg [Fig. 2c)]. MAP_CHANNELS analysis revealed solvent channels of diameter ∼50 Å , and we therefore reasoned that we could adapt this system for the accommodation of a guest protein that is expressed as a genetic fusion to the NCoR1BBD1 sequence.The crystals of the NCoR1BBD2 sequence and the BTB domain within the NCoR1BBD2-BCL6BTB chimera, with the rationale being to extend the interface with the HP region. The modified chimeric protein, NCoR1BBD2(link)-BCL6BTB, was crystallized in complex with the high-affinity NCoR1BBD1 peptide. We solved the structure of the NCoR1BBD2(link)-BCL6BTB/NCoR1BBD1 complex at 2.46 Å resolution; these crystals had a similar solvent content  to the previous complex and formed a similar porous lattice [Fig. 2d)]. As expected, the interface between the NCoR1BBD2 sequence and the HP region of the BTB domain was extended in this structure d). The NCoR1BBD2(link)-BCL6BTB and nebulinSH3-NCoR1BBD1 proteins were expressed and co-purified as a complex using size-exclusion chromatography . Unexpectedly, the BTB dimers did not assemble into filaments; instead, the positioning of the SH3 domains within the lattice precluded the back-to-back associations between BTB dimers, with one of the SH3 domains interacting with the BTB domain HP [Fig. 3c)]. The versatility of the BTB-domain crystal contacts thus allowed the incorporation of the guest nebulin protein even though it drove a change in lattice organization.As proof of principle, we chose to crystallize the SH3 domain of human nebulin by incorporating it as the guest protein within a BCL6 BTB-domain lattice via its genetic fusion to the NCoR14.BBD2 appendage or with a co-crystallized SH3 domain that was recruited via genetic fusion to a natural NCoR1 corepressor BBD1 sequence; from this perspective, the BTB domain served as a chaperone for the SH3 domain and provided an interaction site to facilitate the 3D ordering of the crystal. Although unexpected, the propensity of the HP to interact with a variety of hydrophobic sequences confers versatility upon the organization of BTB-domain lattices, thereby offering advantages over strategies that involve the recruitment of guest proteins into precisely defined scaffolds. For example, the crystallization of ubiquitin by its genetic fusion to RIEN required microseeding with apo RIEN -BCL6BTB/NCoR1BBD1, 6zbuNCoR1PDB reference: BBD2(link)-BCL6BTB/nebulinSH3-NCoR1BBD1, 6y17NCoR1PDB reference: 10.1107/S2052252520015754/lz5043sup1.pdfSupplementary Tables and Figures. DOI:


Callichirus kraussi), which are key ecosystem engineers in South African coasts. We demonstrate experimentally that benthic engineering by sandprawns reduces phytoplankton biomass by roughly 50%. Using long-term estuarine data, we demonstrate similar reductions in phytoplankton biomass (by roughly 70%) in sandprawn-dominated areas. Increased burrow wall chlorophyll-a relative to surface sediments that was evident in experiments suggests that pelagic filtration occurs through bi-directional water pumping and phytoplankton adsorption onto burrow walls. Our findings expand understanding of the ecological relevance of sandprawns and functionally similar organisms, the mechanisms by which they engineer ecosystems and their role in mediating coastal bentho-pelagic coupling. Our findings also highlight the potential for deposit-feeders to be used as nature-based solutions to counter coastal eutrophication.Managing coastal ecosystems and preserving socio-ecological functioning require a comprehensive understanding of ecological services provided by resident organisms. Here, we provide novel information on water-filtration activities of endobenthic sandprawns ( They are critical nursery and feeding grounds for resident and migrant species and subsidise offshore ecosystems through nutrient and trophic transfers5. Coastal ecosystems also supply valuable goods and services that support human livelihoods and local economies6. However, due to their proximity to human settlements and their aesthetic appeal, coastal systems rank amongst the most threatened and degraded ecosystems on earth, owing to multiple human stressors being applied simultaneously10. Nutrient enrichment is arguably the most pervasive driver of coastal deterioration in the 21st century, resulting in notable impairments of ecosystem multi-functionality and declines in the quality of goods and services provided12. Government and management agencies therefore invest heavily in measures to counter socio-ecological losses arising from excessive nutrient inputs.Coastal ecosystems are some of the most productive and ecologically significant habitats on earth15. In this regard, suspension-feeding organisms (e.g. clams and mussels) and coastal vegetation have dominated mitigation narratives, leading to significant investments in conservation, restoration and engineering programs that exploit their water-purification capabilities17. In contrast, burrowing deposit-feeding organisms have been overlooked, despite them being dominant components of sedimentary biotopes 20, which are significant constituents of coastal ecosystems. In particular, there has been a limited appreciation that burrow complexes produced by deposit-feeders can potentially function as water-filtration systems, with burrow-builders providing the water-pumping function22 and burrow walls and adjacent sediments the particle trapping function25.The harnessing of ecological functions and services provided by biological systems to overcome environmental degradation, variously referred to as ecological engineering, nature-based solutions, bioremediation and/or biomanipulation, is increasing in popularity as a sustainable, nature-based tool that can mitigate nutrient enrichment in coastal ecosystemsvia active pumping through complex, sometimes interconnected burrow systems26. Such irrigation oxygenates burrow waters and eliminates faecal material, anoxic/hypoxic waters and potentially toxic compounds (e.g. H2S)21. Through such exchanges, suspended particles, including phytoplankton and other organic matter, can theoretically be adsorbed onto burrow walls and adjacent sediments25 in a manner akin to particle trapping achieved by artificial sediment filters. Particle adsorption is likely to be facilitated by the addition of mucus to burrow linings (for stability), which increases adsorptive properties. Additionally, microbial biofilms that line burrow walls can increase adhesive particle trapping further through exopolymer exudation22. Prior research demonstrating that burrow walls are enriched with microalgae, even at aphotic sediment depths, lends credence to the idea that burrow walls may act as sites of passive adsorption24. Consumption of adsorbed organic particles by burrow-builders, microbes and other infauna likely maintains the functional integrity of burrows as biological filters.Benthic deposit-feeders spend significant portions of their activity budgets on bi-directional water exchanges between pelagic and benthic ecosystems 29, increasing the area of the sediment-water interface several-fold. Secondly, they spend a significant portion of their daily activity budgets on bi-directional water pumping30, given that endobenthic sediments are prone to becoming hypoxic and hypercapnic18. Lastly, they occur as dense assemblages  with spatial occupancy spanning several kilometres22. These features suggest that in the presence of dense assemblages of axiid crustaceans, three-dimensional burrow superstructures may function as elaborate below-ground coastal filtration systems.As is the case with artificial purification systems, pumping rate and filter surface-area are likely key determinants of filtration effectiveness in ecosystems dominated by deposit-feeders and their burrow systems. On this basis, endobenthic axiid crustaceans  are likely to exhibit the strongest filtration effects of extant benthic burrowers for several reasons. Firstly, these organisms produce intricate, interlinked burrow systems that extend several meters deep into sediments (2–3 m in some cases)23, effects on pelagic filtration have not been empirically tested, either in the context of axiid crustaceans or benthic deposit-feeders more broadly. This novel premise thus forms the scientific motivation for this paper, which tests the broad hypothesis that water quality would improve in the presence of the sandprawn Callichirus kraussi, which is a southern African burrowing axiid crustacean. More specifically, we hypothesised that if the burrow systems of C. kraussi function as biological filters, then phytoplankton biomass should decrease in the