The Arrhenius constitutive equation and hot processing map of composites were established, presenting excellent hot workability with low activation energies and broad processing windows. Dramatic discontinuous/continuous dynamic recrystallization (DDRX/CDRX) and grain boundary sliding (GBS) take place in composites during deformation, depending on the Zener-Hollomon parameter (Z) values. It is found that initially uniform TiB2 particles and fine grain structures are beneficial to the DDRX, which is the major softening mechanism in composites at high Z values. With the Z value decreasing, dynamic recovery and CDRX around particles are enhanced, preventing the occurrence of DDRX. In addition, fine grain structures in composites are stable at elevated temperature thanks to the pinning of dense nanoparticles, which triggers the occurrence of GBS and ensures good workability at low Z values.Isothermal compression tests of in-situ TiB Particle-reinforced aluminum matrix composites (PRAMCs) exhibit high elastic modulus and outstanding mechanical properties, which have been widely applied in aerospace, electronics and automotive industries over the past decades ,2. Gener4C particles and Al matrices. Thirdly, particle deformation zones with high stored energy are ideal sites for the recrystallization nucleus (this mechanism is widely known as particle-stimulated nucleation (PSN)), which promotes the local discontinuous dynamic recrystallization (DDRX) during thermo-mechanical processes  and ln[sinh(αpσ)]-1000/T. The values of n1, β, and Q can be acquired from the average slope of fitting lines in pσ)]. Through linear regression analysis, the slope and intercept of fitting line in 1, β, α, Q, n and lnA are calculated as shown in In order to guarantee the safe operation of equipment under the rated maximum load, the peak stress Q is closely related to the thermodynamic mechanism of dislocation motion [−1) because of the dragging forces of particles. However, the average value of Q in this work is 143.11 kJ mol−1 approximately to 142 kJ mol−1, which suggests that thermal deformation of this composites is dominantly controlled by the cross-slip or climbing of dislocations [Q can reflect the hot workability of deformed materials, and the improvement of workability will occur in materials with low activation energy [Q may have better hot workability and the detailed analyses of workability are given in the following sections.It is widely known that the value of activation energy n motion . In prevn motion ,22,30, tocations . In addin energy . Comparen energy ,22,30, tη) that illustrates the degree of power dissipation. Generally, materials deformation with the high value of η will have preferable workability. The efficiency value η is thought to be closely related to the microstructure evolution, such as dynamic recovery (DRV), DRX and superplastic flow. It was reported that [η in the DRV dominating region was about 20–30% and the efficiency in the DRX controlling region should be in range of 30–50%. When the power dissipation efficiency is high, up to 60–70%, superplastic deformation will occur.The processing map based on the dynamic material model , the majority of initial grains are elongated and perpendicular to compression direction as shown in the Zener-Hollomon parameters obtained from Equation (1) represent the combined effects of temperature and strain rate on deformation behavior. The change of Z values usually refer to microstructure evolution and the transformation of deformation mechanisms ,37. Figu2 particles , the deformed grains are divided into several subgrains due to DRV, but the subgrains grow larger b,c and tm obtained from −1 is close to the creeping condition, and the strength of grain boundaries will be decreased at elevated temperature [At low Z value (lnZ = 16.6), the flow stress exhibits low values and the strain rate sensitivity perature ,44. Undeperature . As incr2–Al interface. Actually, the local high strain gradient is often accommodated by geometrically necessary dislocations [2 particles and dislocations during hot deformation. At high Z parameters, the TiB2 particles hinder the motion of dislocations. Thus, localized deformation zones with high stored energy and large misorientation can form around TiB2 particles, providing a large driving force and sites for PSN. At moderate Z values, the activation energy is enhanced at high temperatures and the rate of dislocation motion is small at low strain rates. The recrystallization can be triggered when the dislocation density around partial TiB2 particles reaches the critical level during gradual deformations. However, the stress concentration around partial TiB2 particles can be released by DRV, which hinders the initiation of PSN [It is well known that ceramic particles hardly deform during the plastic deformation of PRAMCs, and thus a relatively high strain gradient exists near the TiBocations . Figure n of PSN . In the n of PSN .From the discussion above, the softening mechanisms of different Z parameters are shown in As shown in the processing map , the PM At high Z parameters, the materials are inclined to exhibit flow localization and deformation instability because the amount of heat energy generated during deformation cannot be dissipated at high strain rates. For the PRAMCs prepared by conventional casting technology, cracks are easily triggered in agglomerated particles to dissipate heat during deformation because of the low bonding strength . NonethemT). Thus, the metallic materials with large initial grains (>10 μm) usually undergo DRX rather than GBS. The alloys with fine initial grains tend to coalesce during high-temperature deformations due to the enhancement of the atoms’ diffusion, causing the GBS severe difficulties geometrically. However, the PRAMCs with fine initial grain may exhibit superplastic deformation at low Z parameters. Wu et al. investigated the isothermal compress deformation of SiC/7075Al composites prepared by spray deposition and noted that the superplastic deformation occurred at 450 °C and the strain rate range of 0.001–0.1 s−1. At that deformation condition, the strain rate sensitivity was about 0.72 [2 particles on grain growth. At low Z parameters, this stable microstructure leads to the occurrence of superplastic flow, which contributes to the excellent workability with the peak η value of 69%. Since the stress in superplastic deformation is lower than the preload of Gleeble-3180 equipment, the superplastic deformation is difficult to test at higher temperatures and lower strain rates. In the following research, we will study the superplasticity of PM composites in detail.Generally, the superplastic deformation is related to the mechanism of GBS. It will occur when the grain size is less than 10 μm and the deformation temperature is higher than half of melting point  The constitutive equation based Arrhenius model is established as η is up to 69%.(2) The processing map of the PM composites based on DMM model is established at the true strain of 0.8. The PM composites exhibit excellent workability with wide safety-processing windows and the peak (3) The Z parameters exert a significant influence on the microstructure evolution and deformation mechanism. At high Z value (lnZ = 30.0), the main softening mechanism is DDRX caused by PSN. At moderate Z values , the effects of PSN on recrystallization become weak and the dominant softening mechanisms transform into DRV and CDRX. At a low Z value (lnZ = 16.6), GBS occurs to promote the superplastic deformation.2 particles promote the effects of PSN on recrystallization and inhibit the grain growth, and the initial fine grains are beneficial to the DRX and superplastic deformation.(4) The uniform particle distribution and fine grain structures contribute to the good workability of the PM composites. The dispersed TiB


However, there is no research to indicate that 12-deacetyl-12-epi-scalaradial exhibited anticancer effect on cervical cancer HeLa cells. The aim of this study was to investigate the anticancer activity of 12-deacetyl-12-epi-scalaradial against HeLa cells and to explore the mechanism. The results from a methylthiazolyldiphenyl-tetrazolium (MTT) assay suggested that 12-deacetyl-12-epi-scalaradial suppressed the proliferation of HeLa cells and flow cytometry analysis showed 12-deacetyl-12-epi-scalaradial could induce the apoptosis of HeLa cells in dose- and time-dependent manner. Western blotting analysis demonstrated that 12-deacetyl-12-epi-scalaradial triggered apoptosis via mediating the extrinsic pathway and was found to suppress MAPK/ERK pathway which was associate with cancer cell death. Nur77, a critical number of orphan nuclear receptors, plays diverse roles in tumor development as a transcription factor and has been considered as a promising anticancer drug target. The dual-luciferase reporter assays suggested that 12-deacetyl-12-epi-scalaradial could selectively enhance the trans-activation activity of Nur77. Furthermore, Western blotting analysis and fluorescence quenching showed that 12-deacetyl-12-epi-scalaradial could induce the phosphorylation of Nur77 and interact with the ligand-binding domain (LBD) of Nur77. Our research confirmed 12-deacetyl-12-epi-scalaradial as a potential agent for cervical cancer therapy and provided a view that 12-deacetyl-12-epi-scalaradial may be a modulator of Nur77.12-Deacetyl-12- Cancer, a hyper-proliferative disorder, ranks as the primary cause of mortality worldwide. Cervical cancer, with an increasing annual incidence and mortality rate, is one of the most frequently diagnosed cancers among women globally . Even thDothiorella sp., was the first natural agonist of Nur77and induced apoptosis via modulating the transactivation of Nur77 [Nur77 , an orphan member of the nuclear receptor superfamily, participates in numerous cellular process such as growth, survival, and apoptosis. The biological effects of Nur77 are regulated by many factors, including its modulators [of Nur77 . In addiof Nur77 ,7,8,9,10epi-scalaradial, a scalarane sesterterpenoid, has been reported to show growth inhibitory activity against hepatocellular carcinoma, breast adenocarcinoma cells, and colorectal carcinoma [epi-scalaradial exhibited cytotoxicity against cervical cancer cells were still unreported so far. Therefore, in this work, we evaluated the antitumor effect and mechanism of 12-deacetyl-12-epi-scalaradial in HeLa cells, and further examined whether 12-deacetyl-12-epi-scalaradial modulated the transactivation activity and phosphorylation of Nur77. Further, we investigated the interaction between 12-deacetyl-12-epi-scalaradial and the ligand-binding domain (LBD) of Nur77.Marine natural products represent an extraordinarily rich source of chemical and biological diversity with unique chemical skeletons and outstanding functionalities ,12. A laarcinoma . Howeverepi-scalaradial was isolated from marine sponge Hippospongia sp and its chemical structure was identified by a comparison with the literature [epi-scalaradial showed cytotoxic effects against human cervical cancer cells. As shown epi-scalaradial. Meanwhile, 12-deacetyl-12-epi-scalaradial exhibited significant inhibitory effects on HeLa