Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are  Acetyl CoA is a major building block of intermediary metabolism and also functions as a substrate or cofactor for enzymes including acetyltransferases, which utilize acetyl CoA as a source of acetyl groups. When acetyl CoA availability is rate-limiting, acetyl CoA generators may have signal transducing capability . Acetyl de novo source of the acetyl donor, acetyl CoA, used by CBP for HIF-2 acetylation also exhibits specificity and is provided by Acss2 rather than ATP citrate lyase (ACLY), the other de novo cytosolic acetyl CoA generator that also is found in the nucleus (Targets for Acss2-dependent acetylation include histones as well as nonhistone proteins such as Hypoxia-Inducible Factor 2 (HIF-2), a heterodimeric DNA-binding transcription factor activated by oxygen or glucose deprivation. Although subject to oxygen-dependent protein degradation, coupled acetylation and deacetylation are required for maximal HIF-2 signaling , 4. HIF- nucleus .We had previously sought to generate a cytosol-restricted, enzymatically active form of Acss2. A directed mutation of a putative nuclear localization signal (NLS) in Acss2 residing in the carboxy terminal portion that changes two basic (RK) to two acidic (ED) amino acid residues abrogates HIF-2 acetylation, blunts HIF-2 dependent signaling, and impairs flank tumor growth in cancer cells expressing mutant Acss2 . IntroduIn characterizing the Acss2 ED mutant mouse strain, we noted that Acss2 ED protein levels were dramatically reduced . StructuTo identify additional elements affecting protein stability in the Acss2 hinge region, we used two types of Acss2 constructs. We introduced deletions or point mutations in full-length Acss2 protein and expressed these constructs transiently in Acss2 null primary mouse embryonic fibroblasts (MEFs). Alternatively, we generated fusion proteins consisting of isolated segments of Acss2 fused to the carboxy terminus of the fluorescent reporter moxGFP  and geneA). For the Acss2 hinge domain carboxy terminal deletions (B), removal of α − 20 through α − 18 destabilized the Acss2 mutant protein . Further deletion of β-E2 stabilized the remaining protein , which was unaffected by removal of the remaining upstream elements of the hinge region .To begin our investigations, we deleted regions from either the amino or carboxy terminus of the Acss2 hinge domain based upon structural features and fused the remaining segments to the amino terminal portion of Acss2 A. For theletions B, removaC), removal of A8 through A10, which truncates the ABC domain mid-way through its sequence, resulted in reduced protein stability . Deleting the remainder of the ABC domain resulted in a stable Acss2 mutant protein . Interestingly, deletion of the segment between α − 19 and α − 20 yielded an unstable Acss2 mutant protein . Removal of the remaining α − 20 segment stabilized the Acss2 mutant protein, which consists solely of the amino globular domain comprising the first ∼80% of the native Acss2 protein .For the Acss2 hinge domain amino terminal deletions C, removaA). With respect to amino localized segments, the moxGFP:A8 fusion protein exhibited modest PDE activity . Deletion of the A8 segment increased stability consistent with A8 functioning as a PDE . However, the carboxy terminal deletion of full-length Acss2 retaining A8 was stable , indicating that A8 does not function as a PDE in this context. Furthermore, isolated deletion of the A8 region resulted in an unstable Acss2 protein , suggesting a masking function of the A8 domain. Thus, A8 exhibits both PDE and masking activity, depending upon the type of mutant protein assay employed.The results observed from these initial experiments are consistent with the DHR and ABC domain functioning as independent PDE . HoweverFig. 2B, construct Ns8) as were moxGFP fusion proteins containing β-E2 compared with those without this segment . In conjunction with our initial data , we conclude that β-E2 is a potent PDE. Several amino-localized segments fused in isolation with moxGFP were highly stable including α − 17, β-E1, and α − 18 . The moxGFP:α − 17 fusion protein was particularly stable and the presence of α − 17 partially offsets instability conferred by β-E2 domain . However, the presence of these segments is not sufficient to override multiple PDE in deletion constructs of full-length Acss2 . In conjunction with earlier data, we conclude that A8 has both PDE and masking capacities, β-E2 is a novel PDE, and α − 17 may function as a stabilizing or masking domain in the hinge region.With respect to other amino-localized segments, the moxGFP fusion protein with β-E2 alone was highly unstable . As we previously reported . Notably, the two segments resulting from partitioning of the ABC domain, A10 and α − 19, retained potent protein destabilization capacity . The region downstream of the ABC domain did not function as a PDE when fused in its entirety to moxGFP , similar to the chimeric Acss2 protein consisting of this region fused to the amino globular portion of Acss2 . However, further partitioning of this region revealed the presence of two functional PDE , which include the α − 20 segment identified earlier in fusions with the amino globular portion of Acss2 . Thus, these two PDEs remain masked when present together in a contiguous manner.Immunoblots of whole cell extracts prepared from stably transformed HeLa cells expressing carboxy terminal fusions revealed that this region contains multiple PDE C. As we reported , the isoS. cerevisiae) or from several prokaryotes  and fused these to moxGFP (A–C). Our assumption was that these elements from Acss2 homologues of diverse organisms are likely to have similar structural features, even though their primary amino acid sequence varies. In the event that overall charge or other physical properties confer PDE activity, we also generated a scrambled mutant (SCRAM) for each PDE that retained the amino acid composition, but disrupted the primary sequence order. These alternative elements were compared to the mouse Acss2 PDE and parental moxGFP in immunoblot analyses.We next sought to assess if there are sequence or structural determinants that confer an ability of the β-E2 region, DHR, and ABC domain to function as PDE. To start, we identified similar regions from a lower eukaryote , the β-E2 element from D. Radiodurans does not function as an effective PDE and the SCRAM mutant likewise loses PDE function. For the DHR constructs (B), DHR elements from two organisms as well as the SCRAM mutant exhibit reduced PDE function. Interestingly, the corresponding DHR element from S. cerevisiae lacks PDE activity. For the ABC domain constructs (C), ABC domains from two organisms lack PDE function including that from H. salinarium, which contains an acidic glutamic acid–aspartic acid (ED) substitution for a basic arginine–lysine pair , and that from M. bovis, which substitutes an aspartic acid residue for a lysine (K669). However, the initial SCRAM mutant for the ABC domain remained a potent PDE, even though it altered the positions of several basic residues.For the β-E2 constructs A, the β-nstructs B, DHR elnstructs C, ABC doH. salinarium and M. bovis exhibited reduced PDE function, which contain at least one acidic amino acid substitution for a basic residue, we considered the possibility that basic charged residues may be important for the ABC domain to function as a PDE. The primary sequence of the ABC domain is enriched in basic residues (A). The secondary structure of the ABC domain contains an alpha-helix with basic residues biased toward one surface (B). We introduced pairwise alanine substitutions of basic residues in the ABC domain, visualized as a linear (C) or amphipathic alpha-helical (D) structure, in moxGFP:ABC domain fusion proteins (E and F) as well as in full-length Acss2 protein (G and H). We also examined a mutant Acss2 protein containing alanine substitutions for all basic residues (K3R4) or one containing the ED substitution mutation based upon the H. salinarium sequence. Isolated ABC domain:moxGFP fusion proteins containing alanine substitutions for all basic residues retained their ability to function as a PDE. In comparison, most of the full-length Acss2 alanine substitution mutants were stable. However, two helical pairwise alanine substitution mutants exhibited reduced stability compared with the parental Acss2 protein. These two mutants,  and  , have one residue in common, R664.Because the ABC domains from residues A. The se surface B. We inta linear C or amph-helical D structuproteins , E and F protein , G and HA) while retaining the overall amino acid composition and varied the charge distribution of the basic residues when viewed in a linear and/or amphipathic helix representation. Notably, all scrambled mutants remained potent PDE when examined as isolated moxGFP:ABC domain fusions (B). Furthermore, and in contrast to the alanine substitutions (C). The results from the pairwise alanine substitutions for basic residues and the scrambled mutations of the ABC domain in total indicated that specific residues in the ABC domain may be important for Acss2 protein stability.The results of the alanine substitutions indicated that a subset of basic residues may be important for stability of the full-length Acss2 protein. To further address determinants that confer PDE function to the ABC domain, we made five scrambled mutants of the ABC domain that varied its primary sequence A while r fusions B. Furtheitutions , all scritutions C. The reA). Substitutions for select residues had significant effects on stability of the full-length Acss2 protein with the most drastic changes occurring with substitutions of three of four leucine and isoleucine residues (B). We examined the sequences of the scrambled ABC domain mutants and noted that they all retained at least three of these four leucine and isoleucine residues in their native positions (A). We constructed moxGFP:ABC domain fusions and full-length Acss2 expression constructs with single alanine substitutions for these residues as well as a construct with alanine substitutions for all four leucine and isoleucine residues (C). Alanine substitutions for any or all leucine or isoleucine residues in the ABC domain did not increase stability of the moxGFP:ABC domain fusion proteins (D). Similar to the scrambled ABC domain mutants, in the context of full-length Acss2 protein, these mutations resulted in instability with two of the isolated substitutions mutants and the pan-substitution mutant having a comparable or greater effect as the ED substitution (E).To assess for the contribution of individual amino acid residues in the ABC domain to Acss2 protein stability, we generated additional mutants of full-length Acss2 protein using alanine scanning mutagenesis A. Substiresidues B. We exaositions A. We conresidues C. Alaninproteins D. Similatitution E.Figure F). The moxGFP:ABC domain fusion proteins containing these mutations had reduced stability relative to moxGFP alone or to the more stable mutant moxGFP:ED ABC domain fusion protein, similar to the moxGFP WT ABC domain protein . Full-length Acss2 proteins containing these mutations exhibited comparable or increased stability relative to WT Acss2 protein .We considered the possibility that posttranslational modifications (PTMs) of amino acid residues in the ABC domain might regulate Acss2 protein stability. Other investigators have shown that the serine at position 659 and the lysine at position 661 undergoes phosphorylation  and acetFig. 