presence of this sandprawn. Our hypothesis was tested using a combination of ex situ mesocosm experiments and comparative in situ approaches in the Zandvlei Estuary Nature Reserve , where it influences multiple resource flows that significantly determine compositional and structural attributes of biotic assemblages34. More broadly, our work was intended to expand understanding of the functional relevance of a globally distributed group of endobenthic organisms that are highly influential ecosystem engineers in coastal habitats22.While passive particle adsorption has previously been hypothesized as an explanation for greater levels of trophic resources along burrow wallsrve Fig. , which w35 located roughly 30 km south of Cape Town, South Africa  and kept open during the winter  months. The lower reaches of the system is occupied by dense aggregations (176/m2–240/m2) of the sandprawn Callichirus kraussi, but this biotope accounts for approximately 4.9% of the total area of the system 2) reported in South African estuaries24.A two-week indoor mesocosm experiment was conducted at the aquarium facility at the University of Cape Town (UCT) to quantify the filtration function provided by sandprawns. Nine mesocosms  were filled with sediment (depth = 20 cm) and water (depth = 20 cm) from the Zandvlei Estuary. Sediment was collected from a sandprawn-dominated biotope in the lower reaches and sieved (2 mm mesh) before being added to each mesocosm. Estuarine water  was collected approximately 200 m upstream of the sandprawn biotope. Individual mesocosms were aerated and left for one hour before initial (Day 0) water quality data were collected and sandprawns added to the designated mesocosms. To ensure uniformity across mesocosms and to maintain conditions to support sandprawn survival, conductivity, dissolved oxygen, pH and temperature were measured in each mesocosm on Day 0 and every 2 days thereafter, using a multi-probe water sampler (Lovibond® Water Testing SensoDirect 150 with PT1000). The experiment comprised three sandprawn treatments - controls lacking sandprawns, 50% natural density (6 sandprawns/mesocosm) and 100% natural density (11 sandprawns/mesocosm), with 3 replicates of each. The 100% density treatment corresponded to upper ranges of sandprawn densities . Only non-ovigerous sandprawns, approximately 4 cm to 8 cm in length (chela to telson), were selected. After extraction from the sediment, the sandprawns were placed in loose, moistened layers of newspaper and transported to the aquarium facility at UCT, where they were distributed amongst the mesocosms according to the designated treatments. Similar sizes of sandprawns were added to each mesocosm to prevent density effects being confounded by size variation. During the experiment, the number of burrow openings in each mesocosm was monitored to approximate the number of sandprawns per mesocosm. Past studies have shown that the number of burrow openings closely corresponds to sandprawn abundancea (chl-a) concentrations. Two water samples were taken from each mesocosm at depths of 6 cm (lower depth) and 12 cm (upper depth) above the sediment surface, using a syringe fitted with a fine tube. This was done to determine if potential sandprawn effects were localised and therefore depth-specific. Chl-a concentrations were determined fluorometrically (Turner Designs Trilogy). On days 1, 7 and 9, 40 mL water samples were collected from the lower and upper depth levels of each mesocosm to measure concentrations of phosphate (PO43−), ammonium (NH4+), nitrate (NO3−) and nitrite (NO2−), using a multiparameter research photometer (Hanna Instruments HI 83203). On day 11, after final pelagic chl-a measurements were made, surface water samples (500 mL) were collected from mesocosms and filtered (Whatman GFF) to quantify total suspended sediment loads. This was followed by filters and sediment being dried (70 °C) overnight. The difference in mass of filters before and after filtration of mesocosm water was used to quantify suspended sediment loads. At the termination of the experiment (2 weeks), each mesocosm was drained completely (four 3 mm holes drilled at mesocosm bases) and 6 randomly interspersed surface sediment cores  were collected from each of the 50% and 100% mesocosms. Three samples were collected from burrow mounds, and the remaining 3 from between mounds; these were pooled to generate a single mean value per mesocosm. Additional samples were collected from the burrow walls by sectioning the burrow to a depth of 1 cm and carefully scraping the sand away to expose the burrow wall38. Three different burrow lining samples were collected per mesocosm and averaged per mesocosm. Three surface sediment cores per mesocosms were also collected from controls. Sediment chl-a concentrations were determined fluorometrically (Turner Designs Trilogy) following extraction in 30 mL of 90% acetone and refrigeration for 46 hours. Chl-a concentrations were expressed relative to mass of sediment sample collected38. Sandprawns were returned to the Zandvlei Estuary at the termination of the experiment.Prior to sandprawn additions to mesocosms (Day 0), and every 2 days thereafter until Day 11, 3 mL water samples were collected from each mesocosm to measure chlorophyll-a biomass differed between sites with and without sandprawns and (2) if trends recorded in mesocosm experiments were also evident in situ. For each year of monitoring, surface chl-a data (depth = 10–20 cm from surface) were collected at 6 sites  for suspended sediment load determinations were collected at Site 5 (n = 5), which occurs within the sandprawn-dominated biotope and Site 4 (n = 5), which is a sandprawn-free site located upstream of the sandprawn biotope  from the Zandvlei Estuary were used to determine (1) whether chl-6–2019 fr39. Linear Models (LMs) were fitted to test effects of sandprawn density and day on conductivity, dissolved oxygen, pH and water temperature in experimental mesocosms using the ‘stats’ package41. Linear Mixed-Effects Models (LMMs) were fitted by restricted maximum likelihood (REML) estimation using the ‘lme4’ package42 to determine effects of sandprawn density, water depth, and sediment position (where relevant) on pelagic and benthic chl-a and nutrient concentrations. Where water depth did not explain significant variation in response variables, surface data were used in subsequent analyses. In cases where data were collected over multiple days, the days were considered a random effect, since concentrations were expected to change over time within each treatment. The significance of main effects in the LMs and LMMs was determined using the ‘car’ package43. In the cases where main effects significantly explained variation (p < 0.05), multiple comparisons of means (Tukey Contrasts) using the ‘emmeans’ package44 were applied to the LMs and LMMs to identify within-group and within-treatment differences. A posterior predictive simulation was used to evaluate model fits. The final LMM was evaluated using interquartile ranges as the summary statistic. The model was deemed to represent the data adequately if the posterior predictive p-value was greater than 0.7842, indicating that at least 78% of observed data fell within the simulated distribution42.All data analyses were performed using the R data analysis platformExperiments were conducted in accordance with guidelines of the University of Cape Town. Approval of the research was granted by the University of Cape Town, Science Faculty Animal Ethics Committee .8,36 = 0.06, p = 1.000), pH  and temperature  did not vary across mesocosms for the study period. Significant variation in conductivity  was detected, which was explained by sandprawn density  and days , but not their interaction . Conductivity generally increased over time and with increasing sandprawn density, but conductivity increases were minor, with mean levels ranging between 26.7 mS/cm ± 0.41 SE and 30.3 mS/cm ± 0.10 SE over time. Conductivity did not differ by more than 2 units between mesocosms with and without sandprawns over the duration of the study.Abiotic conditions were generally uniform among sandprawn treatments across sampling days Table . Dissolv2− , NH4+ , PO43−  and chl-a concentrations in the mesocosms. NO3− levels were below the 0.0 mg/L detection limit for all mesocosms. Sandprawn density significantly explained variation in mesocosm NH4+ concentrations , which were roughly 2.5 times greater in the 100% treatment relative to controls. Variation in PO43−  and NO2−  concentrations were not explained by sandprawn density  and 20% (50% sandprawn treatment) greater