cells under the concentration of 30 μM. Moreover, the IC50 value of 12-deacetyl-12-epi-scalaradial against HeLa cells was 13.74 μM, which is lower than the IC50 values of other cell lines in the reports with 36 μM for MCF-7, 23.4 μM for HepG2, and 27.1 μM for HCT-1116 [epi-scalaradial possessed cytotoxic activities against HeLa cells, while HeLa cells seem to be more sensitive to 12-deacetyl-12-epi-scalaradial.Briefly, 12-deacetyl-12-terature  , and apoptosis was measured by flow cytometry. Indeed, 12-deacetyl-12-epi-scalaradial displayed a robust inducement of apoptotic activity in HeLa cells. epi-scalaradial in the range of 0 to 30 μM  pathway plays crucial roles in various cellular functions, including growth, differentiation, and metastasis. MAPKs consists of three subfamilies including the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. The MAPK signaling pathway was closely involved in tumor cell apoptosis ,28. PI3Kepi-scalaradial on trans-activation of Nur77. As shown in epi-scalaradial. Our previous studies as well used dual-luciferase reporter gene assay to identify small modulators of Retinoid X Receptor-alpha (RXRα) [epi-scalaradial selectively affected trans-activation activity of Nur77, the effect of compound on trans-activation activity of RXRα was measured. As a result, 12-deacetyl-12-epi-scalaradial has no influence on the trans-activation activity of RXRα with the same concentration  ,37,38,39ntration B. Furtheepi-scalaradial was measured by fluorescence quenching technology. As shown in epi-scalaradial treatment. The above results suggested that 12-deacetyl-12-epi-scalaradial could modulate trans-activation activity and phosphorylation of Nur77, and interact with Nur77-LBD.The phenyl alanine, tyrosine, and tryptophan in proteins are the donors of the fluorescence and the interaction between ligands and proteins will lead to the quenching of fluorescence. Thus, fluorescence quenching technology has been an effective way to study the binding of ligand to the target protein ,41,42 anHyatella intestinalis, showed inhibitory activity against human colon cancer HT-29 cells [Hyrtios sp., could induce the apoptosis of cancer cells via modulating the NF-κB and MAPKs signal pathways [Cervical cancer is a growing worldwide health problem for women. There are several therapies for its treatment, such as surgery, radiotherapy and chemotherapy, but side effects remain a rigorous problem ,2. There29 cells . Another29 cells . Heteronpathways ,45. Evenepi-scalaradial is a scalarane sesterterpenoid isolated from a marine sponge Hippspongia sp, which has been reported to inhibit the proliferation of several cancer cell lines containing HepG2, MCF-7 and HCT-116 [epi-scalaradial towards cervical cancer HeLa cells and its possible mechanism of antitumor activity remain largely unknown. Our results confirmed 12-deacetyl-12-epi-scalaradial inhibited the proliferation of HeLa cells  was purchased from Aladdin, China. Annexin V-FITC Apoptosis Detection Kits was purchased from Vazyme, China. Caspase-Glo assay kit was purchased from Promega, USA.epi-scalaradial was isolated from Hippospongia sp. The fresh sponge (1.2 kg wet weight) was extracted with EtOAc. The crude extract fraction (15.3 g) was separated on silica gel and eluted with using the mixtures of n-hexane/ EtOAc to obtain fractions 1-13. Purification from fraction 9 (5.4551 g) through repeated preparative TLC with n-Hexane–Acetone (10:1) afforded 12-deacetyl-12-epi-scalaradial. Briefly, 12-deacetyl-12-2 at 37 °C.HeLa cells were cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% FBS in a humidified atmosphere containing 5% CO3 cells per well and allowed to settle 12 h. Then, the cells were treated with varied concentrations of 12-deacetyl-12-epi-scalaradial. After 48 h, 15 μL of MTT reagent and 60 μL MEM were added and incubated for 4 h. After removing the supernatant, the transformed crystals were dissolved in DMSO (100 μL) and measured at 490 nm using microplate reader . The cell proliferation-inhibition rate (%) was calculated as follows:Anti-proliferative activity of compound was tested by MTT assay. HeLa cells were seeded into 96-well-plate at a density of 5 × 105 cells per well for 24 h. Cells were digested by trypsin with no EDTA and collected by centrifugation and resuspended in 100 μL of Binding buffer (1×) after 12-deacetyl-12-epi-scalaradial treatment. Finally, HeLa cells were stained with Annexin-V/FITC and PI for 10 min at room temperature in the dark. The stained cells were diluted with 1× binding buffer and analyzed by flow cytometry. Data were analyzed with FlowJo 10.The apoptotic rate of HeLa cells was assessed by flow cytometry using the Annexin-V/FITC Apoptosis Detection Kit according to manufacturer’s protocol. Cells were seeded into 6-well plates at a density of 1 × 10epi-scalaradial, HeLa cells were lysed in RIPA buffer. Equal amounts of protein lysates were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% no-fat milk in TBST for 1 h, then incubated with primary antibodies at 4 °C overnight. After washing three times with TBST, the membranes were detected with secondary antibodies for 1 h under room temperature. The final immunoreactive products were detected by using an enhanced chemiluminescence system and examined by densitometric analysis.After treated with 12-deacetyl-12-epi-scalaradial (30 μM). After 12 h, 100 µl Caspase-Glo Reagent were added to each well 12 h later. After 1 h incubation, the cells were measure the luminescence of each sample in a plate-reading luminometer as directed by the luminometer manufacturer.Caspase-3, -8, and -9 activities were measured using a Caspase-Glo assay kit (Promega) according to manufacturer’s protocol. HeLa cells were seeded in 96 well-culture dish and treated with 12-deacetyl-12-4 cells per well in 96-well plates. Three plasmids, namely pGL5-NURE luciferase reporter vector (15 ng/mL), myc-Nur77 vector (15 ng/mL), and pC DNA-Renila (15 ng/mL), were co-transfected by Liposome 2000. As to RXRα transcriptional activities, there are two plasmids, pGL5 luciferase reporter vector (30 ng/mL) and pGAL4-RXRα-LBD vector (15 ng/mL). After 12 hours, cells were incubated with the compounds at different concentrations for 12 h. The activities of firefly luciferase and renilla luciferase were measured using the Dual-Luciferase Assay System Kit (Promega). The 293T cells (293T) were cultured at 37 °C in DMEM with 10% FBS for 24 h and then seeded at a density of 1 × 10600 = 0.6–0.8 and then protein expression was induced by 1 mM IPTG. After sustained at 25 °C for 16 h, cells were harvested and sonicated. The target protein was purified using Ni2+-NTA agarose column at low temperatures. The ligand-binding domain (LBD) of human Nur77 (genes 367–598) was cloned as N-terminal histidine-tagged fusion protein in pET15b vector and transformed to E. coli BL21(DE3). Cells were grown at 37 °C in LB medium until ODThe fluorescence experiment was performed using a Cary Eclipse Fluorescence Spectrophotometer (Varian). Fluorescence spectra (300–500 nm) were conducted via excitation wavelength of 289 nm and slit widths to 10/10 nm at 289T.p < 0.05. The values are presented as mean ± SD and were analyzed statistically with student’s t-tests for simple comparisons between groups, one-way analyses of variance (ANOVA) for more than two groups, and Tukey’s multiple comparisons test using GraphPad Prism 6.0. Differences were considered statistically significant at


Caenorhabditis elegans and other nematodes as important experimental systems for exploring the evolution of ZP modules and their constituent domains.Zona pellucida (ZP) modules mediate extracellular protein–protein interactions and contribute to important biological processes including syngamy and cellular morphogenesis. Although some biomedically relevant ZP modules are well studied, little is known about the protein family’s broad-scale diversity and evolution. The increasing availability of sequenced genomes from “nonmodel” systems provides a valuable opportunity to address this issue and to use comparative approaches to gain new insights into ZP module biology. Here, through phylogenetic and structural exploration of ZP module diversity across the nematode phylum, I report evidence that speaks to two important aspects of ZP module biology. First, I show that ZP-C domains—which in some modules act as regulators of ZP-N domain-mediated polymerization activity, and which have never before been found in isolation—can indeed be found as standalone domains. These standalone ZP-C domain proteins originated in independent  lineages prior to the diversification of extant nematodes, after which they evolved under strong stabilizing selection, suggesting the presence of ZP-N domain-independent functionality. Second, I provide a much-needed phylogenetic perspective on disulfide bond variability, uncovering evidence for both convergent evolution and disulfide-bond reshuffling. This result has implications for our evolutionary understanding and classification of ZP module structural diversity and highlights the usefulness of phylogenetics and diverse sampling for protein structural biology. All told, these findings set the stage for broad-scale (cross-phyla) evolutionary analysis of ZP modules and position  Secreted proteins help cells withstand, react to, and shape external conditions . The extThe zona pellucida (ZP) module is a key component of many secreted proteins . Named ain vitro  and for the replicated loss of ZP-N domains in independent lineages . By taking a comparative, evolutionary approach, this work provides new insights into ZP module biology that should benefit efforts to determine ZP module structure–function relationships, in particular the functional role of standalone ZP-C domains. More broadly, this work provides a foundation for future phylogenetic studies aimed at providing an evolutionary classification of ZP modules and domains across the animal kingdom.Through phylogenetic analysis of 1,783 ZP modules from 59 nematode species, I found that the diversity of ZP modules present in C. elegans ZPD protein sequences and used these to search for homologs in other nematodes. WormBase.org version 259 . When multiple isoforms were available, I selected a single variant, choosing whichever introduced the fewest/shortest indels in preliminary alignments of C. elegans ZP modules. This approach resulted in a data set of 43 C. elegans ZPD proteins  Aside from the already-discounted ZP-N-only protein CUTL-21, these searches did not uncover any additional ZPD proteins (results not shown).I compiled a data set of sion 259  lists 45N domain , whereasproteins  online. C. elegans ZP modules using GISMO (ver 2.0), an alignment program that uses a Bayesian approach to extract and align the homologous core regions of sequences that potentially contain nonhomologous flanks and insertions (n = 5); the positions and lengths of inferred insertions and flanking regions varied among replicates, but all targeted the ZP module, retaining the C-terminal consensus cleavage site (CCS) and excluding upstream domains and the N-terminal trafficking motif. The 43 flank-trimmed (but not insertion-trimmed) sequences from the GISMO run with the longest conserved core .ZPD proteins often include other domains upstream of the ZP module; I isolated sertions . Becauseweblogo.berkeley.edu, last accessed May 23, 2020; The final data set of 1,783 full-length ZPD protein sequences  online wwww.atgc-montpellier.fr/phyml/  web server, with automated SMS-AIC model selection, a BioNJ starting tree, and SPR topology rearrangements  phylogenies were estimated using PhyML via the ngements . Model pee 1.6.0 , using, ee 1.6.0 ; this apee 1.6.0 .Patterns of sequence loss were explored by calculating the amount of missing data within each replicate alignment and mapping these values onto the phylogeny. Gap proportions were estimated separately for the ZP-N and ZP-C domains, with the approximate domain boundaries determined according to cysteine conservation patterns: Using the nomenclature of C. elegans members). In each case, untrimmed protein sequences were realigned using GISMO and the alignment with the top LLR score (out of n = 10 replicates) was used to estimate a subfamily-specific phylogeny  and build a corresponding codon sequence alignment.  