6M. Bovis ABC domain, H. salinarium ABC domain, and the CYT (ED) substitution mutant based on the latter inactivate PDE function of the isolated ABC domain, yet full-length ED Acss2 protein were unstable. These data in total suggested that the ABC domain and other PDE might be normally masked and that masking may depend upon specific basic residues located at precise positions in the ABC domain.The results of the mutational analyses in summary indicate that three elements function as potent PDE in isolation: the β-E2 element, the DHR, and the ABC domain. Amino acid substitutions can reduce the PDE capability of each significantly. The ABC domain was unique in that the isolated ABC domain was resistant to changes in the primary or secondary sequence generated by scramble mutations as well as by alanine substitutions for all basic residues. However, there was discordance when alanine substitution mutants were expressed in the context of full-length Acss2 as only a subset of alanine substitution mutants exhibited instability. In contrast, acidic substitutions for select basic residues as present in the A). Indeed, all PDEs are predicted to be in close proximity to each other on the surface of the hinge domain and in a globular portion spatially distinct from the major body of the Acss2 protein.In the deletion mutational analyses, we identified at least three masking elements present in the hinge region: the A8 domain, the α − 17 domain, and the region downstream of the ABC domain. Because the hinge region forms a globular structure in crystal structures of prokaryotic ACS proteins, we reasoned that masking might be facilitated if the PDEs are in close proximity to each other. To determine if this might be the case, we used a web-based modeling prediction program to map PDE on the predicted tertiary structure of Acss2 A. IndeedB), we identified three basic residues in the ABC domain  that form potential electrostatic interactions with three acidic residues located in A8, α − 17, and the region downstream of the ABC domain . To evaluate whether these residues have a functional role in preventing PDE exposure and therefore Acss2 protein instability, we performed single, double, or triple alanine substitutions for the basic (C) and acidic residues (D) and assessed their effect on stability of full-length Acss2 protein. The results indicated that at least one double and all triple alanine substitutions for either basic or acidic contact points result in marked Acss2 protein instability.We next asked if the PDE might be hidden by recruitment of masking elements we had defined in our mutational analyses. Using the web-based modeling program B, we idehe basic C and aciresidues D and assin silico predictions based upon whole genome sequencing. Because the genomic sequences used in this predictions have not been curated to the same degree as commonly used model organisms, we first performed a quality assessment protocol whenever in silico protein sequences deviated significantly from the consensus protein sequence for Acss2 (eukaroytes) or ACS (prokaryotes) to ascertain if sequences aligned better when potential frame-shift errors were corrected. Final edited protein sequences were aligned and compared using Clustal Omega  are also highly conserved in Acss2 and its homologues.The alignment results indicated that two basic contacts in the ABC domain and their acidic contacts in the A8 and α − 17 elements are conserved across prokaryotes and eukaryotes . The thiin vivo consequences for Acss2 protein stability and HIF-2 signaling (A and B), which results in a carboxy terminal deletion protein similar to one that was unstable in our cell culture experiments. As predicted, the Acss2 protein generated in Acss2 PM mutant mice is undetectable in a tissue survey (C). When assessed by immunohistochemistry, Acss2 PM protein is present at minimal or undetectable levels in mouse kidney and liver, respectively (D). Similar to Acss2 ED mice (E).We have shown that the Acss2 ED mutation has ignaling . The resignaling , A and Be survey C. When aectively D. Simila ED mice E.Figure F), sites of endocrine Epo synthesis in mammals, in anemic Acss2 PM mice relative to anemic Acss2 WT mice.Because of the role of Acss2 in HIF-2 signaling, we asked if Acss2 PM mutant mice had defects in HIF-2-dependent signaling. Acute hemolytic anemia following injection of phenylhydrazine (PHZ) activates HIF-2 signaling in mice. Hematocrit levels in Acss2 WT and PM mice were similar at baseline . WT and PM mice treated with PHZ and with similar degrees of anemia  were chosen for comparison. Gene expression for erythropoietin (Epo), a HIF-2 selective target gene, was blunted in the kidney as well as liver F, sites A). Interestingly and as observed with ectopic mutant Acss2 protein expressed in transformed stable cell lines, ectopic PM and ED Acss2 protein levels were higher when expressed in WT versus KO MEF. Similar to primary MEF derived from Acss2 knockout (KO) mice, MEF generated from Acss2 PM or Acss2 ED mice demonstrated markedly reduced endogenous Acss2 protein levels relative to MEF derived from Acss2 wild-type (WT) mice (B). The reduced expression of endogenous Acss2 protein in KO, PM, and ED mutant MEF resulted in reduced acetate-derived lipid synthesis (C).We asked if the effect of the Acss2 PM and ED mutations is cell-intrinsic. Transient expression of Acss2 PM protein in Acss2 WT or KO MEF resulted in ectopic Acss2 PM protein levels significantly lower than ectopic WT Acss2 protein and even lower than ectopic ED Acss2 protein expressed in a similar manner A. IntereWT) mice B. The reynthesis C.Figure D). Inhibition of protein translation with cycloheximide revealed that the half-life of Acss2 PM or ED proteins (estimated at between 4 and 8 h) is at least three times shorter than that of Acss2 WT protein (greater than 24 h), which indicates that Acss2 PM and ED proteins have reduced stability (E). Finally, degradation of Acss2 PM or ED proteins was not affected by inhibition of ubiquitin-mediated proteasomal degradation with MG132 (F) or by inhibition of autophagy with chloroquine (G).We next addressed the potential reasons for reduced levels of endogenous Acss2 PM and ED protein in MEF. No significant differences were observed in the insoluble relative to soluble fraction of Acss2 PM and ED protein in comparison to Acss2 WT protein D. Inhibitability E. Finallth MG132 F or by ioroquine G.We previously identified two potent PDEs in the Acss2 hinge region, the DHR, and the ABC domain . The ABCOne emerging characteristic for the PDE elements in Acss2 is their short length, but otherwise they lack any clearly defining features. This is a theme that also characterizes degrons recognized by the anaphase-promoting complex, which polyubiquitinates specific cell cycle proteins at precise times determined in part by unique aspects of a given degron . The β-EOther elements in the Acss2 hinge region are noteworthy. A8 exhibits both PDE and masking activities, the latter likely related to the presence of an acidic contact point that interacts with two basic contact points in the ABC domain. The A8 element is located at the most amino terminal portion of the Acss2 hinge region. Crystal structure of a prokaryotic Acss2 homologue indicates that it undergoes a conformational change during enzymatic generation of acetyl CoA involving rotation around the aspartic acid residue that marks the start of the hinge region. If similar conformational changes occur with mammalian Acss2, it is possible that its ability to function as either a PDE or masking element may be related to the spatial orientation of A8 in one of these conformations.The PDEs located downstream of the ABC domain are also noteworthy. The amino terminal segment is at most 15 amino acids in length and is predicted to be an unstructured feature in Acss2 based upon corresponding segments in crystal structures of ACS proteins. The carboxy terminal segment is likewise 15 amino acids in length and is predicted to be an alpha-helical feature. These two segments function as a conditional PDE when present in isolation or in the absence of each other. However, when present together, these two segments effectively mask each other. Given the likely relationship of these two segments in the native Acss2 protein, we refer to this region as a conditionally masked PDE (CMP). Although we have not explored its molecular determinants, the CMP may be a conditional C-degron  given itbona fide elements for several reasons. First, the existence of multiple PDEs is supported by the observation that the ED substitution mutation in full-length Acss2 results in reduced protein stability, whereas this same mutation in the isolated ABC domain impairs its ability to function as a PDE. Second, the PM truncating mutation cleaves off the carboxy terminal CMP, which may mask the ABC domain and other PDE and exposes the amino portion of the ABC domain, which we contend is a potent internal PDE. Finally, the destabilizing effect of amino acid substitutions for putative electrostatic contact points in the Acss2 hinge domain as well as of hydrophobic residues in the ABC domain is profound. We propose that these residues provide critical structural contributions that maintain the hinge region in a tertiary conformation to mask its PDE, which is further supported by the highly evolutionarily conserved nature of these residues.We have identified PDE by their ability to confer instability to a heterologous long-lived protein, moxGFP, the first defining feature of degrons. In some cases, we confirmed the ability of elements to function as PDE by deleting them from the parental Acss2 protein. Because we defined these elements functionally by fusing them to heterologous reporters or by deleting them from the parental Acss2 protein in order to interrogate their effect on protein stability, it is possible that some of these findings are artifactual. However, we argue that the PDEs identified in this study are S. cerevisiae, H. salinarium, and M. bovis are potent PDEs despite the fact that their primary sequence deviates substantially from the mouse Acss2 β-E2 element, particularly the M. bovis β-E2 element. The scrambled mutant for β-E2 is stable, which suggests that there may be positional requirements for some amino acids. However, the limitation for scrambled mutants is that they may lose any conserved structural feature, whereas this is less likely for substitutions that use similar motifs from Acss2 homologues in other species.We found mutations that eliminated protein-destabilizing activity of the isolated PDE, the second defining feature of degrons. However, the molecular determinants of each PDE in the Acss2 hinge region appear to be distinct. For β-E2, it is likely that a major determinant is a structural aspect of the element. This conclusion is supported by our observation that the corresponding β-E2 regions from S. cerevisiae and H. salinarium, reduced the ability of this region to function as a PDE, whereas the DHR from D. radiodurans and M. bovis did not impair PDE function. The DHR scrambled mutant also exhibited enhanced stability, although to a lesser extent than DHR from S. cerevisiae and H. salinarium. The enhanced stability of the DHR from S. cerevisiae was surprising given this region in mouse Acss2 was originally identified by virtue of its homology to this element. However, Acss2 homologues from S. cerevisiae and other lower eukaryotes contain a stretch of amino acids upstream of the DHR that is not present in other Acss2 homologues, which may alter the