than surface values  in overlying waters over the 11-day experimental period, with filtration effects manifesting after roughly 4 days. A decline in chl-a by approximately 70% in the sandprawn dominated biotope relative to the sandprawn-free site immediately upstream in the Zandvlei Estuary, also supports the notion of sandprawn presence being linked to improved water quality. Lastly, the increase in pelagic chl-a in the downstream canalised section of the estuary, where sandprawns are absent, adds further credence to the idea that improved water quality is associated with sandprawn presence. Overall, the broad agreement between findings of the mesocosm experiment and long-term in situ datasets strengthens confidence in ascribing a water-filtration function to sandprawns. Taken collectively, findings support our hypothesis that sandprawn presence would lead to improvements in water quality, by reducing phytoplankton biomass (measured as chl-a). At a broader level, our findings add to knowledge of processes that influence coastal water column productivity, given that physico-chemical processes, and filter feeders from a biotic perspective, have generally dominated perspectives in this field. Our findings however, suggest that engineering activities of sandprawns and similar organisms may be responsible for exerting a form of top-down regulation of coastal productivity.Findings from our mesocosm experiment provide compelling evidence that sandprawn presence is linked causally to improvements in water quality. This is apparent through the near-50% reduction in phytoplankton biomass  burrow walls and with increasing sediment depth. Similarly, studies on Pestarella tyrrhena have reported greater chl-a levels in burrow walls than surface sediments23. Our phytoplankton adsorption hypothesis is additionally supported by prior research in which direct observations of sandprawn (C. kraussi) behaviour within narrow-walled benthic chambers indicated an absence of filter-feeding activity26. Moreover, sandprawn burrows are irregular in shape46, and not ‘U’ or ‘Y’ shaped, which is typical of filter-feeding crustaceans47. Collectively, empirical data from our mesocosm experiment allied with prior research suggests that burrow-wall adsorption of phytoplankton cells is the probable driver of sandprawn-induced declines in phytoplankton biomass.Our findings regarding burrow chl-4+ concentrations in overlying waters increased with sandprawn density is not entirely surprising, given that NH4+ is the most available of the dissolved nitrogen species for exchange across the sediment-water interface18. Increased fluxes of NH4+ in the presence of burrowing organisms are not uncommon and is thought to be driven by burrower excretion, greater diffusive area (burrows) and nitrogen mineralisation either through increased bacterial activity or organic matter content within burrows53. Increasing NH4+ fluxes have on occasion been linked to increases in primary abundance and/or productivity52. In our experiment however, despite the increase in pelagic NH4+ concentrations with increasing sandprawn density, there was no obvious effect on net pelagic filtration by sandprawns, since phytoplankton biomass declined to a steady state after 4 days in the presence of sandprawns, whereas NH4+ levels increased with sandprawn density over time. This would suggest that sandprawn filtration is robust against any potential positive effect of NH4+ fluxes on phytoplankton biomass.Our finding from the mesocosm experiment that NH54. Current paradigms are therefore built around the idea that while resource modulation and emergent engineering effects of deposit-feeders are large, particularly in the case of endobenthic species, they are generally restricted to the benthos54. Our findings show however, that engineering effects may not necessarily be confined to the benthos. While engineering activities are primarily benthic, by virtue of burrow construction within sediments, secondary engineering effects can manifest in pelagic ecosystems due to phytoplankton adsorption onto burrow walls. This secondary engineering function (pelagic filtration) has not previously been ascribed to deposit-feeding organisms and has traditionally been considered the domain of benthic filter-feeders. On a similar note, pelagic filtration by deposit-feeders has not featured in existing paradigms on bentho-pelagic coupling in coastal ecosystems, with narratives emphasising coupling by filter-feeders through phytoplankton consumption and by endobenthic engineers through material fluxes (mainly nutrients) into the water column55. Our findings however, demonstrate that endobenthic burrowers can couple benthic and pelagic ecosystems through pelagic filtration, and highlight the need for further research on this phenomenon to understand its dimensions more broadly. This assumes great significance when viewed in the context of existing knowledge gaps regarding bentho-pelagic coupling processes, particularly those mediated by organisms55. Also relevant are concerns emanating from the latter about limitations in predictive capabilities regarding coastal responses to global change55.At a broad level, the pelagic filtration that we have identified for sandprawns is novel, sheds new light on the functional significance of these and similar deposit-feeders in coastal ecosystems, and expands understanding of mechanisms by which they engineer marine ecosystems. To date, research in these areas has been dominated by bentho-centric ideas, in which ecosystem engineering activities  in sedimentary biotopes have been emphasised56. Such large-scale losses of sandprawn-dominated habitats are likely to have had major repercussions for water quality in the bay, based on our findings. Canalisation, which is common in estuaries locally57, particularly urban ones, need to be reconceptualised to be more ecologically sensitive to burrowing sandprawns and similar endobenthic fauna. As shown by our in situ data, canalisation can lead to increased phytoplankton biomass due to exclusion of sandprawns and their filtration capabilities. Reconfigured canal designs that permit endobenthic burrowing could allow for filtration to occur while fulfilling a stabilisation role. Lastly, unregulated harvesting of sandprawns (and similar organisms) together with trampling-induced destruction of burrows can reduce filtration effectiveness in the long-term.At its broadest level, our findings highlight the potential for ecosystem engineering activities of sandprawns and functionally similar deposit-feeders to increase coastal resilience against nutrient enrichment by limiting phytoplankton biomass. To date, endobenthic deposit-feeders have received relatively little conservation and management attention, partially due to their cryptic lifestyles and a limited appreciation of their functional significance. In this context, our findings provide valuable traction for their conservation and management, given that pelagic filtration can assist in minimising harmful effects of nutrient enrichment. Our findings additionally raise awareness for processes that restrict spatial occupancy and abundance of sandprawns to be managed appropriately to ensure that pelagic filtration and ecosystem resilience are not constrained. At a local level, prolonged mouth closure likely confines marine influence to the lower reaches in the Zandvlei Estuary, thereby imposing spatial limits on sandprawn distribution and filtration effects. Similarly, destruction of sedimentary habitats as part of coastal development, or subsequent canalisation, reduces resilience against nutrient enrichment by eliminating endobenthic filtration. In Durban Bay, on the east coast of South Africa, roughly 75% of intertidal sandflats have been lost to development between the 1890s and 1990s25. Inclusion of sandprawns and similar organisms in coastal management will likely broaden the pool of functional-traits available to combat enrichment, given that they possess unique biological attributes to deal with harsh abiotic conditions, including hypoxia and hypercapnia58. Investments in appropriate management plans for these organisms and their use in bioremediation initiatives have the potential to increase coastal resilience against nutrient enrichment, and thereby enhance ecosystem functioning and the quality of goods and service provision. Overall, our work dovetails with calls that emphasise wilful ecosystem restoration initiatives and the use of nature-based solutions to counter degradation in coastal ecosystems15.Given the value of coastal ecosystems and the ever-increasing threat posed by nutrient enrichment to their socio-ecological functioning, sandprawns and functionally similar deposit-feeders may be useful additions to existing nature-based tools that can counter enrichment. This idea may be extrapolated to suspension-feeding endobenthic crustaceans, given that past work has illustrated the potential for their burrows to act as traps for suspended material