The codon alignments and trees were used to fit codon substitution models via CodeML from the PAML 4.9a package  to synonymous (dS) divergence ratio (=dN/dS), with values near 0 indicating strong purifying selection and values >1 suggestive of positive selection. I fit three codon models: M8, M8a, and M0. M8 and M8a are nested models that were used to test for site-specific positive selection (ω > 1) and to estimate among-site variation in the strength of selective constraint .Homology models were estimated for www.cbs.dtu.dk/services/ProP/, last accessed May 23, 2020; gpcr.biocomp.unibo.it/predgpi/pred.htm, last accessed May 23, 2020; www.ebi.ac.uk/Tools/pfa/pfamscan/, last accessed May 23, 2020; C-terminal R/K cleavage sites and N-terminal signaling motifs were predicted for untrimmed sequences via the ProP 1.0 Server  than Clade III/IV/V species .A data set of 1,783 nematode ZP modules was assembled by BlastP searching the whole-genome predicted protein sets of 58 nematode species for homologs of 43 C. elegans CUT-1 is shown in ZP modules were extracted and aligned using GISMO. Alignment uncertainty is a concern given the short target region and the phylogenetic breadth of the data set. I addressed this by leveraging the stochastic nature of the GISMO alignment procedure, repeating key phylogenetic analyses across 100 replicate alignments. Consistent with expectations for ZP modules, the final GISMO-trimmed alignments were 233–269 aa long, with majority-rule consensus sequences possessing 11–13 cysteines. The percentage of gaps and ambiguous data ranged from 6.7% to 8.6% across alignments. Conservation patterns for the focal alignment  are shown in Evolutionary trees were estimated for the 100 replicate alignments using ML. Alignment variation affected both model selection and the resulting topology. With regard to the substitution model, VT + I + G was favored for 77 alignments, LG + I + G for one, and WAG + I + G+F for the remainder, with the top model receiving an AIC weight of 1.000 in 98/100 cases  online. r = 0.94–0.98).Rather than focusing on the individual ML trees, I constructed a majority-rule consensus tree a, sacrifC. elegans ZP modules into groups based on the number of ZP-C domain cysteine residues present per sequence, and their classification system is broadly congruent with the one provided here  and did not address which cysteine connectivity pattern is ancestral.Visual inspection of the nematode ZP module phylogeny revealed three major groups, which I refer to as Type 1, 2, and 3 ZP modules a. These ded here  online. C. elegans ZP modules were, with few exceptions, distributed broadly across the phylogeny, and similar patterns were seen for the other species  and the C. elegans CUT-1 and CUT-3 modules clearly derive from a recent duplication event. Beyond C. elegans, losses appeared particularly common for Clade I nematode ZP modules (cutl-28 and dyf-7 genes of Clade I (Trichinella) species were fused, resulting in only the DYF-7 sequences ending up in the final data set (results not shown). Putting these few departures aside, the overall pattern is consistent with deep conservation of the ZP module complement across the nematode phylum. Assuming that gene duplication is the primary driver of functional divergence within the ZP module family, these results therefore support efforts to leverage knowledge about cuticular biology in the lab model C. elegans for use in treating or preventing parasitic nematode infections.Although the tree is largely indicative of stable orthology, occasional lineage-specific gains and losses were also observed.  modules  online.  modules  but data modules  and I foC. elegans ZPD proteins, namely the ZP “domain” , two types of PAN domain , two types of epidermal growth factor (EGF)-like domain , and the von Willebrand factor Type A (vWFA) domain . The remaining 9% matched 96 different Pfam entries, with none individually accounting for more than 0.7% of the total; these additional domain predictions were not considered further as nearly half derived from Clade I nematodes  of the input sequences  online. s member  online. s member  online aC. elegans members—showed pronounced signatures of ZP-N domain sequence loss (C. elegans T01D1.8 and F46G11.6 (no results were reported for CUTL-19) but did not explore the issue further.To test for deletions indicative of major structural alterations of the ZP module, I calculated the proportion of missing data for each aligned sequence and mapped these “gap proportions” onto the phylogeny. This was done separately for the ZP-N and ZP-C domains. Three subfamilies—CUTL-19, T01D1.8, and F46G11.6, named for their respective nce loss a. Averagnce loss b: AveragThe phylogenetic distribution of the standalone ZP-C subfamilies indicates that ZP-N loss occurred prior to the emergence of the major nematode lineages, and that it happened at least twice a. The T0N/dS under the M0 codon model was ω = 0.094 for T01D1.8, 0.095 for F46G11.6, and 0.135 for CUTL-19, indicating the action of moderately strong purifying selection acting throughout the history of these subfamilies. Selective constraint was generally strongest within the core regions of the ZP-C domain, especially at sites within predicted β strands (ω > 1) in any of the subfamilies , suggesting that these unusual proteins are still secreted despite the loss of their respective ZP-N domains. However, the three standalone ZP-C subfamilies differed from the norm by generally lacking predicted R/K cleavage sites . Examination of the subfamily-specific alignments and HP motif c–e. FinaC. elegans ZPD proteins using RaptorX , chicken ZP3 (3nk3), human endoglin (5hzv), mouse ZP2 ZP-C domain (5bup), and rat betaglycan ZP-C domain (3qw9). I focused only on homology models generated using the human uromodulin template  to the beginning of the FG loop (position 210), was recovered in 15 of 16 Type 2 modules (and deemed plausible by proximity in the remaining case) . These findings suggest new dimensions of functionality for ZP-C domains. One possibility is that these standalone ZP-C domains perform a regulatory role, but as free-agent regulators of unlinked ZP-N domains rather than of physically linked upstream domains; such proteins might prove useful for remodeling ZPD protein-based extracellular matrices. Another is that ZP-C domains are multifunctional, having some uncharacterized nonregulatory function. Some ZP-C domains have been shown to contribute to protein–protein binding , and it C. elegans genome, less than half have been functionally characterized. Aside from DYF-7, which plays a role in neural dendrite elongation  that are presumably shared across millions of distantly related nematode species. Given the phylogenetic depth of the branch where this reshuffling event is presumed to have occurred, close inspection of ZPD proteins in other invertebrate phyla might plausibly uncover orthologs that share this connectivity pattern. In light of its ancient origin and subsequent conservation across multiple subfamilies, it seems safe to conclude that stabilizing selection has acted to maintain the modified disulfide bond over time. However, it is not obvious whether the modified disulfide’s initial origin was adaptive, and whether its evolution resulted in some novel function. For example, the evolution of an extra cysteine residue in the vicinity of CysB could have rendered CysA redundant, allowing for its exchange by drift. Another possibility is that the novel CysB-partner evolved to compensate for the loss of CysA; here, the novel disulfide would be adaptive only in the sense that it corrected some transient maladaptation, with no net change in overall function. Regardless, this finding speaks to the challenges of categorizing proteins using sequence conservation patterns without a robust phylogenetic framework, and to the importance of utilizing new data to update expectations about protein biology.By combining phylogenetic and structural analyses, I found that disulfide variation among nematode ZP-C domains indeed reflects more than just gains and losses: The CysA–CysB disulfide was modified in Type 3 ZP-C domains, with CysA lost and replaced by a novel binding partner in the adjacent βC strand. The CysB-βC disulfide therefore represents a rare case of disulfide-bond reshuffling . ImportaIn contrast, there are several reasons to suspect that the entirely novel disulfide inferred between the βA and βB strands of Type 2 and 3 ZP-C domains is adaptive. First, it occurs in a region of known functional importance: the βA-IHP. Stabilizing the IHP through a disulfide bond could help maintain the tertiary structure of the ZP-C domain upon protein maturation and activation, during which the cleaved C-terminal tail’s βG-EHP dissociates from the IHP . Second,C. elegans ZPD proteins, I uncovered evidence for 1) the parallel loss of the ZP-N domain in at least two lineages, resulting in the unexpected discovery of standalone ZP-C domains; 2) the modification of a highly conserved ZP-C domain disulfide via a rare example of cysteine replacement; and 3) the convergent gain of stabilizing disulfide bonds in the ZP-C domain’s regulatory IHP motif. As a purely in silico study, it is of course critical that the unusual structural features documented here be confirmed experimentally. Even still, these findings have important implications for our understanding of ZP module structure and function. Moreover, the present study presents a valuable phylogenetic framework for the developmental genetic study of ZPD proteins in nematodes, including the powerful lab model C. elegans. Finally, this work sets the stage for future investigation of ZPD protein diversity in the broad sense. Here, the obvious next step will be to bridge the phylogenetic gap between nematodes, other invertebrates and, ultimately, vertebrates.The present study serves as the largest comparative investigation of ZP module evolutionary diversity conducted to date. By combining the newly estimated nematode ZP module phylogeny with homology modeling of evaa095_Supplementary_DataClick here for additional data file.