boundaries of the DHR in these organisms. Whether the lack of an effect of the S. cerevisiae DHR on protein stability in this study is a result of species-specific differences in recognition of this element, incomplete reconstruction of the S. cerevisiae DHR element, or altered amino acids upstream of the DHR defined by junctions with exogenous reporters remains to be addressed  and HeLa (stable cell lines). All cells were maintained in a cell culture incubator at 37 °C, 5% COn detail . StandarLentiviruses expression constructs were made as previously described in detail . Brieflyg  in an SW28 rotor for 2 h at 4 °C and the virus pellet resuspended overnight in 1 ml PBS. To generate stably integrated cell lines, HeLa was incubated with lentivirus for 24 h, cells were incubated with media containing 5 μg/ml Blasticidin S  for 4 days, followed by 2 weeks of selection at a concentration of 2 μg/ml Blasticidin S. Cells used in experiments were plated in media without Blasticidin S.Lentiviruses and stable cell lines were made as previously described . Brieflyg. To assay insoluble proteins, remaining cell pellets were resuspended in solubilization buffer  and incubated at room temperature for 5 min, followed by centrifugation at 18,000g for 15 min at 4 °C in a microfuge. The supernatant was then heated with in sample buffer  for 5 min at 37 °C. Antibodies recognizing the following epitopes were used for immunoblotting at the indicated dilutions: Acss2, 1:1000 ; GFP, 1:1000 ; α-tubulin, 1:5000 ; V5, 1:1000 ; LC3B, 1:1000 ; ubiquitin, 1:1000 ; HRP-linked anti-mouse IgG ; HRP-linked anti-rabbit IgG .Whole cell extracts and immunoblotting were performed as described earlier . SolubleAcss2 protein sequences from animals were identified manually from the NCBI or UniProt database and the protein identifiers for each organism are provided for review . ProteinThe mouse Acss2 WT protein was modeled using a web-based program, SWISS-MODEL . Defaultad lib throughout the study, and monitored for general health with sentinel mice. All mice used in experiments were generated from breeding colonies in animal facilities located at the University of Texas Southwestern. All animal experiments were approved by the University of Texas Southwestern Institutional Animal Care and Use Committee (APN 2016-101616).The mouse Acss2 gene was targeted for CRISPR/Cas9 by the University of Texas Southwestern Transgenic Core to generate Acss2 ED mice as described earlier . When chKidney and liver samples from Acss2 WT and PM adult mice were formalin-fixed, paraffin-embedded, and used for Acss2 immunohistochemical analysis as previously described . ProteinAcute, hemolytic anemia was induced in mice by phenylhydrazine  injection as previously described . Four daMouse kidney and liver total RNAs were prepared from individual organs using the FastRNA Pro Green kit  on the FastPrep-24 bead grinder and lysis system . Mouse kidney and liver cDNAs were prepared from 1 μg RNA with the iScript cDNA Synthesis kit  for mEpo measurements. Mouse erythropoietin (mEpo), Acss2 (mAcss2), and cyclophilin B (mCyclB) mRNA levels were measured in duplicate or triplicate by real-time PCR (rtPCR) semiquantitative analyses as previously described .MEFs were derived from Acss2 ED, PM, and KO mouse matings as described earlier . MEFs frAcss2 WT, PM homozygous, and ED homozygous MEFs were plated onto 6-well plates 2 days before the start of treatment. Media was changed to standard cell culture media plus vehicle 24 h before harvest. At 24, 12, 8, 4, and 2 h before harvest, media on one well of each genotype was replaced with standard cell culture media plus inhibitor. At harvest, cells including an untreated, 0-h control were harvested and immunoblotted as described earlier with the following modification: protein extract buffer was supplemented with the inhibitor used for each experiment at the same concentration used for treatment. The following inhibitors were used at the indicated final concentrations: 50 μg/ml cycloheximide ; 1 μM MG132 ; 50 μM chloroquine . Stock solutions of cycloheximide and MG132 were prepared in DMSO under sterile conditions. Chloroquine was prepared in molecular grade water and filter-sterilized.14C] acetate  was added to wells in triplicate 24 h before harvest. The following day, one untreated well of each genotype was trypsinized, pelleted, and frozen for protein quantification and western blotting. For the 0-h labeling control, 5.0 μl labeled acetate was added to three wells of WT control cells and swirled to mix, followed by immediate harvest. Each well was rinsed with PBS. Cells were then scraped into 2 ml of PBS and transferred to a borosilicate glass tube. The cells were pelleted at 750g for 5 min at room temperature. The PBS was removed and cell pellet resuspended in 400 μl 0.5% Triton X-100 in water. The following were added to each lysate, followed by 15 s vortexing after each addition: 2 ml methanol, 2 ml chloroform, 1 ml water. The phases were separated by centrifugation at 900g for 10 min. The lower, organic layer was transferred to a new tube with a Pasteur pipette. The aqueous mixture was re-extracted with 2 ml chloroform, adding the organic layer to the first extraction, and dried under a nitrogen stream. Dried lipid extract was resuspended in 200 μl chloroform and transferred to a scintillation vial containing 3 ml Ecolume . Scintillation counts per minute were read on a Beckman LS6500. Counts were normalized to protein content determined with aliquots obtained in parallel.Acss2 WT, PM homozygous, and ED homozygous MEFs were plated onto 6-well plates. Media was changed 6 h before treatment. For 24-h acetate labeling, 5.0 μl (0.5 μCi) of [1,2-Data described in this study are contained within the manuscript or This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The results emphasize the crucial role of nanoscale crystalline defects in determining the AF domains and domain walls, and provide a route to optimizing device performance.Efficient manipulation of antiferromagnetic (AF) domains and domain walls has opened up new avenues of research towards ultrafast, high-density spintronic devices. AF domain structures are known to be sensitive to magnetoelastic effects, but the microscopic interplay of crystalline defects, strain and magnetic ordering remains largely unknown. Here, we reveal, using photoemission electron microscopy combined with scanning X-ray diffraction imaging and micromagnetic simulations, that the AF domain structure in CuMnAs thin films is dominated by nanoscale structural twin defects. We demonstrate that microtwin defects, which develop across the entire thickness of the film and terminate on the surface as characteristic lines, determine the location and orientation of 180 Antiferromagnets offer the potential for higher speed and density than ferromagnetic materials for spintronic devices. Here, Reimers et al study the domain structure of CuMnAs, demonstrating the role of defects in stabilizing the location and orientation of antiferromagnetic domain walls. Moreover, AF order is exhibited in a wide range of materials compatible with the properties of insulators, semiconductors, and metals. Electrical switching has been achieved in several AF systems, with the resulting current-induced domain modifications attributed to spin-orbit torques or thermomagnetoelastic effects7. Spin-orbit torque manipulation of AF domains was first achieved using orthogonal current pulses to induce 90∘ rotations of the AF order parameter, but more recently current-polarity dependent switching of AF order has been achieved and ascribed to domain wall motion8. AF domains and domain walls are therefore the building blocks of AF spintronics, but pinning can limit device performance whilst creep affects long-term memory stability. In ferro- and ferrimagnets, magnetic domain formation has been extensively studied for decades and is well-known to be largely governed by the minimization of the demagnetizing field energy10. On the other hand, domain formation in fully compensated antiferromagnets remains largely unexplored.A key goal of spintronics is the development of high-speed, high-density data storage devices that are robust against magnetic fields. Antiferromagnetic (AF) materials offer a route to realising these goals since they exhibit intrinsic dynamics in the THz-regime, lack magnetic stray fields, and can be electrically switched15. To date, device concepts have considered an ideal AF spin lattice17, but high-resolution AF domain imaging has revealed pronounced non-uniformities and pinning effects during domain switching20.Domain morphologies in AF thin films vary considerably with thickness and nanostructure shape which has been ascribed to strain effects, although evidence for a direct relationship is lacking21 have recently been identified as the most prominent defects. The anti-phase boundaries have been associated with atomically sharp 180∘ AF domain walls22. The microtwin defects, which are the focus of this work, are shown to have a dramatic influence on the AF domain configuration in CuMnAs thin films. We show that microtwin defects largely control the domain structure by generating pinned 90∘ domain walls and confining 180∘ domain walls.Here, we show the relationship between magnetic domain structures and structural defects in the metallic antiferromagnet CuMnAs, which is a focus of AF spintronics research due to its favorable crystal symmetry for spin-orbit torque switching. In CuMnAs thin films, elongated microtwins and atomically sharp anti-phase boundaries21. The CuMnAs layer is a collinear antiferromagnet with a Néel temperature of 485 K23 and a tetragonal crystal structure . Close lattice-matching along the half-diagonal of the cubic GaP substrate unit cell ensures fully strained epitaxial growth with low mosaicity24. The spin axes align in the (001)-plane, i.e., in the plane of the film25 due to a strong magnetocrystalline anisotropy. In the following, the crystallographic axes refer to the orientation of the CuMnAs crystal.The 50-nm-thick CuMnAs(001) films were grown epitaxially on GaP(001)26. Figure E) aligned to highlight the AF domain structure and domain boundaries, respectively. Maximum XMLD contrast is observed between regions with the local spin axis aligned perpendicular and parallel to E. We observe approximately equal populations of light and dark areas in Fig. The AF domain structure was imaged using high-resolution photoemission electron microscopy (PEEM) combined with X-ray magnetic linear dichroism (XMLD)mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimE is at 45∘ to the local spin axis in both AF domains. In this imaging configuration, the domains have the same contrast and the domain boundary contrast dominates. 