M50NiL steel, which belongs to a new generation of case-hardening steels used in aerospace bearing applications, is used mainly for the manufacturing of aerospace transmission components that operate under high temperatures. In this study, the effects of the hot deformation parameters and the initial microstructure on the hot deformation behavior of M50NiL steel were investigated through Gleeble-3500 isothermal hot compression tests. The experimental results demonstrated that the critical stain of dynamic recrystallization and the deformation activation energy of the coarse-grained samples were higher than those of the fine-grained samples. This is attributed to the difficulty of deformation and the dynamic recrystallization behavior of coarse-grained samples. Moreover, fine-grained samples contain a large number of dispersed phases, which can pin the grain boundaries and inhibit the growth of recrystallized grains. Such phenomena are beneficial for obtaining finer and more uniform microstructures in M50NiL steel. The experimental results can provide a useful reference for preparing M50NiL steel with excellent mechanical properties. In future advanced gas turbine engines, the main shaft bearing may operate under extremely high temperature conditions, high loads, and harsh corrosive environments . In ordeHot deformation parameters such as strain, strain rate, and deformation temperature are crucial for controlling the microstructure of the final products. Potential abnormal deformation conditions can induce surface cracks or inhomogeneous grain size distribution . In geneApart from the hot deformation parameters, the initial state conditions, such as grain size and second phase particles, also have an effect on DRX; however, they are often neglected. A few studies ,10 focusIn this paper, the effect of the initial microstructure and hot deformation conditions on the hot deformation behavior of M50NiL steel was investigated to optimize its microstructure and mechanical properties. Different methods, including true stress–strain curves, a critical strain model, and microstructural characterization, were employed to analyze the effect of the initial microstructure and hot deformation conditions on the hot deformation behavior of M50NiL steel. The results can provide a valuable reference for obtaining M50NiL steel with excellent mechanical properties and an appropriate microstructure.In this work, the annealed M50NiL steel  was used to investigate the effect of the initial state and deformation conditions on hot deformation behavior. Its chemical composition is given in −1. The exact experimental procedure is as follows. First, two specimens with different initial states were prepared by heating to 1100 °C for 5 min and to 1150 °C for 30 min, respectively, at a constant rate of 20 °C/s. Next, all specimens were cooled to compression temperature at 10 °C/s for 10 s, and then were compressed until reaching a total true strain value of 1 at strain rates of 0.01, 0.1, and 1 s−1; this was followed by immediate quenching in water. The stress–strain curves obtained during the hot deformation were automatically saved in a computer program. After completion of the hot deformation process, the deformed samples were cut along the longitudinal direction using an electric spark cutting machine. Subsequently, the surface of the samples was ground and polished. Thereafter, the samples were electrolytically etched in a supersaturated solution of oxalic acid to reveal the microstructure of the M50NiL steel. For microstructure characterization, the maximum deformation zone of the samples was observed using optical microscopy, scanning electron microscopy equipped with a backscattered electron (SEM–BSE) detector, and transmission electron microscopy (TEM).First, both the fine- and coarse-grained samples were machined into small cylinders with a diameter of 8 mm and a length of 12 mm. Then, isothermal deformation tests were performed on a Gleeble-3500 thermo-mechanical simulator at 950–1100 °C and a strain rate of 0.01–1 s−1 are presented in The true stress–strain curves of the fine- and coarse-grained specimens under a deformation temperature of 950–1100 °C and a strain rate of 0.01–1 sIn general, stress gradually increases at the initial stage, and then reaches a steady-state without significant softening and clear peaks, which is considered a characteristic of dynamic recovery (DRV) or continuous dynamic recrystallization (CDRX). After reaching a peak stress, the flow stress decreases, which is considered a characteristic of discontinuous dynamic recrystallization (DDRX). It can be seen that in the true stress–strain curves, the DDRX characteristics occupied a dominant part at a higher deformation temperature and lower strain rate d, which It can be seen in −1), σ is the flow stress (MPa), Q is the activation energy (J·mol−1), R is the universal gas constant (8.314 J·mol−1·K−1), and n, As mentioned above, both the temperature and strain rate have considerable effects on flow stress. The effect of both temperature and strain rate can be incorporated in the Z parameter, as shown in Equation (1) :(1)Z= ε˙p, was used to calculate the material constant, and then a logarithm was applied to both sides of Equation (1):In this study, the peak stress, σThen, Equation (2) was partially differentiated at constant T, leading to Equation (3):p and the slope of lnp, respectively. The values of By substituting the corresponding experimental data in Equation (2) and by applying linear regression, the relationship between strain rate and stress was obtained . As per p) versus 1/T, and n can be determined from the slope of ln p). As seen in −1 and 457.16 KJ·mol−1, respectively. Moreover, the obtained activation energy for the coarse- and fine-grained samples was similar to the 481.4 KJ·mol−1 obtained by Ji [For constant The activation energy, Q, is an important material parameter that serves as an indicator of the degree of deformation difficulty during hot deformation . In geneThe critical strain of dynamic recrystallization constitutes one of the important indicators in the analysis of dynamic recrystallization behavior, since it can determine the strain required for the initiation of dynamic recrystallization . The criAs mentioned above, the dynamic recrystallization behavior is affected by the temperature and strain rate. Therefore, the critical strain can be expressed by the Z parameter as:obtained .It can be seen in To investigate the microstructure evolution during hot deformation, optical microscopy images of both samples under different hot deformation conditions were captured, as shown in In both samples investigated here, a typical necklace structure was observed a,c. WithIn −1 on a Gleeble-3500 testing device. Based on the results and analysis, the following conclusions can be drawn:The true stress–strain curve can be considered a macroscopic reflection of the changes in the microstructure of a material. Under most deformation conditions, the flow stress of the fine-grained specimens was higher than that of the coarse-grained specimens, which is attributed to the pinning effect of the fine-grain dispersion on the grain boundary.The critical strain and deformation activation energy of the fine-grained samples were lower than those of the coarse-grained samples. It was proven that a microstructure with fine grains is beneficial to the dynamic recrystallization of M50NiL steel.The original microstructure with fine grains and dispersed phase is more favorable for the hot deformation of M50NiL steel. This is due to the fact that the dispersed phase can pin the grain boundaries and hinder the growth of recrystallized grains. Consequently, a more uniform and finer microstructure can be obtained after recrystallization.The aim of the present study was to assess the effect of the initial microstructure and hot deformation parameters on the evolution of dynamic recrystallization of M50NiL steel. To this end, hot deformation experiments were performed at temperatures between 950 and 1100 °C and strain rates between 0.01 and 1 s