Self-raising flours were significantly higher in sodium (p < 0.001) and gluten-free products were lower in protein and dietary fibre, making legume, buckwheat and quinoa flour a better choice. Sustainability principles in fruit and vegetable production and novel insect products have driven new product development. There is a clear opportunity for further on-pack promotion of whole grain and dietary fibre within the category via food product labelling.Flour, typically derived from wheat, rye, corn and rice is a pantry staple, providing structure to bread and baked goods. This study aimed to provide a cross-sectional analysis of flour for home baking, highlighting the nutrition composition of whole grain flour and identifying novel categories. An audit was undertaken in February 2020, in four major supermarkets in metropolitan Sydney . Ingredient lists, Nutrition Information Panel, claims, and country of origin were collected. The median and range were calculated for energy, protein, fat, saturated fat, carbohydrate, sugars, dietary fibre and sodium. Overall, 130 products were collected, including 26 plain flour, 12 self-raising, 17 plain wholemeal, 4 wholemeal self-raising, 20 bread-making mixes (4 were whole grain), 20 other refined grain (including corn and rice flour), 17 gluten-free, 3 legume, 4 fruit/vegetable, 4 coconut and 3 other non-grain  products. Plain wheat flour dominated the category, while whole grain  made up 19% of products, yet they contained significantly more dietary fibre ( According to Food Standards Australia New Zealand (FSANZ), flours are defined as ‘products of grinding or milling of cereals, legumes or other seeds’ . Flour lFlour is a universal pantry staple, supplied as part of rations in the early colony of Australia, with as little as 2 1/2 pounds 1.3 kg) supplied in the ‘hungry years’ (1788–1792), and up to ten pounds 4.5 kg) in the 1830s  with the sources of our food’ and sustainable practices within the food supply system , for exaLess than 20% of the flour products were considered whole grain. In diets, bread and breakfast cereal are known to be the main two food categories contributing whole grain . HoweverUnlike other nutrients, whole grain claims are not regulated by FSANZ in Australia and New Zealand, rather, they fall under a voluntary Code of Practice for Whole Grain Ingredient Content Claims (The Code), introduced in 2013 by GLNC to encourage evidence-based promotion of whole grain foods. GLNC utilises audits of grain-based foods to monitor the operation of The Code and provide feedback to industry . While 2With the sodium content of diets as the leading issue in relation to the Global Burden of Disease , the sigThere are a range of alternative flours launched locally and globally, made from legumes, ancient grains, indigenous wheat and insects featuring extra protein and dietary fibre, appealing to the ‘natural’ consumer . Many ofCoconut flour, derived from the dried, ground flesh, absorbs a great deal of liquid in baking, and recipes would need to be adjusted. While the product is very high in dietary fibre, this may be the only nutritional attribute worth promoting due to the higher fat, saturated fat and sugars. Blended flour products previously on the market containing a combination of wheat, millet, oat and coconut flours were no longer available on shelf during this audit. This perhaps points to problems in exchanging wheat flour for the blended products containing coconut, as they suggested, ‘cup-for-cup’ exchange on the packaging, and this may not have been precise enough for home bakers. Coconut flour currently on the market suggests on the packaging that 1C of wheat flour can be replaced by 1/3C of coconut flour and extra liquid or egg may be needed to compensate, and in addition to this, the overall volume of the mix would be affected.Among the novel flour products, broccoli, sweet potato and hemp flour were also very high in dietary fibre, even higher than legume flour. Legume flour has been investigated for suitability in baking ,35, and Brassica, it is rich in vitamins  and minerals , and a range of other protective compounds. A disadvantage of this crop is that as much as 70% is commonly discarded in the field [The products using ‘powder’ as a descriptor included broccoli, sweet potato, cricket and hemp products. Broccoli powders have recognised potential as a natural food supplement. Belonging to the family Cruciferae and the genus he field , and as he field . These qhe field . In the he field , and on-Bananas are the second most popular global crop, after citrus, although large portions of a crop may be rejected if they are immature, cracked or irregular ,40. GreeDespite its name, tiger nut flour is derived from a starchy root rather than a nut, although it was not as high in carbohydrate as the green banana flour. Tiger nut flour was high in fat and saturated fat, and this gluten-free flour alternative was promoted as great for baking sweet or savoury treats, with twice as much dietary fibre as wholemeal flour. In the scientific literature, it has been tested in biscuits , noodlesCannabis sativa were approved as a food by FSANZ in April 2017 and both the seeds and the oil are used in Europe, Canada and the USA [New Nutrition Business has identified plant protein as a key driver of food choice in recent years and this trend is apparent in this food category . Hemp se the USA . Contain the USA ,50. In a the USA ,52. Like the USA , buns [5 the USA , muffins the USA  and brea the USA , with gen = 31). As previously stated, reporting of dietary fibre and whole grain within the ingredients and in the Nutrition Information Panel is not mandatory in the absence of an on-pack claim, so this information was not always declared, and thus there was some missing data. Finally, we did not conduct an independent nutrition analysis of products and were reliant on manufacturer information to draw comparisons between product categories. Strengths of this study include its comprehensive nature and, to our knowledge, it is the first study that has compared the range of flour products and novel entrants to the market. While all efforts were made to capture the category in its entirety, differences may exist between geographic areas. In addition to supermarkets, there are a range of bulk food stores selling alternative grain flours including teff, sorghum and millet. However, these are not often contained within packaging and were excluded from this analysis . Although less than 20% of products in this analysis were whole grain, a simple swap to wholemeal flour in suitable recipes would more than double dietary fibre in home-baked products. The packaging claims on novel products are compelling, and for mainstream grain products, improved on-pack information, clearly articulating the benefits of dietary fibre, including descriptions of how to exchange white for whole grain, could attract more consumers. The added directions for consumers may in turn help flour manufacturers and health professionals alike encourage the use of whole grain and higher-fibre grain-based flour products in baking.


A domain wall‐enabled memristor is created, in thin film lithium niobate capacitors, which shows up to twelve orders of magnitude variation in resistance. Such dramatic changes are caused by the injection of strongly inclined conducting ferroelectric domain walls, which provide conduits for current flow between electrodes. Varying the magnitude of the applied electric‐field pulse, used to induce switching, alters the extent to which polarization reversal occurs; this systematically changes the density of the injected conducting domain walls in the ferroelectric layer and hence the resistivity of the capacitor structure as a whole. Hundreds of distinct conductance states can be produced, with current maxima achieved around the coercive voltage, where domain wall density is greatest, and minima associated with the almost fully switched ferroelectric . Significantly, this “domain wall memristor” demonstrates a plasticity effect: when a succession of voltage pulses of constant magnitude is applied, the resistance changes. Resistance plasticity opens the way for the domain wall memristor to be considered for artificial synapse applications in neuromorphic circuits. By changing the density of conducting ferroelectric domain walls that straddle the interelectrode gap, it is shown that a large number of different direct current resistance states can be created, in parallel‐plate thin film lithium niobate capacitors. Surprisingly, such microstructural manipulation can result in colossal changes in device resistance (over twelve orders of magnitude). Early‐stage devices have recently emerged: for example, studies on bismuth ferrite have revealed domain‐wall enabled memory, where binary states have been switched by AFM contact to an electrode; other research, on lead zirconate titanate, has demonstrated transient conducting domain wall conduits, providing a volatile (non‐remnant) resistance change in a transistor‐like device. In both of these studies, only two or three resistance states have been accessed, based on a single pair of conducting domain walls straddling an inter‐electrode gap. Conceptually, however, the number of distinct resistance states in such devices could be almost without limit: as long as different populations of conducting domain walls are injected in a controllable and repeatable manner, then there should be no problem in developing domain‐wall‐enabled multi‐resistance devices, or domain wall memristors . A proof‐of‐concept demonstration has already been performed in BiFeO3, by Siedel et al., where different numbers of conducting domain walls were manually written in pairs between electrodes, using an atomic force microscope (AFM). While illustrating the viability of the concept, such experiments are clearly a long way from practical devices.Domain walls in ferroelectrics and multiferroics have functional properties that are often distinct from those of the domains that they surround.5, 6° domain walls, the density of which can be controlled by the extent to which ferroelectric switching has progressed. Since this is, itself, a function of the electric field (the polarization‐field hysteresis loop is a defining characteristic of a ferroelectric), a large range of resistance states can be accessed by applying voltage pulses of different magnitudes: indeed, we explicitly demonstrate that at least 100 stable conductance states can be induced within a two order of magnitude window in conductance, using writing pulses of fixed length and variable amplitude. Conductance of the device can also be progressively changed by applying multiple successive voltage pulses of the same magnitude: a phenomenon known as “plasticity” which can, in principle, be utilized in artificial synapse development. While the details of the conduction mechanisms involved are radically different, the domain wall memristors that we demonstrate herein are reminiscent of those based on asymmetric ferroelectric tunnel junctions: in both cases different conductivity states are made by controllably changing the relative volume fraction of differently oriented domains.In this letter, we report a major step forward in harnessing conducting domain walls as the active components in new forms of memristors. We demonstrate devices, based on simple parallel‐plate capacitor structures, in which thin film ion‐sliced single crystal congruent lithium niobate acts as the dielectric (ferroelectric) layer. Partial reversal of the polarization in the ferroelectric causes the steady‐state resistance of the capacitor to change by up to twelve orders of magnitude. This change is caused by the injection of strongly inclined conducting 180unctions:28 in 23) is an archetypal trigonal uniaxial ferroelectric, used extensively for applications in non‐linear optics and in surface acoustic wave devices. It has a high Curie temperature (between 1141 and 1210 °C) and a commensurately large room temperature spontaneous polarisation (≈70 µC cm−2) that varies significantly with stoichiometry. Under ambient conditions, the bulk single crystal is considered to be a perfect insulator and this makes the contrast in transport behavior between domains and charged conducting domain walls particularly stark. LiNbO3 is usually non‐stoichiometric, as single crystal growth from the congruent melt leads to lithium deficiency and subtle associated defect chemistry. Even in bulk, the coercive field needed for ferroelectric switching can be as high