90∘ domain walls appear as well separated black or white lines, depending on the average direction of the spin axis across the domain wall, with typical width (400 ± 50) nm. Adjacent black and white lines in Fig. ∘ domain walls  combined with PEEM is sensitive to local changes in the charge anisotropy and can therefore act as an indicator of local crystallographic variations, i.e., structural defects. Figure mentclass2pt{minim∘ domain walls are found to be confined between two parallel defects. These domain walls extend over several microns and can be seen as the long, thin light and dark lines in Fig. ∘ domain walls form corners in areas where two defects are orthogonal, as for example in the bottom half of Fig. Long straight 18027. Crystallographic defects lead to specific contributions to a reciprocal space map (RSM). A three-dimensional RSM of the CuMnAs (003) Bragg peak, generated from two-dimensional diffraction images for several sample tilts CuMnAs 00 Bragg peq-dependence of the scattering revealed by SXDM indicate defect orientations along four different crystallographic directions in the bulk, while the XLD-PEEM images only show their surface termination.Mapping the intensity of these wings generates real-space images of the defects. Maps produced using the intensity of one of the wings yield bright defect lines, on a homogeneous background, which run perpendicular to the direction of the selected wing, see Fig. mentclass2pt{minimc-axis is tilted away from the film normal by ~82∘, as shown in Fig. 21 which can be distinguished in SXDM, but not in XLD-PEEM.High-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) reveals that the defects are slabs of a microtwinned phase, in which the lattice is rotated so that the c-axis25, the ~82∘ rotation of the c-axis in the microtwin region will have a profound effect on the local spin orientation. The microtwin region and surrounding bulk film share only one magnetic easy axis, determined by the intersection of the easy planes  substrate and capped with a 3 nm Al film to prevent surface oxidation for the PEEM imaging.∘ to the film surface. Images were recorded using the ELMITEC-III PEEM on I06 with a hybrid-pixel electron detector . Magnetic contrast with ~30 nm spatial resolution was obtained from the asymmetry between images recorded using photon energies corresponding to the maximum and minimum of the Mn L3 XMLD spectrum (see Supplementary Note E) was in the plane of the film, and the largest magnetic contrast was obtained between AF domains with spin axes parallel and perpendicular to E. The XMLD spectrum has a similar lineshape, but opposite sign, for E∥[110] and E∥[010] (ref. 23). PEEM images with sensitivity to the microtwin configuration were obtained from the asymmetry between images recorded at photon energies corresponding to the maximum and minimum of the Mn L3 non-magnetic XLD spectrum  measured from the direction of the incident beam in the vertical plane, Θ which defines the angle of incidence with respect to the sample surface in the vertical diffraction plane and ϕ which defines the sample azimuth. The sample was scanned laterally over a 2D-mesh at a fixed sample orientation with a diffraction image recorded at each position. The imaging was performed using a Merlin Si Quad area detector with 512 × 512 pixels, each 55 × 55 μm in size. The distance between the detector and sample was 0.65 m.SXDM was performed on the NanoMAX-beamline at MAX IV Laboratory∘ increments. For these measurements, ϕ was chosen such that the X-ray beam impinged along the CuMnAs [110] direction. Analysis was performed using the xrayutilities toolbox described in reference32.RSMs were constructed via several 2D-mesh scans with a stepsize of 200 nm at different Θ angles around the (003) Bragg reflection in 0.02ϕ chosen so that the beam impinged along the CuMnAs [100] direction. The microtwin configuration was mapped with the sample at an angle ΔΘ = ± 0.4∘ from the Bragg angle. For each angle, the detector plane sliced through two of the microtwin-related wings in reciprocal space. Consequently, if a microtwin was in the illuminated area, significantly higher intensity was recorded on the corresponding area of the detector, depending on the microtwin orientation. Mapping these areas of high intensity thus revealed the spatial pattern of microtwins with a specific orientation. The SXDM map shown in Fig. Bragg − ΔΘ and ΘBragg + ΔΘ. For details see Supplementary Note SXDM imaging of the microtwin configuration was performed with 33.For the HAADF-STEM measurements, the CuMnAs samples were capped with an additional 10–15 nm of carbon ex situ and tungsten in situ. Thin lamellae were prepared by a Ga-focused ion beam. The lamellae were polished at 2 kV and 25 pA. The lamellae were investigated using a FEI Titan Themis 60–300 cubed high-resolution (scanning) transmission electron microscope at 300 kV. The atomic model overlay shown in Fig. n in the presence of different microtwin configurations was simulated using the Matlab PDE Toolbox to solve the micromagnetic equationA is the magnetic stiffness, ∇2 is the Laplace operator, andwan, wtw, and wme are the densities of magnetic anisotropy energy of the bulk film, the magnetic anisotropy energy of the microtwin, and the magnetoelastic energy, respectively. Details can be found in Supplementary Note The distribution of the Néel vector Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Video 1


This study proposes the use of a mix composed of chickpea flour and chestnut flour in cookies, aiming to improve their acceptability. Cookie properties and nutritional value were also analysed. The gluten-free cookies were made by using different mixes of chickpea and chestnut flours . Dough rheology and cookie dimensions, texture, external colour and acceptability were evaluated. The presence of the chestnut flour increased the values of G’ and G”, but reduced the loss factor (tan δ) when compared with the doughs made with chickpea flour. Chestnut flour also decreased the diameter and the spread ratio of the cookies, while increasing the hardness and darkening of the cookies. Furthermore, adding chestnut to the flour mixture increased the nutritional quality of the cookies by adding unsaturated fatty acids and fibre. The use of reduced percentages of chestnut flour (25%) resulted in masking the off-flavour of the chickpea flour, which improved the cookie’s acceptability without significantly changing the dough rheology, cookie dimensions, hardness, or lightness. In recent years, there has been an increasing demand on the market for tasty and affordable foods containing nutritional and health benefits. Interest in the use of legume-based ingredients in food formulations is growing, mainly because of their nutritional properties , but alsLegumes are known for their high protein content in comparison with vegetable products, and they are also rich in fibre and bioactive compounds, including enzyme inhibitors, lecithins, folates and phenolic compounds . Legume Unfortunately, there is a lack of information in the literature as regards the reduction or elimination of these off-flavours in pulse-based food products, and traditional methods generally consist of adding sugars, salt, acids or aromas  to mask Cookies could represent a good opportunity to use these flours due to their high level of acceptance by consumers and them having a comparatively long shelf-life. In general, this type of product does not contain a large amount of protein, and so a naturally rich protein flour could be almost exclusively used to improve the nutritional value of this product. Furthermore, a high gluten content and gluten strength may not be necessary, and sometimes not even desirable, to determine the good quality of cookies as regards their textural properties . Research on the incorporation of different pulse flours, namely pea flours, in cookies has been conducted for a long time . Still, Chestnut flour also has nutritional advantages, especially because of its high content of resistant starch  and bioaThis study aims to demonstrate that sugar-snap cookies are a good product to apply chickpea flour in, since this allows for the production of gluten-free cookies with higher protein contents. Meanwhile, the use of chestnut flour can help to mitigate residual off-flavors. For this purpose, cookies were created with different mixes of chickpea flour (CPF) and chestnut flour (CNF) . Chemical analysis was performed to determine the nutritional composition, free sugars, and fatty and organic acids present in the flours and different cookie formulations. Additionally, dough rheology and cookie dimensions, texture and external colour were evaluated to determine the physical characteristics of the cookies. A consumer sensorial test was also carried out to evaluate the acceptability of the different cookies . The fibre enzymatic kit was obtained from Sigma-Aldrich , whereas all the chemicals and reagents were acquired from scientific common suppliers and were of (at least) analysis purity. Chickpea flour was supplied by Molendum Ingredients  and chestnut flour was supplied by Sortegel . The other ingredients were acquired from retail stores and consisted of white sugar , 100% vegetable margarine , sodium bicarbonate  and potable water.Five different cookie samples were made using the following formulation (as g/100 g dough basis): flour (42.8), sugar (30.8), margarine (19.2), water (6.2) and sodium bicarbonate (0.9).The ingredients of the five formulations were added in the same portions, except for the water, which was added to adjust flour moisture content to 15.0%, and the proportions of the flour. One cookie formulation was created with 100% of chickpea flour (100CPF), another with 100% of chestnut flour (100CNF) and the others were produced with flour mixes using different proportions of CPF and CNF: 75%CPF + 25%CNF (75CPF/25CNF), 50%CPF + 50%CNF (50CPF/50CNF) and 25%CPF + 75%CNF (25CPF/75CNF).The margarine was heated in a microwave for 1 min at 1000 W, then, the melted margarine and the sugar were mixed in a Kitchen Aid 5KPM50 mixer  using speed 4 for 3 min, scraping down the batter every 60 s. Then, the water was added and mixed at speed 4 for 2 min. At the end of this phase, flour and sodium bicarbonate were added and mixed at speed 2 for 2 min, stopping every 30 s to scrape down the batter. Afterwards, the dough was allowed to rest at 24 °C for 30 min, prior to laminating (6.00 mm gap) in pieces by a Salva L-500-J sheeter  and cut with a circular cookie shaper with a diameter of 40 mm. Next, the cookies were baked at 185 °C for 14 min. Finally, the cookies were left at room temperature for 60 min and stored at 24 °C in plastic bags for further analysis. All the cookies were prepared in triplicate.