Interestingly, each MCS displays a unique molecular signature, adapted to organelle functions. This review will explore the literature describing the molecular components and interactions taking place at ER-PM contact sites, their functions, and implications in eukaryotic cells, particularly neurons, with emphasis on lipid transfer proteins and emerging function of SNAREs.The compartmentalisation achieved by confining cytoplasm into membrane-enclosed organelles in eukaryotic cells is essential for maintaining vital functions including ATP production, synthetic and degradative pathways. While intracellular organelles are highly specialised in these functions, the restricting membranes also impede exchange of molecules responsible for the synchronised and responsive cellular activities. The initial identification of contact sites between the ER and plasma membrane (PM) provided a potential candidate structure for communication between organelles without mixing by fusion. Over the past decades, research has revealed a far broader picture of the events. Membrane contact sites (MCSs) have been recognized as increasingly important actors in cell differentiation, plasticity and maintenance, and, upon dysfunction, responsible for pathological conditions such as cancer and neurodegenerative diseases. Present in multiple organelles and cell types, MCSs promote transport of lipids and Ca A distinctive property of eukaryotic cells is the compartmentaliasation of the cytoplasm by intracellular membranes. In addition to the plasma membrane (PM), phospholipid bilayers and monolayers form complex internal networks, defining individual organelles specialised for specific functions. While this is useful for separating incompatible biochemical reactions and restricting specific conditions such as low pH, and/or redox potential, compartmentalisation can impede the transfer of molecules which instead relies on vesicular trafficking . Recent 2+ homoeostasis  between membranes, then recruit partner proteins which perform contact specific functions . In MCSs2+ signalling; compromising cellular integrity, and activating ER unfolded response  in the PM, via a C-terminal basic stretch of 69 amino acids Pno acids . This inno acids . Furtherno acids .2+ activated Cl– channels  and stretches of basic amino acids at the C-terminus  domain, a coiled-coil domain involved in dimerisation  motifs, the final two of which are separated by a joining region  mediates PM targeting, though the direct involvement of MORN repeats has not been demonstrated  and PM . While ig region . These Megion  domain and three to five C2 domains (2+-dependent manner (2+ level (Extended synaptogamins (E-Syts) are evolutionarily conserved proteins shown to function at ER-PM membrane contact sites in multicellular organisms. Sequence and domain analysis revealed homology with the yeast Tricalbins family, also identified at ER-PM MCSs. The deletion of all three known Tricalbin isoforms in combination with deletion of other associated tethers reduces the number of contact sites . Tricalb domains , which bt manner . Indeed,t manner , enablin2+ level .2+ regulation. E-Syts2/3 bind PIP2 at the PM via the C2C domain  constitute the basic molecular machinery of intracellular membrane fusion. Positioned on opposite membranes, vesicular SNAREs on one side and target SNAREs on the other assemble in a ternary complex which allows for close docking and subsequent fusion as exemplified in the case of synaptic vesicle fusion with the presynaptic PM  channel clustering at the PM in hippocampal neurons  freely diffusive channels in PM and b) micron-sized clusters present on the soma, dendrites, and axon initial segment . Kv2 vol neurons , cerebel neurons , and eve neurons  due to a neurons . While e neurons , where Lt firing . Furthert firing . Howevert firing . Furthert firing .+ entry upon ischaemic injury  activation by mediated via G-protein coupled receptor (GPCR)   led to tr (GPCR) . In addiA domain . FurtherA domain . Here, Sand PIP2 . A role osophila  and mammosophila  because dynamics . Furtherdynamics , a recepamics  and membrane-binding domains, most commonly a Pleckstrin homology (PH) domain (Another major class of LTPs are ORPs . There a) domain . Recentl) domain  and ER-P) domain . The fun) domain . In yeas) domain  where th) domain . Althoug) domain . This la) domain . In the ) domain  could beN-terminal to the StARt domain, thought to be involved in targeting, although unable to bind lipids  to facilitate lipid transfer  domain (StARD) proteins , also knd lipids . In mammd lipids . For exatransfer . While c2 in the PM by promoting transfer from the ER, offering a feedback loop for receptor-induced Ca2+ signalling  indicates extensive functional redundancy, highlighting the significance of lipid homoeostasis in biological membranes. Enhancement of ER-PM MCSs by E-Syt recruitment also induces translocation of Nir2 to ER-PM MCSs, potentially via interactions with VAP isoforms . Here, Ngnalling . Abnormaynthesis . In accor stress . Followi2+ is an essential second messenger utilised ubiquitously in eukaryotes. Early work by 2+ signaling. A prerequisite for Ca2+ signaling is a low baseline Ca2+ concentration in the cytosol; in resting cells, Ca2+ levels are actively maintained in the nanomolar range, several orders of magnitude below typical extracellular concentrations   , directlel Orai1 . These f ER Ca2+  P2-dependent, highlighting the MCS for recruitment and translocation of STIM1 post depletion of Ca2+ -enriched phagophore, which requires progressive vesicular fusion to form the maturing structure (Lipid transfer is essential to maintain cell homoeostasis and simultaneously mediate dynamic changes upon stimulus. Indeed, transfer can modulate lipid synthesis, cell growth and is also vital for organelle biogenesis and function, as each membrane possesses specific lipid compositions, including asymmetric distributions between leaflets. This requires targeted and often large-scale transport of lipids throughout the cell . Lipid ttructure  and alsotructure . Furthertructure . SiRNA-mtructure . It is tThe study of MCSs has grown tremendously in the last 20 years, however, our understanding of their structure and functions still shows many gaps. Much of this difficulty arises from the inherent features of MCSs; they are minute, dynamic structures with diverse molecular compositions between organelles and organisms and thus can be difficult to study. ER-PM MCSs, while relatively well characterised, exemplify the complexity surrounding MCSs, with multiple tethering complexes and binding partners defining the structure’s performance as a signalling hub and as key homeostatic regulators, responsive to physiological and stressful conditions. What remains a major question in the field is the relationship between MCS and particularly their lipid transfer activity with membrane fusion events. The fact that SNAREs, which constitute the basic membrane fusion machinery, would interact with LTPs and be found both at MCSs suggest that their regulation might be key to either generate a contact or fuse. In such scenario, we propose an important function for the Longin domain of Sec22b. In other words, could membrane contact correspond to a membrane docking with frustrated fusion? An evolutionary view of these processes would certainly solve this question.BioRender. TG supervised and wrote final draft.BH wrote initial version and revised last version. NS and CV extensively rewrote and completed. NS designed the figures using 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.