as 220 kV cm−1 and imprint, due to defect‐related internal fields, is common. In thin films, coercive fields increase and imprint effects are often exaggerated: Volk et al. found coercive fields in 300 nm thick ion‐sliced layers to be ≈670kV cm−1 (around three times that of bulk), while Jiang et al. found such strong imprint that only one remnant polarization state could be stabilized. For the research described herein, commercially obtained thin film material was used. The as‐received structure consisted of a 500 nm thick single crystal lithium niobate layer (ion‐sliced from a congruent boule) bonded to a chromium–gold–chromium thin film electrode (150 nm thick) above a silica adhesion layer (2000 nm), on top of a bulk single crystal lithium niobate wafer. The ion‐sliced layer was such that the polarization was oriented perpendicular to the film surface (z‐cut).Lithium niobate (LiNbOhiometry.30 Und for example), but is now a well‐established phenomenon: pioneering work by Lukas Eng and coworkers initially observed that domain wall conduction could only be induced by photoexcitation, but they later found that, even in the absence of light, walls that were inclined with respect to the polarization axis would conduct. Inclined walls are necessarily charged, as they create discontinuities in polarization, seen in other ferroelectric systems to be critical for generating or suppressing domain wall currents. In lithium niobate, head‐to‐head polar discontinuities cause enhanced domain wall conduction, but the inclination angles seen vary widely: in some studies, complete “inversion” domain walls can be created, with effectively 90° inclination angles between the domain wall and the polarization vector. However, “inversion” layers require exotic processing routes, such as extreme heat treatments , partial melting, or ion diffusion, sometimes in the presence of molten salts. While worth noting their existence, it is clear that the conditions needed to make them are incompatible with standard electronic device operation. When device compatible electric‐field switching processes are used, the maximum inclination angles reported to date have been modest and of the order of 1–3°; nevertheless, this still allows significant domain wall conduction to occur.Enhanced conductivity along 180° domain walls in lithium niobate had been suspected for some time . This is commensurate with reports in established literature. Surprisingly, though, when nanodomains were written, using a stationary AFM tip, the measured domain wall currents . This result is very surprising, given that previously considered “high” inclination angles had a mean value of ≈1° but it is also exciting, as it suggests that the conductivity of the domain walls in these ion‐sliced systems can be particularly high.As a general observation, 180° domain walls, written into the as‐received monodomain LiNbOts Figure1a–c wer3.23 film. The simulation starts with a single domain state with upward polarization. A local bias is then applied from a tip at the surface, and the resultant domain switching process is recorded and visualized , allowing switching pulses of up to 100 kV mm−1, which were sufficient to traverse almost the entire polarization‐field hysteresis loop for the lithium niobate films  was monitored. Its form is as might be expected . Images confirm that microstructures which support the largest steady‐state dc currents are highly mixed . Switching fields, above and below the coercive field , still produce mixed microstructures , the dc current as a function of time for a partially switched state is shown in Figure 48 giv3.4Figure3 films . Plasticity in the conductance, as a function of the number of pulses applied to these domain wall memristors, could be relevant in applications such as artificial synapses, so that they could influence aspects of future neuromorphic computing. There are, of course, potential limitations: for example, the domain microstructures are hundreds of nanometers in length scale and this would currently prevent significant electrode miniaturization. However, we expect that much thinner LiNbOThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Hematopoietic adaptor containing SH3 and SAM domains-1 (HACS1) is a signaling protein with two juxtaposed protein–protein interaction domains and an intrinsically unstructured region that spans half the sequence. Here, we describe the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif (ITIM3) of the paired immunoglobulin receptor B (PIRB). From surface plasmon resonance binding assays using a mouse and human PIRB ITIM3 phosphopeptides as ligands, the HACS1 SH3 domain and SHP2 N-terminal SH2 domain demonstrated comparable affinities in the micromolar range. Since the PIRB ITIM3 sequence represents an atypical ligand for an SH3 domain, we determined the NMR structure of the HACS1 SH3 domain and performed a chemical shift mapping study. This study showed that the binding site on the HACS1 SH3 domain for PIRB shares many of the same amino acids found in a canonical binding cleft normally associated with polyproline ligands. Molecular modeling suggests that the respective binding sites in PIRB ITIM3 for the HACS1 SH3 domain and the SHP2 SH2 domain are too close to permit simultaneous binding. As a result, the HACS1-PIRB partnership has the potential to amalgamate signaling pathways that influence both immune and neuronal cell fate. Kwan et al. show the interaction between the HACS1 SH3 domain and a sequence near the third immunoreceptor tyrosine-based inhibition motif of the Paired immunoglobulin receptor B (PIRB). This study suggests that the HACS1-PIRB partnership has the potential to unite signaling pathways that regulate both immune and neuronal cell fate. By virtue of its location on chromosome 21, there is interest in exploring the role of HACS1 in Down’s syndrome5 and late-onset Alzheimer’s disease6. Sequence analysis of human HACS1 and its homolog, HACS2/SLy111, reveal juxtaposed SAM12 and SH313 domains that are well-known mediators of protein–protein interactions. Since over half of HACS1 is predicted to be intrinsically disordered, this adaptor protein has the potential to serve as a platform for many yet to be discovered protein partners and post-translational modifications. This report presents a biochemical and structural study of an interaction between the HACS1 SH3 domain and the cytosolic region of the Paired immunoglobulin receptor B (PIRB) receptor that has not been explored since its original identification by yeast two-hybrid methods.HACS1  is a signaling adaptor protein that was first associated with leukemia. Since that discovery, HACS1 has been recognized further in blood vessel development14. Thus, if either HACS1 or PIRB is inactivated, the immune system becomes hypersensitive. In HACS15 and PIRB knockout mice16, hypersensitivity is manifested by enhanced T-helper cell response and increased cytokine signaling. Since HACS1 and PIRB mutants have similar phenotypes, it supports the idea that their interaction may be required in certain immune signaling pathways.Positive signals arising from antigen receptors are antagonized by HACS1 and PIRB17, platelet activation18, brain repair24, and Alzheimer’s disease25. As a result of this interest, HACS1 represents one relatively under-investigated member of a large repertoire of PIRB/LILRB protein partners that include major histocompatibility complex related proteins26, and oligomeric ß-amyloid25.The murine PIRB receptor and its five human leukocyte immunoglobulin-like receptor (LIRLB) orthologs have received considerable research interest not only to due their participation in immune signaling, but also in infection27. Normally, SHP phosphatases are autoinhibited until they are phosphorylated themselves or bind a partner like PIRB. Once activated, SHP phosphatases inactivate downstream kinases including Syk, Btk, and PLC-γ that act as sentinels for pathways involved in proliferation, survival, and the production of cytokines in the immune response. The discovery of HACS1 as a protein partner binding to PIRB therefore creates a new mode of regulation at or near ITIMs that may either complement or compete with SHP family proteins.The PIRB cytoplasmic domain contains four ITIMs (intracellular tyrosine-based inhibitory motifs) which, upon phosphorylation, are able to recruit SHP family phosphatasesIn this present investigation, we have used a combination of nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, circular dichroism spectroscopy, and surface plasmon resonance methods to identify new structural and biochemical features of the HACS1-PIRB interaction. An amalgamation of these experimental observations with molecular modeling suggests that the PIRB-HACS1 relationship represents an example of an atypical ligand binding an SH3 domain with a correspondingly atypical binding cleft. Molecular modeling is also used to explore the potential for cooperation or competition with SHP family phosphatases.Human and mouse HACS1 are 86% identical over 372 aa. When the SH3 domain is considered separately, the human and mouse sequences are essentially identical, differing only by one conservative substitution of arginine for lysine Figure .15N-T2 relaxation times in portions of the RT-loop were larger than the average of 130 ms and had larger associated errors associated with fits to a monoexponential decay model. Heteronuclear 15N{1H} NOEs for Y179 and T181 were lower than the average value of 0.81. The 15N-T1 relaxation times for the RT-loop, in contrast, were consistent with the average value of 620 ms. Taken together, the 15N relaxation data suggests that the RT-loop is dynamic relative to the rest of the SH3 domain. A rotational correlation time of 5.2 ns was determined from a global fit of the relaxation data and corroborates the conclusions of the multi-angle laser light scattering study that the HACS1 SH3 is monomeric.The NMR structure of the human HACS1 SH3 domain was determined using a combination of NOE distance restraints, torsion angle restraints derived from backbone chemical shifts and amide residual dipolar couplings Table . InclusiThe structure of murine SAMSN1/HACS1 has been solved as part of an NMR-focused structural proteomics program (PDB: 2EBP) but has not been described in the literature. The two structures superimpose with a 1.7 Å RMSD across all amino acids. When the RT and N-Src loops are removed from the comparison, the backbone RMSD is 1.0 Å.1H-15N HSQC spectrum of the HACS1 SH3 domain  that in B-cell receptors, for example, are phosphorylated by tyrosine kinases such as Syk that promote cell maturation32. The interaction between HACS1 and PIRB cytosolic region may bypass SHP phosphates and introduce a new junction in signaling pathways governing immune- and neuronal cell fate. Building upon a yeast two-hybrid study that first identified a possible HACS1-PIRB partnership, we extended this observation by demonstrating that the HACS1 SH3 domain was sufficient for direct interaction with PIRB. In Fig. PIRB is a receptor consisting of six immunoglobulins (Ig) repeats in its extracellular segment, a thirty amino acid hydrophobic transmembrane segment, and a 190 aa. cytosolic domain with four immunoreceptor tyrosine-based inhibition motifs (ITIMs). Each ITIM is consistent with the consensus sequence [SIVL]-Y-xx-[IVL] Fig. . When PI33. The HACS1 SH3 domain; however, was not among the proteins that was surveyed.An array of 12-mer peptides spanning the entire PIRB cytosolic domain at a three amino acid resolution was synthesized on a cellulose membrane and challenged with a 6xHis-tagged HACS SH3 domain. As many SH3 domains participate in low-affinity interactions with their ligands, the blot was probed with protein at a concentration of 5 µM. After stringent washing and chemiluminescent detection with an antibody against the 6xHis tag on the protein, a series of positive binding signals were observed overlapping ITIM3, but not the other ITIMs  to alanine was tolerated. In contrast, the substitution of cysteine at either to alanine . If both arginines were substituted with either alanine  deviates from cysteine that was shown to be an important determinant of binding in ITIM3. An ITIM2 sequence substituted with cysteine at the first position  was determined. As shown in Fig. The region of PIRB ITIM3 that interacts with the HACS1 SH3 domain is near a regulatory tyrosine. When this tyrosine is phosphorylated, ITIM3 becomes a ligand for the two SH2 domains of SHP2 phosphatase, differentiated by the names, SH2(N) and