−1 of dry weight (DW). Crude protein was estimated as nitrogen content via the macro-Kjeldahl method . Crude protein was expressed as g 100 g−1 of DW. Ash content was determined by incineration at 550 ± 10 °C (AOAC 923.03) in a muffle . The total mineral content was expressed as g 100 g−1 of DW. The carbohydrate content was determined by the difference. The total available carbohydrates were expressed as g 100 g−1 of DW. Dietary fibre content was determined using the Megazyme kit based on the AOAC Method 985.29 and was expressed as g 100 g−1 of DW. Finally, the total energy was calculated using the European Parliament and Council Regulation No. 1169/2011 Formula (1):The nutritional values of the studied flours and cookies were determined based on their fat, protein, ash, carbohydrate, and dietary fibre contents, obtained following the AOAC official methods . Crude fv/v) solution during 30 min at 80 °C. Melezitose was added as an internal standard . The extracts were centrifuged  and the supernatants were collected and concentrated under reduced pressure in a rotatory evaporator. Finally, the samples were defatted in ethyl ether (three times) and filtered using 0.2 µm Whatman’s nylon filters for HPLC analysis. The quantification was based on the RI signal response of each standard, depending on the IS method, and using calibration curves from commercial standards of each compound. The results were expressed in g 100 g−¹ of DW.Soluble sugars were determined using a High-Performance Liquid Chromatography (HPLC) system coupled to a refraction index  detector. Chromatographic separation was achieved with a Eurospher100-5 NH2 column  as comprehensively described by Reis, Barros, Martins, Vasconcelos, Morales and Ferreira . The samFatty acid analysis was performed via gas chromatography coupled to a flame ionisation detector (GC-FID). Fatty acids obtained were subjected to a transesterification procedure  and filt−1 of DW.Organic acids were determined by ultra-fast liquid chromatography  coupled with a photodiode array detector (PDA), which used 215 nm and 245 nm as preferred wavelengths. Samples were subjected to metaphosphoric acid extraction, as previously described by Barros, Pereira and Ferreira . A SpherChickpea and chestnut flours were characterised by their hydration properties. Water holding capacity (WHC) is defined as the grams of water retained per gram of sample when not submitted to stress. WHC was evaluated following the standard method . An amouParticle size distribution was determined using a Mastersizer 3000 particle size analyser . Values of D, which represents the equivalent spherical diameter of the particles, and D(90), which represents the maximum particle diameter, below which 90% of the sample falls, respectively, were obtained. Measurements were carried out in triplicate.Rheological measures of the cookie dough were conducted using a controlled strain rheometer  at 25 °C. Circular dough pieces (3 mm height and 60 mm diameter) were placed on titanium parallel-serrated plate geometry PP60 Ti and compressed with a gap of 3 mm. Vaseline oil  was added to the exposed sample surface to avoid drying during the analysis. For the identification of the LVR (linear viscoelastic region), the first measurement involved a strain sweep test performed a constant frequency (1 Hz) in the range of 0.1–100 Pa. The stress value obtained from the first test was used in a frequency sweep test with a frequency range from 10 to 0.1 Hz, to obtain the values of the elastic modulus (G’ (Pa)), viscous modulus (G” (Pa)), complex modulus (G* (Pa)) and the loss factor (tan δ) (G”/G’). Every sample was evaluated twice.*a*b* colour space. Measurements were made at the centre of the upper surface. The texture of the cookies was measured by a “three-point bending test” with the use of a TA-XT2 texture analyser  running the “Texture Expert” software. The conditions used during the experiment were as follows: supports 30 mm apart, a 20 mm probe travel distance, a trigger force of 5 g and a test speed of 2.0 mm/s. The hardness (N), which is the maximum force used to break the cookies, was obtained.Cookie characteristics were evaluated 1 day after baking. Six cookies of each batch were evaluated. Cookie dimensions were obtained using a calliper. The diameter of each cookie was measured three times, perpendicularly, to obtain the average diameter. To obtain the spread factor, the average diameter was divided by the thickness. The surface colour was measured using a Colorimeter PCE-CSM 1  with the D65 standard illuminant, LED light source type, blue light excitation and a 10º standard observer. The results were expressed in the CIE LA sensory analysis was performed in two parts. In the first part, volunteers evaluated the cookies acceptability according to 5 attributes  using a hedonic scale from 1 (extremely dislike) to 9 (extremely like). The second part aimed to quantify how many consumers would identify the flavour of chickpea or chestnut in the cookies. They received a paper with a table containing six different ingredients  and they were instructed to evaluate the perception of these flavours in each sample by using a scale composed by 0 (“I do not taste this flavour”), 1 (“I taste this flavour and it is slight”) and 2 (“I taste this flavour and it is intense”). These analyses were carried out according to the protocol previously approved by the Committee of Tests and Research from the Hospital Rio Carrión . The tests were made in a tasting room at the university with appropriate light and available potable water. The volunteers were a total of 93 usual cookies consumers whose range of age 18 to 64 years of age. Samples were analysed one week after baking. The five cookies, one of each type, were placed on white plastic plates coded with four-digit numbers and served in random order.t-test for the flour samples after confirming their homoscedasticity. The statistical analysis for the acceptance tests, flour properties, dough, and cookie characteristics were performed using Statgraphics Plus 5.1 software . Differences between the parameters of the different formulations were studied by analysis of variance (One-way ANOVA) using Fisher’s least significant difference test as a post hoc to determine the significant differences between the respective means. All analyses were performed using 95% confidence intervals and represented as average ± SD (standard deviation).Statistical tests for the chemical analysis were using SPSS Statistics . The results were analysed using a one-way analysis of variance (ANOVA one-way) followed by Tukey’s HSD post hoc test for the cookie samples, and a Student’s The nutritional profiles of the flours, the soluble sugars, organic acids, and fatty acids are shown in In terms of the soluble sugars, glucose and trehalose were only detected in chestnut flour, which also recorded higher values of fructose and sucrose, and consequently total sugars.Four organic acids were identified, namely oxalic, quinic, malic and fumaric acids. Quinic was only found in CNF, while malic was only found in chickpea flour. Oxalic was detected in a higher quantity in CPF and fumaric was only found in traces. Overall, chickpea flour showed the highest organic acid concentration.Chestnut flour presented higher quantities of fatty acids, mainly polyunsaturated fatty acids, than chickpea flour. However, chickpea flour exhibited a greater variety of fatty acids and a higher quantity of monounsaturated fatty acids than chestnut flour. The most abundant individual fatty acid in the CPF was oleic acid, while linoleic was the most abundant in the CNF, which did not present any monounsaturated fatty acids, but rather 48% of saturated fatty acids, while chickpea showed 45% and 43% of monounsaturated fatty acids, and 45% of saturated fatty acids, making it a healthier flour. Chickpea and chestnut are good sources of nutritionally important unsaturated fatty acids. Jukanti, Gaur, Gowda and Chibbar  also desAs regards the hydration properties (WHC and SV), no significant differences were found between CPF and CNF, or between these samples and the different flour mixes . Values The rheology of the dough is a characteristic that requires important attention when preparing a cookie formulation. In fact, if the dough is too soft or too firm it is not easy to manipulate, thus, the dough must be sufficiently cohesive to remain united during the process and to be easily laminated, without being so sticky that is becomes attached to the rolling mill. According to Belorio et al.  to make The results obtained from the rheological analysis are shown in The G’, G” and G* values showed a tendency to increase due to an increment in the percentages of chestnut flour in the mixes. However, no significant differences were observed for G’ when doughs were created with mixes containing chickpea flour. As regards G” and G*, no significant differences were found for doughs containing up to 50% CPF. Values of tan δ reduced with increasing percentages of CNF in the formulations. Demirkesen et al.  found a −1 of DW. As expected, the cookies with CNF showed a higher quantity of carbohydrates, while the cookies with CPF had a higher concentration of proteins and fibre. Once again trehalose and glucose were only detected in the cookies with 100% CNF, and the chestnut cookies had the highest soluble sugar quantity with significant differences in comparison with all other samples. In terms of the oxalic acids, the same ones were identified in the cookies, and thus, once again, fumaric acid was only detected in trace amounts or was not detected at all, while malic acid was only detected in the cookies with higher concentrations of CPF and quinic acid in samples with CNF. Oxalic acid was found in higher amounts in CPF cookies, as expected. As regards the fatty acids, contrarily to the flours, the most abundant fatty acid in all cookies was palmitic acid due to its high presence in the margarine that was used to make the cookies. Still, the highest amounts of saturated fatty acids were sought for all the cookies with CPF, while the unsaturated fatty acids were quite similar in all cookies, exception made to the monounsaturated fatty acids of the cookies made with 25% CPF and 75% CNF.The cookies, baked with varying concentrations of CPF and CNF, also underwent a nutritional profile analysis as well as individual compounds such as soluble sugars, organic acids and fatty acids, and are presented in As can be seen in In general, most studies find correlations between the dough rheology and the spread factor, so that when values of G’ and G” are increased, the diameter of the cookies is reduced ,35, simiAdditionally, there is a tendency for the hardness to increase as the content of chestnut flour increases. This finding is also consistent with the result of Dokić et al. , who sho*)  and b* decreased (less yellowish) as the amount of CNF increased. Other authors found similar results when substituting wheat flour [* and an increase in a*, these authors also found an increase in b*. These differences could be related to the different formulations used in these studies. The colour of cookies mainly depends on Maillard reactions, which occur between proteins and reducing sugars, and on sugar caramelization [*)  decreaseat flour  or rice at flour  for CNF.lization . In addilization , which ilization .The results of cookie acceptability are shown in Consumers were asked about the identification of different flavours in the cookies, among different options. They then had to establish if this was slight or intense (data not shown). In the case of CPF cookies, more than 15% of the consumers identified the chickpea flavour and they qualified it as intense in 90% of the cases. The identification of chestnut flavour was smaller (10%) and it was identified as intense in 66% of the cases. Nevertheless, the number of consumers that identified the flavours in cookies produced with flour mixes was even smaller and in most of cases it was qualified as a slight flavour. The percentage of consumers that correctly identified the flavour mix was not greater than 3% for all the cases. In samples with 75% of CPF, 12% of the consumers identified the chickpea flavour, but only 40% qualified it as intense. In mixes made with 50% of CPF and CNF, only 5% of the consumers identified an intense flavour of chickpea. Thus, the use of CNF contributed to masking and mitigating the chickpea flavour, which can contribute to improve cookie quality.The combination of CPF and CNF can generate cookies with better overall acceptability than cookies produced with only one of these flours. The incorporation of CNF generated cookies with a small diameter, that were darker and harder. However, in small percentages, it contributed to improving the acceptability of cookies produced with CPF. Beyond this, the use of CPF and CNF allow for the production of cookies considered as containing “high in dietary fibre”. Finally, the use of reduced percentages of chestnut flour (25%) improved the acceptability of chickpea cookies without significantly changing dough rheology, cookies dimension, hardness, or the lightness of these cookies.