This included, for example, a key RBD residue ARG357 in the Up-Down interaction and a three residue sequence ALA520-PRO521-ALA522, associated with a turn structure in the RBD of the Up state protomer, acting as a stabilizing interaction with the NTD of its neighbor protomer. Additionally, our intra chain dominant energy mappings within each protomer, identified a significant “glue” point or possible “latch” for the Down state protomer between the S1 subdomain, SD1, and the RBD domain of the same protomer that was completely missing in the Up state protomer analysis. Ironically, this dominant energetic interaction in the Down state protomer involved the backbone atoms of the same three residue sequence ALA520-PRO521-ALA522 of the RBD with the amino acid R-group of GLN564 in the SD1 domain. Thus, this same three residue sequence acts as a stabilizer of the RBD in the Up conformation through its interactions with its neighboring NTD chain and a kind of latch in the Down state conformation through its interactions with its own SD1 domain. The dominant interaction energy residues identified here are also conserved across reported variations of SARS-CoV-2, as well as the closely related virions SARS-Cov and the bat corona virus RatG13. We conducted preliminary molecular dynamics simulations across 0.1 μ seconds to see if this latch provided structural stability and indeed found that a single point mutation (Q564G) resulted in the latch releasing transforming the protomer from the Down to the Up state conformation. Full trimeric Spike protein studies of the same mutation across all protomers, however, did not exhibit latch release demonstrating the critical importance of interchain interactions across the S1 domain, including RBD-NTD neighboring chain interactions. Therapies aimed at disrupting these noncovalent interactions could be a viable route for the physico-chemical mitigation of this deadly virion.The SARS-CoV-2 virion responsible for the current world-wide pandemic COVID-19 has a characteristic Spike protein (S) on its surface that embellishes both a prefusion state and fusion state. The prefusion Spike protein (S) is a large trimeric protein where each protomer may be in a so-called Up state or Down state, depending on the configuration of its receptor binding domain (RBD) within its distal, prefusion S1 domain. The Up state is believed to allow binding of the virion to ACE-2 receptors on human epithelial cells, whereas the Down state is believed to be relatively inactive or reduced in its binding behavior. We have performed detailed all-atom, dominant energy landscape mappings for noncovalent interactions  of the SARS-CoV-2 Spike protein in its static prefusion state based on two recent and independent experimental structure publications. We included both interchain interactions and intrachain (domain) interactions in our mappings in order to determine any telling differences  between residues in the Up and Down state protomers. The S2 proximal, fusion domain demonstrated no appreciable energetic differences between Up and Down protomers, including interchain as well as each protomer’s intrachain, S1-S2 interactions. However, the S1 domain interactions  SARS-Cov-2 is a positive-sense RNA virus responsible for the disease COVID-19 which was recently declared a world-wide pandemic by the WHO. SARS-CoV-2 virion enters human epithelial cells by attachment of it’s prefusion-form Spike protein (S) with a specific cell surface receptor ACE2 (Angiotensin Converting Enzyme 2) . The Spiβ corona viruses [The specific structural details of the RBD in the Up versus Down states in SARS-CoV-2 have recently been elaborated and compared across the S proteins of  viruses  and Up v viruses  for SARS viruses , based o viruses  noted th viruses  recently viruses  also detIn order to better understand the differences between the Up and Down protomer states and the possible transitions between them, we conducted an all-atom interacting energy landscape mapping of the entire Spike protein from their *.pdb (Protein Data Bank) structure files (6VSB and 6VYB), where one of the protomers is in the Up state and two are in the Down state. This allows us to identify key interaction energy “glue” points (both interchain and intrachain) associated with relatively strong non-covalent atom-atom interactions between residues that may be responsible for specific persistent domains of this complex trimeric protein. In doing so, we are able to identify some unique and critically different glue point residues between the Up and Down protomers within the overall trimeric structure. This analysis was then used to guide molecular dynamic studies incorporating single point mutations of the S protein in order to investigate the local dynamic stability between the Up and Down states. We note that these key residues will be shown to be conserved across the closely related virions SARS-CoV and the bat corona virus RatG13, as well as known variations of the novel corona virus. Comparative analyses between Up and Down state protomers, such as those given here, may provide insights useful for vaccine development in COVID-19, for example via multiple and single point mutations to favor the Up conformational state , potentiintrachain interactions) for the Up and Down state protomers, including S1 and S2 domain interactions and sub domains of S1 that include the RBD domain.The SARS-CoV-2 Spike protein structures considered here consist of three chains or protomers  of which one chain is given in the so-called “Up” state of its RBD and the remaining two chains are in their “Down” state. We energetically mapped the interchain interactions “Up-Down” and “Down-Down” and specific domain interactions  criteria and −0.3kT units for Coulombic interactions, although lower values can also be specified in the analysis part of the mappings in order to further refine the results [Following our recently published study on  fibrils , we anal fibrils . This ma results .Explicit solvent molecular dynamics (MD) simulations of the novel coronavirus Spike protein were performed using the NAMD2 program . We usedMappings of the dominant atom-atom interactions among the residues for the Up-Down (A-B) and Down-Down (B-C) states are illustrated in Figs We specifically examined the interchain interactions between the RBD with the NTD of its neighbor protomer for both the Up-Down (A-B) and Down-Down (B-C) interactions for 6VSB and 6VYB, shown in Figs intrachain interactions for both the Up and Down protomers in order to attempt to discern any telling differences in these two states.We next looked in detail at We first energetically mapped the S1 domain  to the S2 domain  of both the Up protomer and Down protomer separately, as shown in S5 and S6 Tables in Overall the energetic mappings between Up and Down states for these two S1 domains (RBD-SD1/SD2) appear very similar for any structure, as shown, for example, by the dominant partial charge interactions , we eliminated the R group glue point atoms of GLN 564 by replacing it with GLY 564, which is deemed one of the essential residues for the latch system, and did not alter in any way the three residue sequence ALA520-PRO521-ALA522 in the RBD domain or any other residues throughout the S1 chain. This mutation then effectively removes the R-group interaction of the backbone atoms of ALA520-PRO521-ALA522 thereby unhooking the latch. Note that ALA screening may not be effective at unhooking the latch in this case, since the R-group of ALA would still interact with its partner backbone atoms. Our trajectory analysis revealed the early release of the RBD from SD1 (within a few nanoseconds) and overall hinge opening and distal release of the RBD for this single point mutant, whereas the hinge angle and RBD relative positions were preserved in the wild state  of the complete trimeric Spike protein for a total period of approximately 1.0 microseconds. The results are shown in the supplemental movie S2 Video in The all-atom energetic mappings are shown to be useful in attempting to understand the overall functionality of the Spike protein of the novel corona virus. The S2, fusion machinery domain, for example, also acts as a rigid base for the more flexible S1, prefusion domain, where flexibility is an important part of its function. The RBD is tethered to the SD1/SD2 domain by two peptide strings: one from the NTD (approximately residues 327-335) and the other connected to the SD1 domains (approximately residues 527-535). These peptide “strings” are stabilized or “tied together” by beta strand structures near the S1/S2 interface. The RBD domain in the Down state protomer is partially stabilized by the dominant energetic interactions of the backbone atoms of three of its residues (in sequence) with the R-group of a single residue from the SD1 domain, which we have called a possible latch. Ironically, these same three residues are involved in helping to stabilize the Up state protomer by interactions with the NTD of its Down state neighbor. This study raises a number of new questions about the origins of the Up and Down state protomers, including the reasons behind the occurrence of the Up state protomer, since as we have shown here, the Down state is partially stabilized by not only the potential latch, but also neighboring chain interactions between the RBD and it’s NTD neighbor. It seems reasonable, however, that the process of “Down to Up” is irreversible in the absence of deliberate outside interference. We further note that the possible three residue latch of the RBD Down state (ALA520 PRO521 ALA522) along with GLN654 of SD1 are conserved across reported variants for SARS-CoV-2 [S1 File(ZIP)Click here for additional data file.