SH2(C). The affinities of the HACS1 SH3 domain and SHP2 SH2-N domain for PIRB were determined using surface plasmon resonance-based assay. Biotinylated murine and human phosphopeptides were immobilized on a streptavidin-coated gold sensor and varying concentrations of the protein analyte solutions were applied and then washed from the sensor at a controlled flow rate. On- and off-rates were simultaneously fit to the resulting sensorgrams and a dissociation constant , the observed affinity of 7 µM would be consistent with the prediction that ITIM3 would be a low-affinity SHP2 SH2 domain ligand.The pY+4 position is an important determinant of affinity for both the SH2-N and SH2-C domains38 and three programs designed to identify secondary structured regions41. The lack of consensus among the secondary structure prediction programs combined with consistently strong predictions by the intrinsically unstructured region prediction programs suggests that the cytoplasmic region including the ITIMs, serve as a large display site42 for the transient binding of protein partners  as a protein partner of Paired immunoglobulin receptor B (PIRB). Using a combination of pull-down assays, peptide arrays, and binding assays with purified proteins, we have shown that the interaction is direct and mediated by the SH3 domain of HACS1 and a sequence in the PIRB cytosolic domain that overlaps ITIM3, a well-established SH2 domain binding site. Chemical shift mapping identified a surface on the HACS1 SH3 domain that is known for binding both canonical proline-rich ligands and noncanonical ligands such as the RxxK motif of SLP-7644. It is worth noting that the way in which the SLy1 SAM domain dimerizes is unique among the myriad of homo- and hetero-oligomeric SAM domain structures that have been described.The NMR and SEC-MALS analyses indicate that the HACS1 SH3 domain is monomeric over a wide range of concentrations. The SAM domain that closely follows the SH3 domain in HACS1 is monomeric, as well (PDB: 1V38). HACS1 has a homolog SLy1/SASH3 that also participates in immune cell developmental programs, but cannot substitute for HACS1 despite having over 40% sequence identity. These functional distinctions appear to extend to the architecture of SLy1 since its SAM domain forms homodimers45 and multiple myeloma2, HACS1 is underexpressed, while in glioblastoma3, it is overexpressed suggesting that the function of HACS1 adaptor protein is complex. As a result, the identification and characterization of protein partners that work with HACS1 represent an important first step towards understanding its precise role in cancer. In this report, we have demonstrated how the SH3 domain serves one such role through its association with PIRB, although it is important to be aware that the remainder of this adaptor protein is also a platform for numerous partners, including 14–3–3 proteins46. Genomics studies have implicated HACS1 in Alzheimer’s disease associated pathways, although its role has not been established6. In summary, renewed interest in HACS1 and its relationship with PIRB may reveal new ways how extracellular signals affect innate immunity and synaptic signaling.In lung cancerMolecular modeling suggests that SHP1 and HACS1 cannot simultaneously bind PIRB. With relatively modest and similar affinities for PIRB, concentration changes of HACS1 and SH2-domain-containing proteins would change the occupancy on PIRB and downstream signaling outcomes. In Fig. 46. Cortactin, an adaptor protein that is recruited to sites of actin reorganization, has also been identified as a ligand of HACS1 SH3 domain although the specific nature of this interaction has not been investigated further47. When all of the known and potential partnerships are considered, we believe that HACS1 has the potential to be a important signaling node in processes governing immune and neuronal cell fate.While the interaction with PIRB receptor places HACS1 in signaling processes occurring at the cell membrane, HACS1 is also found in the nucleus where it has the potential to interact with components of the SAP30-HDAC chromosome remodeling complexE. coli BL21(DE3) and plasmids regulated by a T5 promoter were transformed into E. coli BL21. A 1.5 L fermentation at 37 ˚C in a minimal medium containing 1 g of 15NH4Cl as the sole nitrogen source and 3 g of 13C-glucose as the sole carbon source was sufficient to manufacture a sample for NMR structural studies. Otherwise, fermentations were carried out in standard LB media. For all protein expression studies, a similar protocol was followed: Briefly, when the bacterial culture reached an A600 of 0.6, soluble protein expression was initiated by the addition of IPTG to 1 mM and the culture was incubated further for three hours. After centrifugation, the cell pellet was resuspended and lysed in a French pressure cell. The soluble 6x-His-tagged proteins were purified by affinity chromatography on a Nickel-NTA column (Qiagen), incubated with in-house made 6xHis tagged TEV protease to remove the MBP carrier protein if required, and purified further by gel filtration chromatography . Unless otherwise indicated, proteins were used and stored in a buffer consisting of 20 mM sodium phosphate, pH 7.8, 0.15 M NaCl, 5 mM DTT, and 0.05% NaN3.A set of human HACS1  clones were provided by Jaime Claudio (University of Toronto) as a starting point for deletion analysis. This set was used to make a number of SH3 domain (155–228) containing gene fragments that were cloned via the Gateway system into the expression vector pDEST-586 . This vector appends an amino-terminal 6xHis tag, maltose-binding protein (MBP) tag, and a tobacco etch virus (TEV) protease recognition site. In later studies, the MBP-fusion set was replaced by codon-optimized, 6xHis-tagged HACS1 SH3 domain that was gene synthesized and provided in a pD441NH expression vector from ATUM . Gene synthesis was also used to manufacture a 6xHis/Flag-tagged human SHP2 N-SH2 domain  and a 6xHis/Protein G B1 domain tagged murine PIRB fragment  separated by a random 13 amino acid linker. Expression plasmids regulated by a T7 promoter were transformed into 15. To 500 µg of lysate, 10 µg of purified 6xHis-MBP-HACS1 SH3 domain or 6xHis-MBP protein were mixed and incubated overnight with 50 µL Nickel-NTA resin. After washing and elution with TBST  Tween-20, pH 7.4), proteins were resolved on a 10% SDS polyacrylamide gel and transferred to a PVDF membrane. After blocking the membrane in TBST supplemented with 5% skimmed milk, it was probed with a mouse anti-PIRB antibody MAB2754  and detected by chemiluminescence.Mouse bone marrow and splenocyte lysates were prepared following a previously described method48 with an Intavis MultiPep instrument. A crude estimate of the peptide content in each spot was made by staining the array with Fast Green FCF. The array was probed with 5 µM of 6xHis-MBP-HACS1-SH3 in PBST . Following blocking and washing with 5% skimmed milk and 2.5% bovine serum albumin  in PBST, bound protein was identified by incubating the array with a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated 6xHis monoclonal antibodies (Qiagen) in PBST and a chemiluminescent reagent (Santa Cruz Biotechnology).A set of 12-mer peptides on a 150 × 100 mm cellulose membrane arranged in a 20 × 30 array were synthesized using the SPOT method3). This analyte buffer was selected to minimize nonspecific binding effects. The addition of either competitor protein or detergent alone did not mitigate nonspecific binding effects completely. Flow rates for binding and washing phases of the experiment were performed at 40 µL/min. The chip was regenerated after each binding experiment with 10 mM HCl. Data were processed with TraceDrawer  and exported to proFit  for presentation.A streptavidin-derivatized gold sensor chip for an OpenSPR instrument  was coated with either biotinylated human (GGQDVT{pY}AQLHSLTRLREATE) or murine (GGQDVT{pY}AQLCSRTLRQGAAAS) PIRB derived phosphopeptide . Pure, 6xHis-tagged HACS1 SH3 domain protein or 6xHis-tagged human SHP2 N-SH2 domain protein was prepared at concentrations ranging from 20 µM to 0.1 µM in analyte buffer  and AdvanceBio SEC300A column (Agilent). To ensure optimal stability, the chromatography system was equilibrated with the sample buffer for 18 h before the first injection. The detection system consists of a MiniDAWN TREOS MALS and OptiLab T-rEX refractive index module (Wyatt). Prior to the analysis, a 2.0 mg/mL bovine serum albumin standard was injected to calibrate the peak and retention time characteristics of the flow cells. Following calibration, a 20 µL HACS SH3 domain protein sample at 2.0 mg/mL was injected. Chromatograms were analyzed and molecular masses determined with ASTRA software (Wyatt).3, pH 7.5). After the first injection of 2 μL, the titration followed with successive injections of 8 μL with an equilibration delay of 300 s. The heat of dilution was determined by titrating the PIRB-GB1 solution into buffer alone. The raw data were corrected for heat of dilution and fitted to a single-site binding model using Origin v5.0 .Measurements were performed on VP-ITC calorimeter . An optimal titration was observed when 37 μM 6xHis-tagged HACS1 SH3 domain was in the sample cell and a 600 μM solution of PIRB-GB1 was in the syringe. Prior to the experiment, proteins were exchanged by gel filtration chromatography into identical buffers NH, HNCO, HNCACO for backbone assignments, H(C)(CO)NH, C(CO)NH, HCCH-TOCSY, HB(CBCG)CD, HB(CBCGCD)CE for side-chain assignments and 15N-edited 1H-1H NOESY and 13C-edited 1H-1H NOESY experiments (100 ms mixing time) for distance restraints. All assignments were made manually. Amide residual dipolar couplings from IPAP-HSQC spectra were acquired using a 15N-labeled protein sample supplemented with 10 mg/mL Pf1 phage . Binding experiments using 15N-labeled HACS1 SH3 domain and Protein GB1-tagged PIRB were acquired on a Bruker Avance 700 MHz instrument equipped with a nitrogen chilled probe. All datasets were processed with NMRPipe49 and interpreted with CCPN Analysis50. A relaxation study at 700 MHz was performed to determine 15N T1 relaxation times , 15N T2 relaxation times  and heteronuclear 15N{1H}NOEs. Using a freshly prepared and reduced 0.3 mM sample of 15N-labeled in a similar NMR buffer except DTT was replaced with 2.5 mM TCEP. 6xHis-tagged HACS1 SH3 domain, relaxation data were acquired as interleaved pseudo-3D spectra. Peak integrations were made with the nLinLS module of NMRPipe, followed by curve fitting to an exponential decay function with LMQUICK51. A correlation time was calculated by a global analysis of the relaxation data52.To assign and determine the structure of the HACS1 SH3 domain, a conventional heteronuclear, triple-resonance strategy was followed using datasets acquired at 25 ˚C on an Agilent 600 MHz NMR spectrometer equipped with a helium-cooled probe and a uniformly 53. From an ensemble of 500 structures calculated with CYANA54, the top structure was selected among twenty that had no NOE violations > 0.2 Å and no torsion angle violations > 5˚. This structure served as input for refinement with XPLOR-NIH v2.5855. The supplied refine.py script was used as supplied except the maximum force constant for the RDC energy term was set to 0.2 kcal•mol-1•Hz-1. Inferred hydrogen bonds from backbone NOEs were explicitly defined for the HBDA energy term56 and other hydrogen bonds were determined during the refinement by the HBDB energy term57. From 400 structures, a final ensemble of twenty structures was selected based on the lowest energy and no NOE violations > 0.2 Å and no torsion angle violations > 5˚. Structure quality was assessed by PSVS58 and PROCHECK59. From a Ramachandran analysis of residues 167–174,184–196, 202–207, 210–214, and 216–221 in the ensemble, 93.4% residues were located in the most favored region, 6.5% were located in additional allowed regions, 0.1% were located in generously allowed regions, and no residues were located in disallowed regions.NOE distances were calculated from peak volumes with CCPN Analysis. Backbone torsion angles were predicted from chemical shifts using TALOS-N1H,15N-HSQC spectrum was acquired with a sample of 15N-labeled HACS1 SH3 domain in a matched NMR buffer containing an excess Protein G B1 domain tagged PIRB. A weighted chemical shift perturbation, d, was calculated using the following relationship60.A 61 and then modified to match PIRB with Rosetta Remodel62.The coordinates of the SHP2 SH2-N domain crystal structure (PDB: 4QSY) bound to a GAB1 phosphopeptide was used as a starting point to make a molecular model of the SH2 domain bound to PIRB ITIM3. The GAB1 peptide was extended several amino acids with SWISS-MODEL63. Specific secondary structure prediction programs used were PSIPRED39, SPIDER340, and DeepCNF-SS41. Specific intrinsically unstructured region prediction programs used were IUPred36, SPOT-Disorder37, and DISOPRED338.To identify potential structured and intrinsically disorder regions of the murine PIRB cytosolic domain, the protein sequence was analyzed by Quick2D utility of HHPREDNMR solution structure statistics are expressed as the root-mean-square deviation from the average structure or value.Further information on research design is available in the Supplementary InformationReporting summary