Droplet microfluidics provides a versatile tool for measuring interfacial tensions between two immiscible fluids owing to its abilities of fast response, enhanced throughput, portability and easy manipulations of fluid compositions, comparing to conventional techniques. Purely homogeneous extension in the microfluidic device is desirable to measure the interfacial tension because the flow field enables symmetric droplet deformation along the outflow direction. To do so, we designed a microfluidic device consisting of a droplet production region to first generate emulsion droplets at a flow-focusing area. The droplets are then trapped at a stagnation point in the cross junction area, subsequently being stretched along the outflow direction under the extensional flow. These droplets in the device are either confined or unconfined in the channel walls depending on the channel height, which yields different droplet deformations. To calculate the interfacial tension for confined and unconfined droplet cases, quasi-static 2D Darcy approximation model and quasi-static 3D small deformation model are used. For the confined droplet case under the extensional flow, an effective viscosity of the two immiscible fluids, accounting for the viscosity ratio of continuous and dispersed phases, captures the droplet deformation well. However, the 2D model is limited to the case where the droplet is confined in the channel walls and deforms two-dimensionally. For the unconfined droplet case, the 3D model provides more robust estimates than the 2D model. We demonstrate that both 2D and 3D models provide good interfacial tension measurements under quasi-static extensional flows in comparison with the conventional pendant drop method. Interfacial tension (IFT) between immiscible liquids plays an important role in determining the morphology, stability, structure and dynamics of emulsions and multiphase systems that are used in various scientific and industrial applications, such as oil recovery, cosmetics, pharmaceuticals and food processing ,2,3,4,5.For microfluidic approaches relying on the dynamic droplet deformation to measure IFTs, both empirical scaling analysis  and theoHomogeneous extension for dynamic droplet deformation can be approximated with an optimized microfluidic constriction geometry, however, it requires robust optimization procedures through computational fluid dynamic (CFD) simulation . An alteh are the continuous phase viscosity, the strain rate, the interfacial tension, the initial droplet radius and the channel height, respectively [For confined droplets in a shallow microchannel, the fluid flow with a confined droplet can be described by the Darcy approximation in which the droplet deformation is associated with the Capillary number ectively . All theectively ,33,34. Wh the channel height.For the 2D confined droplet deformation model, we consider the droplet deformation in the presence of quasi-static extensional flows at the cross junction, wherein the depth-averaged velocity of the fluid sed flow . The drolaxation ,35. The l and b are the largest and smallest distances of the droplet surface from its center, p is the pressure. For an incompressible flow, the Stokes equation can be solved by using a complex potential z on the interface Taylor’s pioneering work described the deformation of unconfined droplets in shear flows, with the droplet deformation being described by ribed as (1)u→=−w−1D and confinement ratio D can be measured experimentally by using optical microscopy, and D, Equation  can be sEquation . The intEquation  containsEquation  and compD is the droplet deformation, t is the time, An early study for the droplet deformation under extensional flows was established by Taylor where a four-roller apparatus was used to achieve a distortion of a droplet by viscous forces associated with a continuous phase fluid . The defThe instantaneous deformation of a droplet can be expressed in a convenient form by considering time-invariant extensional flows with the extensional strain rate D < 0.15 for modest deformations before droplets break or burst [The first term on the left hand side (LHS) in Equation  accountsor burst ,33. The We use standard multilayered soft-lithography protocols for fabricating the microfluidic device ,38. A moh = 80 h = 140 Emulsion droplets are first generated at the flow-focusing region by manipulating the flow rates of the continuous  (Qc/Qd) b. The di (Qc/Qd) a, drople (Qc/Qd) c. The th (Qc/Qd) d. Two flh = 80 When the droplets of a dispersed phase are produced at the flow-focusing region, they travel downstream towards the cross junction. The droplets produced in the channel are confined into the channel walls as the confinement ratio  control . For the channel a. At strx and y are the spatial coordinates. For the droplet trapping with the active feedback control, we can transfer the minimum zero-velocity-potential point into the user-defined trapping position along y-direction. The center of the region of interest , wherein the velocity potential ε˙x,ε˙y) ,40. Here˙=83 s−1 b. With tD is dependent on the viscosities of dispersed and continuous phases, interfacial tension, strain rate and droplet confinement. D is measured by varying the strain rate and droplet confinement at fixed viscosities and interfacial tension of fluids. A recent report has shown that droplet shape relaxation in the moderately confined regime exhibited strong dependence on the droplet radius but not on the ratio of dispersed to continuous phase viscosity [D increases with increasing droplet radius at fixed D increases with increasing D typically follows a linear trend on a log–log scale of v = 0.009–0.018 m/s, D on the droplet size  and a lower branch  with a critical D between them. The solution does not exist when D and We perform numerical predictions of the deformation of confined droplets by using the 2D Darcy approximation model at a fixed confinement ratio on of Ca . The uni droplet ,36. At lmentally ,41. Howeormation ,41, whicThe droplet deformation in a microfluidic platform allows one to measure IFTs of immiscible liquid pairs by considering the droplet deformation, confinement ratio, continuous and dispersed phase viscosities based on the force balance between extensional stress and liquid–liquid interfacial tension. We validated the quasi-static 2D model for the measurement of IFTs and compared the results with a conventional pendant drop method .D. The strain rate D measured under the different strain rates in For the 2D Darcy approximation model, we calculated the IFT based on Equation . The keyFor the 3D small deformation model, the IFT of the droplet is measured by using Equation . The IFTTo summarize, we employed the microfluidic approach with confined and unconfined droplet deformations under the quasi-static 2D and 3D extensional flows to measure interfacial tensions of two immiscible fluids. The droplets undergo deformation upon the extensional flows. The 2D Darcy approximation model with the effective viscosity gives a reasonable IFT value even though the model cannot describe the complex 3D effects that occur at the interface between immiscible fluids. The 3D small deformation model also showed good estimation of IFT values when compared to the conventional pendant drop result. These models are based on the force balance between the interfacial tension force and the drag force acting on the droplet. It is advantageous that the influence of confinement extends the utility of this approach to IFT measurements since a single device can be used to study a wide range of droplet sizes. Practically, droplets confined in shallow geometries can be simplified as a disc shape in which the confinement effect plays a key role in the deformation and dynamics of droplets. The 2D deformation model is also useful to understand the confinement effect since the presence of the confinement alters the droplet deformation. However, the 2D model is not desirable when there is a strong interaction between the confining walls and droplets or strong charges exist on the walls. On the other hand, the 3D model requires that the microfluidic channel dimensions are larger than the droplet size. If the droplet size is close to the channel height, it can lead to incorrect prediction of low IFT . In addiThis work investigated the droplet deformation in both quasi-static 2D and 3D extensional flows in a microfluidic platform and measured the interfacial tensions of immiscible liquids based on droplet deformations. The droplets are trapped at the stagnation point with the aid of the active feedback control of the pressure valves, and stretched along the outflow direction upon the extensional flow. IFT calculation based on the 2D Darcy approximation with the effective viscosity shows good agreement with the pendant drop method. This 2D droplet deformation model is a useful tool to measure interfacial tensions in-situ and in real time in certain applications where the channel geometry is fixed and the droplet sizes are comparably larger than the channel height. The 3D small deformation model captures the interfacial force balance on the droplet surface, which gives more robust theoretical calculation than the 2D model. We confirmed that both 2D and 3D models provide good interfacial tension measurements in comparison with the conventional pendant drop method. This work will help develop not only the generation of emulsions on chip but also incorporate knowledge to conduct in-situ, real time interfacial tension measurement on a single platform. Further, it will provide valuable insights to understand the confined and unconfined models for the droplet deformation and interfacial tension dynamics of immiscible liquids in droplet microfluidics.


Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD–PCα region in solution, at the single residue level by NMR and at lower resolution by complementary biophysical methods. We probed POD–PCα inter-domain contacts and showed that small molecules were able to modify the dynamics of PCα. These structural properties are fundamental to the peculiar binding mode of RSV phosphoprotein to L, where each of the four protomers binds to L in a different way.The phosphoprotein P of  A number of viral proteins are intrinsically disordered proteins (IDPs) or contain large intrinsically disordered regions (IDRs) ,2,3. ComMononegavirales (MNV) order, i.e., non-segmented negative strand RNA viruses, has been widely investigated from a functional and a structural perspective ,41Cα, beOD and PCα domains in unbound RSV P. PCα is a neither fully disordered nor globular region, but situated in the continuum between these two extremes (OD is the only stably folded part of the protein. However, even the boundaries of POD are fluctuating. Structure resolution by solution NMR showed that the C-terminal end of the coiled coil tetramer may extend up to residue V154 (Cα domain was necessary to promote α-helicity, since PCα did not adopt its native conformation in the truncated P127-184 fragment (C1 region displayed the highest α-helical propensities (C1 was also singled out in high-pressure NMR experiments by higher linear and quadratic pressure coefficients (C1 region is the only one adopting α-helical secondary structure in all four protomers in the RSV L—P complex [C1 pre-exists in unbound P, in equilibrium with a more disordered state shifted towards the ordered state.Here we investigated into more details the structure and dynamics of the consecutive Pextremes . POD is due V154 , whereasfragment . The bouensities . αC1 wasficients . The αC1 complex ,42. FromCα domain makes transient contacts with POD, leading to compaction of the P127–205 fragment. The overall size of the structural models derived from PRE analysis (Ctail, are fully disordered [By using paramagnetic labels, we showed that the Panalysis  agrees wsordered , we may Cα domain might be targeted by small molecules to modify its dynamics Cα domain, plays a crucial role for the L—P complex formation, i.e., for a functional viral polymerase. This now needs to be verified for the full-length P protein using molecules with demonstrated antiviral activity.Finally, we showed that the P


Using speckle patterns formed by an expanded and collimated He-Ne laser beam, we apply DIC  methods to estimate the deformation of LLDPE (linear low-density polyethylene) film. The laser beam was transmitted through the film specimen while a tensile machine applied a load to the specimen vertically. The transmitted laser light was projected on a screen, and the resultant image was captured by a digital camera. The captured image was analyzed both in space and frequency domains. For the space-domain analysis, the random speckle pattern was used to register the local displacement due to the deformation. For the frequency-domain analysis, the diffraction-like pattern, due to the horizontally-running, periodic groove-like structure of the film was used to characterize the overall deformation along vertical columns of analysis. It has been found that when the deformation is small and uniform, the conventional space domain analysis is applicable to the entire film specimen. However, once the deformation loses the spatial uniformity, the space-domain analysis falls short if applied to the entire specimen. The application of DIC to local (windowed) regions is still useful but time consuming. In the non-uniform situation, the frequency-domain analysis is found capable of revealing average deformation along each column of analysis. Commercial shipping facilities commonly use linear low-density polyethylene (LLDPE) for packaging freight. For efficiency, wrapping machines are responsible for packaging shipments by rotating thin LLDPE film around the objects. These machines perform this action as quickly as possible to optimize both time and cost of the process. At increased wrapping rates, however, LLDPE film stretches more, ultimately leading to its failure.The unique characteristics of this film make the material advantageous and multi-functional across industries. LLDPE is a thermoplastic polymer characterized by a predominantly linear backbone and a high proportion of short branches. Due to the material’s structure, it cannot be packed tightly, which gives it a hazy, transparent appearance. In comparison to traditional low-density polyethylene, LLPDE has a crystalline structure with little to no elastic memory or recovery, resulting in increased tensile strength and elongation . During While many non-destructive optical testing methods are available to evaluate material deformation ,3, speckTraditionally, the subtraction method used in speckle pattern studies ,3 retrieThe present study applies conventional DIC methods ,11 usingWe find that we can use the periodicity of the grooves to estimate the film’s deformation. In the frequency domain, this quasi-constant interval produces fairly sharp peaks in the Fourier spectrum. As the film stretches, the interval between linear dark-fringes increases, which shifts the peak frequency on the Fourier spectrum. From this shift, we can estimate the amount of stretch ,14. HereThe linear backbone and short branches of the LLDPE molecules align in the direction of spooling during the extrusion manufacturing process, resulting in periodic structural grooves observable on the film’s surface, as illustrated in When an external load stretches the film, the linear dark-fringes and speckles change independently. Recall, The random nature of speckle patterns on the projected image makes application of traditional optical interferometric techniques difficult based on the phase of the source-light such as holographic inteferometry ,17. SpecThe experimental setup used for data collection consisted of several parts. Initially, several LLDPE film samples with thicknesses of 10 µm and 20 µm were cut into 4 cm squares and attached to an ADMET tensile machine’s stationary and dynamic grips. Recall that the film’s surface structure contains grooves in line with its spooling direction. Thus, in separate experiments, the samples were oriented so that these striations were perpendicular  and parallel to the pulling direction .A 17 mW Helium-Neon laser provided the light source for experiments. The laser beam expands as it propagates, and collimation is necessary for obtaining accurate interferometric patterns. Initially, we suspected that polarization could affect the film’s transmission characteristics, which was investigated by varying the polarization. We did not find a noticeable effect of polarization, which lead us to the experimental setup shown in Additionally, the spot size of the beam is increased by a factor of 10. The beam expander consists of two lenses with focal lengths of 25 mm and 250 mm. The radius of the laser beam on the specimen is 5 mm and covers approximately 20% of the film’s surface. Additionally, the beam’s waist size is 0.5 mm. Multiple studies including beam spot size measurements along the propagation axis and cross-sectional beam profile measurements using a charge-coupled device camera verified that the laser beam is in the Gaussian mode (TEM00) . We adjuDigital images were taken as the tensile machine pulled the specimen. The digital camera was set to take images as the material stretched in elongation intervals of 50 µm. The image correlation procedure, described in the next section, compares pairs of images to evaluate the corresponding deformation. The camera specifications follow in l is the initial length [The following method finds the average stretch, or normal strain, over a column (or row) of a projected image. Here, the normal strain is defined by Equation , where dl length .(1)ϵ=dlThe four steps below describe the analytical process for 1D analysis in the space-domain.1.Stretch the original, un-stretched image by artificially stretching the axis for a given scaling factor. 2.Compare the physically stretched image with the artificially stretched image by computing the correlation coefficient Equation  over a ca and b are the column (row) vectors containing gray-scale values of un-stretched and stretched image. In Equation cov as the stretch factor that maximizes the cross-correlation.4.Repeat 1–3 for all the columns (rows) to find the average stretch  for each column (row).x before the stretch and x after the stretch.y-axis by This method is generally known as the convolutional DIC technique . A smallstretch.(f★g)(px)e kernel . By examinput and output signals, respectively, where the input function is the image before elongation, and the output function is the image after elongation.For clarification, the DIC process using Equation  in a numThe one-dimensional DIC method discussed above uses the speckle pattern along the entire length of a column of interest. This method works well when the speckle patterns change in the same fashion as the artificial elongation, which assumes uniform stretch. In reality, the deformation of the film specimen becomes non-uniform at a low level of elongation. This situation makes it difficult to apply the one-dimensional DIC method to a realistic situation where deformation of the wrapping film undergoes non-uniform stretching. The convolutional DIC method is applicable in this situation; however, it is unrealistic and time-consuming.The frequency domain method solves the problem by utilizing differentiation and scaling properties of the Fourier transform. When deformation of the film specimen is not uniform at the local level so that the one-dimensional space domain method is not applicable, it is often the case that the deformation is uniform enough at the global scale. The periodic groove-like structures can serve as a gauge to evaluate overall deformation. Under this condition, the periodicity of dark-fringes due to the groove-like structures form a quasi-single peak in the frequency domain. As the film stretches, the peak shifts on the frequency axis, which can be interpreted as scaling in the space domain. In this situation, we can estimate the scaling factor in the frequency domain as follows.x-axis, Let function actor α.Gα(ω)=FdfEquation  indicateWe took the following steps to implement the above algorithm.1.Gaussian filter the optical image projected on the screen. In this algorithm, the local speckles due to the scattering of the film material become noise while the periodic pattern of the linear dark-fringes produces the signal. Since the local speckle pattern has higher spatial frequency than the linear dark-fringes, low-pass filter the optical image to increase the signal-to-noise ratio.2.Numericaly differentiate the gray-scale value of the original image with respect to the coordinate variable that is set parallel to the tensile axis. This step is to evaluate Equation .3.Take the FFT of the