Background: Educational Climate (EC) may determine teacher and student behaviour. Our aim was to evaluate EC longitudinally in a period of ‘curricular transition’ from traditional (teacher-centred learning) to Bologna curricula (interactive student-centred learning). Methods: The ‘Dundee Ready Education Environment Measure’ (DREEM) questionnaire was completed by 397 students from a Spanish School of Dentistry. Students’ perception was assessed in different courses and academic years. Results: The overall EC scale average was 115.70 ± 20.20 (57.85%) and all domain values showed a percentage > 52%, which were interpreted as ‘positive and acceptable’. The EC mean was: 118.02 ± 17.37 (59.01%) for 2010–2011; 116.46 ± 19.79 (58.23%) for 2013–2014; 115.60 ± 21.93 (57.80%) for 2014–2015; 112.02 ± 22.28 (56.01%) for 2015–2016, interpreted as ‘more positive than negative EC’. The worst Learning domain scores corresponded to later academic years and may reflect the Bologna curriculum’s more intensive clinical training involving greater responsibility and self-learning. Conclusions: EC and its domains were perceived more positively than negatively. The Social domain was the most positively evaluated, while the Learning domain was the worst.  The educational environment is defined as students’ perceptions of their influences and pressures, and how this perception is aligned with the curriculum’s educational aims . The perSeveral qualitative and quantitative methodologies have been used to evaluate students’ perception of EC ,7,8,9. IAs a result of the Bologna reform process in the European Union , it was We carried out a prospective longitudinal study applying the DREEM questionnaire to undergraduate dental students from the Medicine and Dentistry School, Universidade de Santiago de Compostela (USC), Spain. Ethical approval for the study was obtained from the Universidade de Santiago de Compostela. The students in the sample were from 3rd, 4th, and 5th year courses. Before participating in the survey, students were informed regarding the data processing characteristics, the importance of voluntary participation, and the anonymity of the process. The average questionnaire completion time was approximately 7 min. Questionnaires were distributed to students at the end of several academic years , which had different curricular configurations: in the 2010–2011 academic year, all courses were taught using a teacher-centred approach, in the 2013–2014 academic year, the 3rd and 4th year courses used a student-centred approach, while 5th year courses used a teacher-centred approach. Finally, in the last two academic years , all courses used a student-centred approach . The instrument used in this study was the DREEM questionnaire  which coThe DREEM scale provides results for each item, for each domain (the sum of the scores of the corresponding items) and total EC score (the sum of the scores of each domain). The maximum possible scores for the different domains are: Learning Perception: 48; Teacher Perception: 44; Academic Perception: 32; Atmosphere Perception: 48 and Social: 28. The maximum score for EC is 200. The data can be expressed as percentages of maximum scores in the respective subscale or the overall scale. Therefore, in relation to the general interpretation of the scale, the higher the score (or percentage) obtained in the different parameters, the more positive perception about the aspect evaluated. p ≤ 0.05. In the case of multiple comparisons between academic years, the Bonferroni correction was applied, establishing a value of p < 0.008 as significant. In the case of multiple comparisons between the teaching courses in different academic years, the Bonferroni correction was applied, establishing a p-value < 0.016 as significant.The data obtained were processed with the PASW Statistics program (SPSS version 2.1) for Windows. The data in the overall assessment of EC, for each domain, and each questionnaire item were expressed as averages. The data in the overall assessment of the EC and for each domain were also expressed as percentages in relation to the maximum score . Non-parA total of 397 (70%) dentistry students completed the DREEM questionnaire. There were one hundred and eighteen (29.7%) males and two hundred and seventy-five (69.3%) females (gender data were unavailable for four subjects). The average age was 23.19 ± 4.62 years. Regarding the different courses, there were 117 students in the 3rd course, 119 in the 4th course and 161 in the 5th course.The overall EC mean was 115.70 ± 20.20 (57.85%), which was interpreted as “more positive than negative EC”. According to domain values, all were interpreted as “positive and acceptable”. The mean obtained for Learning domain was 25.12 ± 6.04 (52.12%), for Teacher domain was 25.15 ± 5.79 (57.15%), for Academic domain was 19.65 ± 4.11 (61.40%), for Atmosphere domain was 28.44 ± 5.87 (59.25%) and for Social domain was 17.21 ± 3.59 (61.49%) . Social p = 0.013) when academic year 2015–2016 was compared to 2010–2011 (p = 0.003) and 2014–2015 (p = 0.009) (The mean EC by academic year were as follows: 118.02 ± 17.37 (59.01%) for 2010–2011; 116.46 ± 19.79 (58.23%) for 2013–2014; 115.60 ± 21.93 (57.8%) for 2014–2015; and 112.02 ± 22.28 (56.01%) for 2015–2016. In all academic years, the interpretation for EC was more positive than negative. Although we observed a decrease in EC mean value, the only significant difference was observed in ‘Learning domain’ (= 0.009) . The ave= 0.009) .p = 0.001), 2013–2014 (p = 0.001), and 2014–2015   compared to previous years, although differences were not significant. In addition, this 4th course corresponded to 5th course in the 2015–2016 academic year, where all domains showed significantly lower values in Learning domain as compared to academic years 2010–2011 (= 0.057) .To the best of our knowledge, the present study represents the first longitudinal analysis of EC, in a period of ‘curricular transition’ in Dentistry. EC is considered the expression and manifestation of the curriculum. It represents a critical element of the analysis of the quality of the teaching-learning process ,24. Our Although the DREEM survey evaluates the perception of teaching in five aspects of learning, it is not designed to analyse specific clinical or laboratory lessons, nor type of dental treatments performed by the students during their training. According to Miles et al.  the asseIn the present study, all domain values showed a percentage >52%, which were interpreted as “positive and acceptable”. The best score was for Social domain. However, Edgren et al.  stated tWe found students’ perceptions of EC to be higher (59.01%) in the traditional curriculum (2010–2011) compared to the Bologna curriculum 2015–2016) (56.01%), although differences were not significant. This means that the development of the new curriculum did not have a significant negative impact on CE in its early years. Only Learning domain showed significant differences between 2010–2011 and 2015–2016 academic years. A higher percentage (30%) of items were identified as ‘problematic educational areas’ in the 2015–2016 academic year. While Tomás et al.  found a  (56.01%)With respect to courses and academic years, all domains presented higher values in the academic year 2010–2011. Interestingly, the Learning domain presented a value of 46.25% in the 4th course of the 2014–2015 academic year and 45.35% in the 5th course of the 2015–2016 academic year, reflecting a negative perception of the learning process in courses adapted to the Bologna education reform. Moreover, this domain was the only one that showed statistically significant differences between the 5th course in different academic years. Student perception in the final course is in line with the findings reported by other authors ,37. It mOne of the notable strengths of this study was its prospective longitudinal design. Nevertheless, the limitation that it was conducted at a single institution with a limited sample size should be kept in mind. This study was designed to reveal the problematic educational areas related to the idiosyncrasy of our own institution in order to improve several curricular aspects.Overall, EC and its domains were perceived more positively than negatively by dental students during a period of ‘curricular transition’. The Social domain was the most positively evaluated, while the Learning domain was the worst. Our analysis revealed problematic educational areas during the transition from traditional to Bologna curricula, especially related to the Learning domain. The identification of problematic educational areas through the DREEM scale has potential for assessing the educational needs of higher education students to develop strategies for enhancing the teaching-learning process.