There is an increased interest and need to make our economy more circular and our diets healthier and more sustainable. One way to achieve this is to develop upcycled foods that contain food industry by-products in their formulation. In this context, the aim of this study was to develop muffins containing upcycled sunflower flour (a by-product from the sunflower oil industry) and assess the effects of sunflower flour addition on the fibre, protein, amino acid, mineral content, and antioxidant activity measured by a Ferric Reducing Antioxidant Power (FRAP) assay and Photo chemiluminescence (PCL) assay. Results show that the sunflower flour inclusion significantly improved all the parameters analysed as part of this study. A more balanced muffin amino acid profile was achieved, thanks to the increased levels of lysine, threonine, and methionine, the limiting essential amino acids of wheat flour. We can conclude that upcycled ingredients, such as sunflower flour, could be used for the nutritional improvement of baked goods, such as muffins. Their addition can result in several nutritional advantages that could be communicated on packaging through the use of the appropriate EU nutrition claims, such as those on protein, fibre, and mineral content. In order to make our economies more circular and our diets more sustainable, there is an increased need to valorise food industry by-products into ingredients that can re-enter the food chain as part of new foods.Sunflower cake is a by-product of the sunflower oil industry which has been traditionally used as animal feed . Rich inCircular economy principles should push us to valorise food industry by-products as ingredients for human diets, rather than just as animal feed, as explained in the food recovery hierarchies developed in the EU and in US ,8. This Popular baked goods, such as muffins, cakes, or biscuits, are usually high in sugar and fat but low in fibre, antioxidants, and minerals , so theyThe aim of this short communication was to partially replace wheat flour with sunflower flour (at 15% or 30%) in muffins and evaluate the effects of this replacement on their fibre, mineral, and amino acid content, as well as antioxidant activity. No attempts have been made so far to investigate these parameters in the development of muffins with upcycled sunflower flour, while an investigation on the proximate composition , physical analyses, and sensory quality of muffins with sunflower flour can be found in a study by Grasso, Liu, and Methven .Muffins were manufactured according to the recipe and procedure shown by Ateş and Elmacı  and the Dietary fibre content was evaluated according to Grasso, Pintado, Pérez-Jiménez, Ruiz-Capillas, and Herrero . DuplicaProtein content was measured in duplicate with a Nitrogen Determinator LECO FP-2000 . The factor used to convert nitrogen content to protein was 6.25, and the results were expressed as g/100 g of the sample.For mineral content determination, freeze-dried samples  were prepared by acid digestion with nitric acid in a microwave digestion system . The minerals were quantified on a ContrAA 700 High-Resolution Continuum Source spectrophotometer  equipped with a Xenon short-arc lamp . Three determinations were carried out per sample to measure Calcium (Ca), Magnesium (Mg), Sodium (Na), Potassium (K), Phosphorus (P), Iron (Fe), Zinc (Zn), Copper (Cu), and Manganese (Mn). The determinations were made in duplicate, and the results were expressed as mg/100 g of the sample. More information on the mineral content analysis can be found in a study by Sánchez-Faure et al. .Amino acid content was determined and measured using ninhydrin derivative reagent and separated by means of cation-exchange chromatography, using a Biochron 20 automatic amino-acid analyser  where we injected the extract of samples that was dried and hydrolysed in vacuum-sealed glass tubes at 110 °C for 22 h in the presence of 6 N HCl containing 0.1% phenol and nor leucine  as the internal standard. After hydrolysis, samples were again vacuum-dried, dissolved in application buffer, and injected onto a Biochrom 20 amino-acid analyser . A mixture of amino acids was used as the standard . The determinations were made in duplicate, and the results expressed as mg/g of the sample.For the determination of antioxidant capacity, an aqueous-organic extraction was carried out in duplicate following the methodology of Jiménez et al. .FRAP reagent, freshly prepared and warmed to 37 °C, was mixed (150 μL) with distilled water (15 μL) and the test sample, Trolox, or appropriate blank solvent (5 μL). Readings at 595 nm in a Synergy MX  spectrophotometer after 30 min were selected to calculate the FRAP values. Results were expressed as µg eq Trolox/mg after interpolating in the calibration curve.This assay was used to determine antioxidant capacity using an automated photo chemiluminescent system , which measures the capacity to quench free radicals. This method is based on controlled photochemical generation of radicals, part of which is quenched by the antioxidant, and the remaining radicals are quantified by a sensitive chemiluminescence-detection reaction. Results were expressed as µg eq Trolox/mg sample (liposoluble fraction) and µg eq ascorbic acid/mg (hydro soluble fraction).p < 0.05) between formulations.The baking experiment was repeated twice on two different days. One-way analysis of variance (ANOVA) was carried out to evaluate differences between formulations using the SPSS program . To compare mean values between formulations, least squares differences and Tukey’s HSD tests were used to identify significant differences (The fibre and protein content of the sunflower flour and the three muffins are shown in The mineral content of the sunflower flour and the muffins is shown in . SunflowThe use of upcycled ingredients in baked goods, such as sunflower flour in muffins, could result in several nutritional advantages as here shown, such as improved fibre content, mineral content, amino acid profile, and antioxidant activity. The development of baked goods with a balanced amino acid profile through the use of upcycled ingredients is of particular interest and should be explored in further research. Upcycled ingredients could be promoted on the packaging if they are used at sufficient levels to make nutrition claims, such as those on fibre, protein, or mineral content. The sensory quality of muffins with sunflower flour was investigated through a Quantitative Descriptive Analysis by Grasso, Liu, and Methven . Results


Plexins are receptors for semaphorins that transduce signals for regulating neuronal development and other processes. Plexins are single-pass transmembrane proteins with multiple domains in both the extracellular and intracellular regions. Semaphorin activates plexin by binding to its extracellular N-terminal Sema domain, inducing the active dimer of the plexin intracellular region. The mechanism underlying this activation process of plexin is incompletely understood. We present cryo-electron microscopic structure of full-length human PlexinC1 in complex with the viral semaphorin mimic A39R. The structure shows that A39R induces a specific dimer of PlexinC1 where the membrane-proximal domains from the two PlexinC1 protomers are placed close to each other, poised to promote the active dimer of the intracellular region. This configuration is imposed by a distinct conformation of the PlexinC1 extracellular region, stabilized by inter-domain interactions among the Sema and membrane-proximal domains. Our mutational analyses support the critical role of this conformation in PlexinC1 activation. Plexins are the receptors for the guidance molecules semaphorins and regulate immunity and the development of the nervous and cardiovascular systems. Here authors present a structure of full-length human PlexinC1 in complex with its ligand A39R, which reveals how inter-domain interactions couple extracellular ligand binding to receptor activation and signaling. Semaphorins transduce signal through the plexin receptors, which are single-pass transmembrane proteins localized on the cell surface4. The essential common downstream signaling pathway shared by all plexins is mediated by their GTPase Activating Protein (GAP) activity, which specifically catalyzes GTP hydrolysis for the small GTPase Rap and thereby converts Rap into the GDP-bound inactive state5. In addition, individual plexin family members can trigger unique signaling pathways by interacting with different downstream signaling proteins3. In general, these signaling pathways collectively lead to changes in cell adhesion and cytoskeleton, etc, ultimately resulting in repulsive guidance of plexin-expressing cells, including neurons, endothelial cells and immune cells3.Semaphorin is a large family of guidance molecules that play important roles in many physiological processes, including neuronal axon guidance, cardiovascular development, and immunity1. Ectromelia virus secrets a protein named A39R that is a Sema7A mimic6. Semaphorins all contain an N-terminal Sema domain that adopts a 7-bladed β-propeller structure2. The Sema domain in most cases is followed by a plexin–semaphorin–integrin (PSI) domain, an Ig-plexin-transcription factor (IPT) domain and a C-terminal region that is highly divergent among different family members. Most semaphorins form stable dimers through the homotypic interaction of the Sema domain, which in some cases is further stabilized by the interaction between the IPT domain and an inter-chain disulfide. The extracellular region of plexins also contains an N-terminal Sema domain, a number of PSI and IPT domains2. The intracellular region of plexins is composed of a juxtamembrane region, the RapGAP domain and a RhoGTPase-binding domain (RBD), which are all important for signaling9.Semaphorins and plexins are classified into seven (classes 1–7) and four classes (classes A–D), respectively, which show different specificities to one another12. Consistently, the RapGAP activity of the plexin intracellular region is activated upon formation of a specific active dimer5. Crystal structures of the zebrafish PlexinC1 intracellular region have revealed the conformation of the active dimer in both the apo and Rap-bound state8. These structures show that two protomers of the PlexinC1 intracellular region interact in a side-by-side mode to form the active dimer, with the two juxtamembrane regions arranged in parallel to make major contributions to the dimer interface. This conformation of the juxtamembrane region enables it to interact with the activation loop in the RapGAP domain from the dimer partner in trans, switching the RapGAP to the active conformation that can bind Rap and catalyze its GTP hydrolysis8. These observations together suggest that semaphorin-induced plexin dimer needs to have its two copies of the extracellular membrane-proximal and transmembrane regions placed close to each other in order to drive the formation of the intracellular active dimer. However, all of the reported structures of the semaphorin/plexin complexes only contain the Sema domain and one or a few following domains in plexin13. The C termini of the two truncated plexin molecules in these structures point away from each other and are separated by a large distance. Therefore, it had been unclear how this configuration of the complexes could induce the formation of the intracellular active dimer. Recent structural studies of the intact extracellular region of class A plexins in the apo state show that the three PSI domains and six IPT domains together adopt a highly curled shape, leading to an overall ring-like architecture15. Based on these structures, a docking model of the entire extracellular region of class A plexins in complex with semaphorin has been