differentiated gray-scale obtained in step 2. Call this resultant spectrum the original Fourier spectrum. Numerically integrate the original Fourier spectrum for a selected spatial frequency range. This step is to evaluate the area enclosed by the Fourier spectrum and the frequency axis. Call the resultant value the original spectrum-frequency area. The frequency range for this integration should contain the spectral peak and exclude the high frequency region removed by the Gaussian filter. Since the optical intensity varies at each image captured for various reasons such as the change in the background optical intensity, normalize the spectrum-frequency area for the selected frequency range by dividing it by the Fourier spectrum-area of the entire frequency range.4.Repeat steps 1–3 after the specimen stretches to the current elongation. This process yields the Fourier spectrum for a given stretch factor and the corresponding (current) spectrum-frequency area. Call the resultant spectrum-frequency area the current normalized spectrum-frequency area. Iterate this step for other stretch factors by further elongating the specimen. This procedure yields multiple current normalized spectrum-frequency areas.5.Compare the current normalized spectrum-frequency areas obtained in step 4 with the original normalized spectrum-frequency area. From the ratio of the current spectrum-frequency area to the original spectrum-frequency area, determine the axis compression factor We present the actual process of the above steps in Prior to the application of the above method, we estimate the horizontal and vertical displacement of the image The difference in the coordinates of each reference point gives us the displacement vector. From The correlation data in According to These observations indicate that the intensity correlation in the space-domain is not a good method to evaluate the stretching factor for an entire column or row.It is seen that along column 200 the displacement vectors are oriented at approximately 45The differences in orientation between vectors can be attributed to a variety of factors, including error. However, preliminary results from our thermal imaging study, presented in High frequency speckles (the speckle noise) superposed on the linear dark-fringes compromises the accuracy of this method. Gaussian filtering reduces the speckle noise and, therefore, is an effective way to process the image prior to applying this technique. Choosing a good frequency range for the analysis is not straightforward. One idea is to use the frequency range that the filtering does not alter. It is seen that the frequency range of 1–10 pixelAccording to the above argument, we use the frequency components in the range of 1–10 pixelUsing the analytical steps described in The above procedures yield Since the y-intercept of this line represents the FFT spectrum area before the specimen stretches, the division of all other data points by the y-intercept provides us with the axis compression factor In summary, this study applies DIC and speckle pattern techniques to characterize unique patterns observed in LLDPE film undergoing tensile deformation. This insight is of particular interest when considering desirable financial outcomes in industries like commercial shipping that aim to optimize time, cost, and efficiency. Images of the film’s surface undergoing deformation were projected onto a screen using a linearly polarized, collimated Helium-Neon laser beam. Resulting digital images contain speckle and diffraction-like fringe patterns and were used in both the spatial and frequency domain analyses as well. In the space domain, the random speckle pattern was used to register local displacement generated from the deformation. The fringes, however, exhibit periodic features consistent with the structural grooves due to the polymer arrangement on the film’s surface, and compromises the registration of local displacement. These periodic groove patterns are used in the frequency domain analysis. Conventional DIC in the space domain applies when deformation is uniform over the entire specimen or analysis is limited to small, localized regions. Overall, spatial DIC is found to be unreliable and inefficient for the present application. The frequency domain analysis, however, is found to be capable of revealing average global deformation under non-uniform conditions. Ultimately, we conclude that analysis in the frequency domain using the linear groove patterns is superior to the traditional methods because it is capable of revealing the global average deformation.More quantitatively, the study has led to the following findings.1.The space-domain one-dimensional DIC exhibits an order of magnitude smaller  strain than the expected value at the 1% or lower strain level. The speckle patterns lost correlation when the strain level becomes approximately four times higher. We suspect the reason behind this finding is as follows. The speckles are formed by the diffusive nature of the transmitted light due to the randomness of the short branches that form the polymer. When the film specimen stretches these short branches shift depending on their original orientations. Therefore, the shift of the associated speckle patterns are not necessarily in line with the direction of the stretch. Hence, at a certain point of elongation, the speckle pattern starts to change randomly.2.Frequency domain analysis appears superior to its space-domain counterparts. Unlike the speckle patterns due to the random structure of the polymer, the frequency domain analysis uses the periodic structural grooves. Consequently, the linear dark-fringes resulting from this periodic structure correlates well with the stretch of the film specimen. The random speckle patterns compromise this correlated change in the linear dark-fringes. Proper low-pass filtering diminishes this compromising effect. In the present case, Gaussian filtering with the standard deviation of 20 is found effective. With this configuration, the estimated global strain shows reasonable agreement with the expected strain level at least up to 1.4%.3.The two-dimensional DIC based on the convolutional algorithm is found effective to some extent. However, some of the vectors in the resultant local displacement field exhibit seemingly incorrect directions. Whether these vectors are incorrectly produced by the algorithm or possibly representing the actual material’s behavior is an open question. The preliminary result of additional thermal imaging experiments indicate a similar behavior of deformation. It is the subject of our future research.


Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors. Patients with hemophilia A are prone to uncontrolled bleeding events and require regular infusions of recombinant or plasma-derived fVIII to maintain functional coagulation , 2. In ain vitro . Immune in vitro , but canin vitro .Coagulation fVIII is a multidomain glycoprotein that circulates in the bloodstream as a heterodimer of the heavy chain A1-A2) and light chain (A3-C1-C2) while bound to von Willebrand factor (vWf) to prevent premature clearance and/or degradation  and ligh. Once clPrevious studies indicate the A2, C1, and C2 domains to be highly immunogenic , 12–15. AB). Our structure represents the first crystal structure of a fVIII replacement therapeutic bound to an anti-C2 inhibitor, providing insight into how non-classical inhibitors alter the C2 domain conformation of mature fVIII and allosterically influence the thermal motion of nearby epitopes. These results illustrate how fVIII replacement therapeutics can be designed to reduce inhibitor binding and fVIII antigenicity.The pathogenic, non-classical murine monoclonal antibody inhibitor G99 is a group BC inhibitor with specific inhibitory activity of 15,000 Bethesda units per mg IgG . The G992 and 350 mM NaCl at -80°C.ET3i was expressed and purified as previously described , 26 to aAB fragments were prepared as previously described  according to the manufacturer’s protocols. Purified FAB fragments were stored at a final concentration of 10 mg/mL at -80°C in FAB storage buffer .G99 monoclonal antibodies were expressed and purified in hybridoma cell lines and Fescribed , 20. BriAB complex was formed at a 1:1.2 stoichiometric ratio in 50 mM Tris HCl (pH 7.4), 200 mM NaCl, and 2.5 mM CaCl2 and purified using a 100 kDa MWCO spin column (Amicon) to 1 mg/mL. Initial crystal conditions were determined via high-throughput microbatch crystallization using the Hauptman-Woodward High-Throughput Crystallization Center   . Diffracalo, NY) . Phasingalo, NY) , 30. Modalo, NY) . All figwork/Rfree values of 0.2998/0.3384 (PDB ID: 7KBT)   Table S1D: 4KI5)  with a r4KI5)    suggests2). In addition to the free ET3i and ET3i:G99 crystal structures, we also included the crystal structure of ET3i complexed with the anti-C1 domain inhibitor 2A9 (PDB ID: 7K66) in our analysis as the C2 domain in this structure undergoes a similar translocation (B’) were calculated for each atom using the following equation:To further investigate how inhibitor binding influences the flexibility of C domain epitopes, we performed a comparative B-factor analysis on the C1 and C2 domains from several crystal structures of ET3i bound to antibody inhibitors as well as the free ET3i structure  and provlocation . To bettB is the raw atomic B-factor from the crystal dataset, Baverage is an average of the atomic B-factors of the ET3i molecule in the crystal asymmetric unit, and Resolution is the reported atomic resolution for the respective structure. Model A of the free ET3i crystal structure, which has two ET3i molecules in the ASU   can be found in the article/ER planned experiments, performed experiments, analyzed data, and assisted in writing the manuscript. SP, JG, and CW performed experiments and assisted in analyzing data. HS, CD, and PL developed expression and purification procedures for ET3i and G99. PS and KC planned experiments, analyzed data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The Pilatus detector on 5.0.1. was funded under NIH grant S10OD021832. The ALS-ENABLE beamlines are supported in part by the National Institutes of Health, National Institute of General Medical Sciences, Grant P30 GM124169. This Work Was Supported by the Dreyfus Foundation (Henry Dreyfus Teacher-Scholar Award), the National Science Foundation (MRI 1429164) and the National Institutes of Health/National Heart, Lung and Blood Institute .PL is inventor on a patent application describing ET3i and is an inventor on patents owned by Emory University claiming compositions of matter that include modified fVIII proteins with reduced reactivity with anti-fVIII antibodies. CD, PL and HS are cofounders of Expression Therapeutics and own equity in the company. Expression Therapeutics owns the intellectual property associated with ET3i. The terms of this arrangement have been reviewed and approved by Emory University in accordance with its conflict of interest policies.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.