The resulting inflammatory reaction to polyethylene (PE) wear debris, which may result in osteolysis, is still considered to be a main reason for aseptic loosening. In addition to the primary wear in hip joint replacements caused by head-insert articulation, relative motions between the PE liner and the metal cup may cause additional wear. In order to limit this motion, various locking mechanisms were used. We investigated three different locking mechanisms  to address the resulting relative motion between the acetabular cup and PE liner and the maximum disassembly force. A standardized setting with increasing load levels was used in combination with optically based three-dimensional measurements. In addition the maximum disassembly forces were evaluated according to the ASTM F1820-13 standard. Our data showed significant differences between the groups, with a maximum relative motion at the maximum load level (3.5 kN) of 86.5 ± 32.7 µm. The maximum axial disassembly force was 473.8 ± 94.6 N. The in vitro study showed that various locking mechanisms may influence cup-inlay stability. Total hip arthroplasty (THA) is one of the most successful orthopedic procedures in joint replacement ,2. The TThe aim of this study was to evaluate specific locking mechanisms regarding the resulting relative motion between the acetabular cup and the PE liner. In addition, the maximum dissembling forces were investigated. For this purpose, three implant designs were tested, to determine if different types of locking mechanisms affect the magnitude of relative motion and dissembling force.®-S Alloclassic® with a cross-linked Durasul®-PE liner , (B) Pinnacle®-Multihole combinates with a cross-linked Marathon®-PE liner , (C) Plasmafit® Plus7 with a conventional ultra-high-molecular-weight polyethylene (UHMWPE) liner . All hip cup systems were non-cemented with an outer cup diameter of 52 mm and a corresponding 32 mm PE liner. In each group, six acetabular hip cups with corresponding PE liners were used.Three acetabular titanium hip cup designs, with different inner designs to fixate the PE liner, were investigated : (A) All®-S Alloclassic® is fixed by a circular press fit mechanism. In addition, two spikes in the inner polar region of the cup on which the PE liner is inserted improve rotational stability. The inner anchoring surface of the cup is spherical, and has a smooth surface  [The PE liner of the hip cup system Allofit 6–8 µm) .®-Multihole is realised by a central dome region and a taper lock mechanism. Additionally, 12 grooves are placed at the rim of the shell. Corresponding to the grooves, six tabs on the liner were found to improve rotational stability. The inner anchoring surface of the cup showed values of Ra = 0.2 µm and Rz = 1 µm.The anchoring of the PE liner of the hip cup system Pinnacle® Plus7 is fixed by a conical locking mechanism. The cup shows a rough inner surface  [The PE liner of the hip cup system Plasmafit= 25 µm) ,15.An overview of the locking mechanisms and the surface specifications of the different cup systems are shown in In order to carry out a standardized PE liner stability test, the cups were fixed according to Braun et al. in polyurethane  at an angle of 30 degrees to the vertical load axis . A loadiA rigid connection was generated between femoral head and PE liner. This, in combination with a 2 mm offset b L betweThe determination of relative motion was performed by placing optical markers  along the rim of the hip cup and PE liner a. These  3.5 kN) . The cooIn addition, the maximum disassembly force of the PE Liner was determined according to the ASTM F1820-13 standard. The PE liners of the investigated cup designs were assembled in the shell with a mean peak load of 2002.2 N ± 1.1 N using a material testing machine . Therefore, a displacement control rate of 0.04 mm/s was used. The force was applied by a femoral head coincident with the polar axis of the liner. The assembled liner shell constructs were placed in a fixture frame, with continuous support of the cup as shown in A repeated measures analysis of variance (ANOVA) was conducted to test for significant differences in relative motion depending on the load level for all investigated cup designs. Additionally, we conducted a one-way ANOVA to assess the effect of cup design on the maximum pull out force and relative motion. To identify which particular differences between pairs of means were significant, a post hoc analysis was performed. We used a Bonferroni Test as post hoc analysis to explore differences between the three group means while controlling the experiment wise error rate. Pre-analysis, the normal distribution of the data was evaluated using a Shapiro–Wilk-test and the homogeneity of variance was verified using the Levene-test. The results allowed for the use of the ANOVA test. For the repeated measures ANOVA, a Greenhouse–Geisser adjustment was used to correct for violations of sphericity.p < 0.05.Additionally, the data were evaluated descriptively using the arithmetic mean, standard deviation, minimum and maximum. The data were analysed using SPSS 25  with a significance level of The mean resulting maximum relative motion in XYZ-direction for each load level and all investigated cup designs are shown in p = 0.001) for mean resulting maximum relative motion of the Zimmer Biomet and the Aesculap group  and also between the Zimmer Biomet and DePuy group (p = 0.004) . There was no significant difference (p = 1.0) for the relative motion between the Aesculap and DePuy groups. The load level of 1.5 kN showed a significant difference (p < 0.001) for the relative motion between the Zimmer Biomet and the Aesculap group . There was no significant difference between the Zimmer Biomet and DePuy groups (p = 0.082) or between the Aesculap and DePuy groups (p = 0.070). The load level of 2.5 kN showed a significant difference (p = 0.001) between the Zimmer Biomet and Aesculap groups  and between the Aesculap and DePuy groups (p = 0.017) . There was no statistically significant difference for the relative motion between the Zimmer Biomet and DePuy groups (p = 0.718). For the load level of 3.5 kN, the Bonferroni-adjusted post-hoc analysis revealed a significant difference (p = 0.006) for the mean resulting maximum relative motion between the Zimmer Biomet and Aesculap groups  and between the Aesculap and DePuy groups (p = 0.014) . There was no statistically significant difference for the relative motion between the Zimmer Biomet and DePuy groups (p = 1.0). The repeated measures ANOVA with a Greenhouse–Geisser correction determined that the load levels showed a significant influence on the relative motion, F  = 309.886, p < 0.001.For the load level of 0.5 kN the Bonferroni-adjusted post-hoc analysis revealed a significant difference  = 29.25, p < 0.001.The mean maximum axial disassembling forces showed the highest values for the Aesculap group at 473.7 ± 94.6 N, followed by the Zimmer Biomet group at 294.8 ± 48.2 N and the DePuy group at 146.8 ± 49.8 N . The meap = 0.002),  and between the Aesculap and DePuy groups (p < 0.001), . There was also a significant difference between the Zimmer Biomet and DePuy groups (p = 0.011), .The Bonferroni-adjusted post-hoc analysis revealed a significant difference for the mean maximum disassembling force between the Aesculap and Zimmer Biomet groups . The DePuy system with a central dome region and a taper lock mechanism plus grooves at the rim of the shell showed relative motion between 15.2 ± 8.3 µm and 81.1 ± 18.5 µm (0.5 and 3.5 kN). For the Aesculap system with a conical locking mechanism and a locking free contact with the base of the cup we found relative motion between 6.2 ± 1.1 µm and 38.2 ± 9.9 µm (0.5 and 3.5 kN).p = 1.0). The Zimmer Biomet cup showed significantly higher values compared to the Aesculap and DePuy cups . At the highest load level of 3.5 kN, the Zimmer Biomet group and DePuy system no longer showed any significant difference (p = 1.0). However, the Aesculap system still showed significantly lower values compared to the Zimmer Biomet and DePuy cups . The locking mechanism of the Aesculap system showed the lowest relative motion between cup and PE liner for the investigated load levels compared to the Zimmer Biomet and DePuy cup. A retrieval study conducted by Wasielewski et al. evaluated the backside of 55 polyethylene liners [For the initial load level of 0.5 kN, the DePuy and the Aesculap cups showed no significant differences . There was also a significant difference between the Zimmer Biomet and DePuy groups (p = 0.011). In a comparative study of eight different cup systems, Tradonsky et al. determined a disassembly force between 129 N and 2950 N [Another aspect besides the relative motion between cup and PE liner was the maximum disassembly force with regard to the risk of dislocation. Therefore, the maximum disassembly force was determined according to ASTM F1820-13 standard . The maxd 2950 N . Our datd 2950 N .The results of our investigation showed that a tapered locking mechanism and non-locking contact with the bottom of the cup (Aesculap) showed a significant improvement in relative motion and disassembly force compared to a system with a central dome region and a taper lock mechanism (smooth surface) plus grooves at the rim (DePuy) and a hip cup system with a circular snap-fit mechanism in combination with two spikes in the inner polar region of the cup (Zimmer Biomet).® Plus7) compared to the Zimmer Biomet system (Allofit®-S Alloclassic®) using an identical experimental setup [This relative motion between cup and PE liner could be a predictor of backside wear. However, features such as the surface structure, specifically roughness of the inner geometry of the acetabular cup, could also have a major impact on backside wear behaviour. Braun et al. showed 45% higher backside wear particles for the Aesculap system  of the PE particles that may arise and prove their influence on the human body.From these findings, and the measured results in our study, it could be concluded that there is not a one dimensional relation between a tight fit of the inlay inside the cup, which is reflected in a low relative motion and increased disassembly forces, and the amount of backside wear. In addition, a closer look will be needed at the inner structure of the cup. It is necessary to distinguish between a smooth and a rough surface. A smooth surface with a high rate of relative motion could generate fewer backside wear particles than a rough surface with less motion.Initially, it should be noted that the fixation of the cups by fixating them in polyurethane does not correspond to the physiological fixation in vivo. However, the advantage of this connection was standardization of the fixation and the focus on the locking mechanisms of the cup systems. Other influencing factors could be excluded. There was a defined impaction force applied on the inlay, but no peak force as it occurs at the impaction by the surgeon.In the analysis of motion, this work deals with relative motion. The relative motion between cup and inlay, however, could be a combination of plastic and elastic deformation, migration, rotation, creeping and many other factors.This experimental study showed significant differences in relative motion at the cup–liner interface and in the disassembly forces for the three investigated locking mechanisms. How relative motion and assembly force affect backside wear in combination with different roughnesses should be analyzed in more detail in a further investigation.