constructed, showing that the ring-shape of plexin brings the two copies of the membrane-proximal IPT6 domain in the dimeric complex to close proximity, compatible with the formation of the intracellular active dimer15. Classes B and D plexins may use the same mechanism, as their extracellular regions have the same domain composition as class A plexins. However, the dimer of intact semaphorin/plexin complexes in the active state has not been observed directly through structural analyses to date.Structural analyses have shown that a dimeric semaphorin binds two plexin molecules through interactions mediated by their respective Sema domains, supporting the ligand-induced dimerization model for plexin activation16. To address these questions, we purified full-length human PlexinC1 and reconstituted the 2:2 dimeric active complex with its high affinity ligand A39R from ectromelia virus. We determined the structure of the complex to 2.9 Å resolution by using cryo-electron microscopy (Cryo-EM). The structure reveals a distinct architecture of the PlexinC1 extracellular region, which is stabilized by extensive interaction among the Sema domain and the three membrane-proximal domains (IPT2–4). This architecture in combination with the particular A39R/PlexinC1 binding mode juxtaposes the two protomers of the PlexinC1–IPT4 domain in the dimeric complex properly for inducing the dimerization of the transmembrane and cytoplasmic region. The importance of these structural features for PlexinC1 signaling is confirmed by our structure-based mutational analyses. Our findings provide a structural basis for the activation of PlexinC1 and shed light on the arrangement of the transmembrane region in the ligand-induced dimer of plexins.The class C plexin PlexinC1 is unique in that it only has two PSI and four IPT domains in the extracellular region, rather than three and six, respectively, in classes A, B, and D plexins Fig. . The sman-Dodecyl β-D-maltoside (DDM) formed the complex with A39R that remained intact through gel filtration chromatography  for cryo-EM analyses. Full-length PlexinC1 solubilized in buffers containing 12. The binding interface between A39R and the Sema domain of PlexinC1 is virtually identical in the cryo-EM structure and the crystal structure of A39R in complex with the isolated Sema–PSI domains of PlexinC1 , the Sema and IPT domains interact through a small interface, leading to a closed ring-shape  Fig. . Similar15. However, the curved rod shape of the IPT2–4 domains in PlexinC1 does not resemble the membrane-proximal IPT4–6 domains of in any of the structures of class A plexins  Fig. . The relThe inter-domain interactions described above keep the rigidity of the PlexinC1 extracellular region, particularly the membrane-proximal IPT2–4 domains, which is likely required for the proper positioning of the IPT4 domain for ligand-induced dimerization of the transmembrane and intracellular regions of PlexinC1. In support of this idea, most of the residues involved in the inter-domain interactions are highly conserved in PlexinC1 from various species, suggesting that they are functionally important  and class A plexins (IPT6) suggest that the C termini in the different plexins are related to their dimer partner differently  and a C-terminal His8-tag. All primers used in the study are listed in Supplementary Table The cDNA of A39R  was synthesized by Integrated DNA Technologies and cloned into a modified pEZT-BM vectorPseudomonas aeruginosa28 and a His8-tag at the C terminus in tandem was inserted into the pEZT-BM vector. The plasmid was used to generate recombinant baculovirus for protein expression in HEK293 GnTI− cells  with the BacMam system29. GnTI− cells were then infected by the baculovirus at 1:50 (v/v) ratio. Sodium butyrate at 5 mM was supplemented 24 hrs after infection to enhance protein expression. Ninety-six hours after infection, cells were pelleted and resuspended in a lysis buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM CaCl2, DNase I, serine protease inhibitor AEBSF , and a protease inhibitor cocktail . Cells were lysed by a cell disruptor and centrifuged at 35,000 rpm for 1 h at 4 °C. Membrane pellet was resuspended by a homogenizer in the lysis buffer without DNase I. Membrane proteins were solubilized by stirring at 4 °C overnight after adding 2% n-dodecyl-B-d-maltopyranoside  and 0.4% cholesteryl hemisuccinate tris salt . The mixture was centrifuged at 45,000 rpm for 1 h at 4 °C. The supernatant was cleared by passing through 0.45 μM nylon membrane. Purification of Tsi3-tagged PlexinC1 was carried out in a similar manner as previously described30. Briefly, the protein was captured by Sepharose 4B resin  conjugated with the T6SS effector protein Tse3 equilibrated in buffer B containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM CaCl2, 0.05% DDM, and 0.01% CHS. After thorough washing with buffer B, the PlexinC1 protein was cleaved from the Tsi3-tag by incubating overnight with His6-tagged rhinovirus 3C protease in the presence of 25 mM imidazole. The elutant was passed through Ni-NTA Sepharose resin  equilibrated with buffer B containing 25 mM imidazole to trap the Tsi3-His8-tag and His6-tagged protease. The flow-through was further purified by a Superdex 200 Increase 10/300 GL gel filtration column  equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.03% DDM. Purified protein was concentrated and stored at −80 °C.The cDNA of human PlexinC1 was from ThermoFisher (UT Southwestern human cDNA library). The coding region of the full-length PlexinC1 (residues 1–1568) fused to a cleavage site for the human rhinovirus 3C protease, T6SS secreted immunity protein 3 (Tsi3) from 8 tag. The construct contains a segment that encodes a human rhinovirus 3C protease cleavage site inserted between Glu649 and Asn650 of PlexinC1, introduced by polymerase chain reactions. The protein was expressed in Expi293F cells with PEI transient transfection. The purification procedure was the same as that for A39R. The purified protein was concentrated to 3.3 mg/ml in the buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl.The extracellular region of human PlexinC1 (residues 1–942) was cloned into the pRK5 vector (BD Biosciences) with a C-terminal HisProtease digestion of the PlexinC1 extracellular region containing the 3C protease cleavage site in the linker was carried out by incubating the protein (10 µg) with sumo-tagged 3C protease (0.1 µg) in a 10 µl reaction system at room temperature for 1 h. Protein samples were mixed with loading dye with or without dithiothreitol and subjected to electrophoresis on a 4–20% gradient gel (Bio-rad).https://peptidisc.com). The sequence of peptidisc peptide is FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD 17. The peptidisc peptide powder was dissolved at 2 mg/ml using 20 mM HEPES pH 7.5 and 150 mM NaCl. To help solubilization, the peptidisc solution was incubated at 50 °C water bath for 20 min and vortexed. Reconstitution of the PlexinC1/A39R complex into peptidisc was carried out by following the protocol as described in17. Briefly, 300 μg of PlexinC1 was mixed with A39R at 1:1 molar ratio and incubated for 10 min at 25 °C. 60X molar excess of peptidisc peptide (460 μg) was then added to the PlexinC1/A39R mixture and incubated at 4 °C with constant rotation overnight. The mixture was centrifuged at 15,000 rpm for 10 min at 4 °C to remove precipitated protein and then loaded to a Superose 6 Increase 10/300 GL column equilibrated with a buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl. Fractions containing the PlexinC1/A39R complex were pooled, concentrated to ~0.5 mg/ml, and stored at −80 °C.The peptidisc peptide was purchased from PEPTIDISC LAB  and Alexa-488-coupled anti-mouse IgG secondary antibody  were used to stain His8-tagged A39R. Nuclei were stained with DAPI . Images were taken by a DeltaVision Core microscope and processed with deconvolution and Z-stack quick projection using the SoftWoRx 7.0 software package provided by DeltaVision. To test A39R-induced COS-7 cell collapse, cells expressing PlexinC1 wild type or mutants were seeded in four-well cell culture chamber slide  at 50,000 cell/well. Twenty-four hours after seeding, cells were washed three times with HBHA buffer . Cells were then treated with A39R at a final concentration of 50 nM in HBHA buffer for 90 minutes to induce collapse. For better visualizing of the morphology, cells were stained for PlexinC1 and nuclei with an anti-FLAG antibody  and DAPI, respectively. Images were acquired at 200X magnification with a ZOE fluorescent cell imager (Bio-Rad). Collapsed cells were identified based on substantially reduced area and star-like morphology as compared with the characteristic large and round shape of normal COS-7 cells31. Counting of cells were carried out in a blinded manner. Data were plotted in GraphPad Prism 8.FLAG-tagged PlexinC1 wild type or mutants were cloned into a modified pLVX vector . Lentivirus were produced by transfection of HEK293T cells with the plexin constructs and packing plasmids. Supernatants containing viral particles were filtered with 0.45 µm syringe filter and concentrated by centrifuging at 26,000 rpm for 2 h. After centrifugation, the supernatant was aspirated and viruses were resuspended in PBS. To generate COS-7 cells stably expressing PlexinC1, COS-7 cells  were infected with the virus and selected with 5 µg/ml puromycin. To visualize the binding of A39R by PlexinC1 expressed on the cell surface, cells were incubated with His32. The GIF-Quantum energy filter was set to a slit width of 20 eV. Images were recorded in the super-resolution counting mode with the pixel size of 0.828 Å. Micrographs were dose-fractioned into 32 frames with the dose rate of 1.5 e−/Å/frame.The A39R/PlexinC1 complex in peptidiscs at 0.5 mg/ml was applied to a glow-discharged Quantifoil R1.2/1.3 300-mesh gold holey carbon grid , blotted under 100% humidity at 4 °C and plunged into liquid ethane using a Mark IV Vitrobot (FEI). Micrographs were collected on a Titan Krios microscope (FEI) with a K3 Summit direct electron detector (Gatan) operated at 300 kV using the SerialEM software33. CTF correction were carried out using GCTF 1.0634. The following image processing steps were carried out in RELION 3.0 or 3.135. Auto-picked particles were extracted and binned by 3 or 4 times and used in 2D classification. Particles in good 2D classes were selected and subjected to 3D classification using an initial model generated from a subset of the particles. A total of 184,225 particles from two good 3D classes were re-extracted to the original pixel size. 3D refinement of these particles with the C2 symmetry imposed led to a 3D reconstruction to an overall resolution of 3.4 Å, although the transmembrane and intracellular region of PlexinC1 were completely missing in the density map. Based on this reconstruction, two strategies were used to improve the density for the membrane-proximal and transmembrane domains. In the first, the particles of the initial 3D reconstruction were subjected to 3D classification with local angular search. One class  that showed stronger density for the membrane-proximal and transmembrane region was chosen for the final 3D refinement and postprocessing. The resolution of the resulting 3D reconstruction was similar to the previous one, but had better local resolution for the membrane-proximal region and show weak density for the transmembrane region  0.143 criterion. Local resolution was calculated in Relion.The procedure of image processing and 3D reconstruction is outlined in Supplementary Fig. 12. Additional domains were manually built in Coot 0.936. Determination of sequence register of the IPT domains in PlexinC1 was facilitated by secondary structure predications, comparisons with the IPT domains of class A plexins, bulky residues, and glycosylation groups. Real-space refinement of the model against the map was carried out in the Phenix package (V1.7), with secondary structure restraints and Ramachandran restraints37. Two copies of the half complex were docked into the map of the intact complex and subjected to real-space refinement with twofold symmetry constraints to generate the final 2:2 PlexinC1/A39R complex. Model validation were carried out by using MolProbity as a part of the Phenix validation tools  or Chimera 1.339.The 3D reconstruction of the half complex from the focused refinement shows excellent density for A39R, the Sema, PSI1, and IPT2 domains of PlexinC1 for model building (Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary