Structural insights into the cPLA2α preference for phosphatidylcholine (PC)-enriched membranes have remained elusive. Here, we report the structure of the cPLA2α C2-domain (at 2.2 Å resolution), which contains bound 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and Ca2+ ions. Two Ca2+ are complexed at previously reported locations in the lipid-free C2-domain. One of these Ca2+ions, along with a third Ca2+, bridges the C2-domain to the DHPC phosphate group, which also interacts with Asn65. Tyr96 plays a key role in lipid headgroup recognition via cation–π interaction with the PC trimethylammonium group. Mutagenesis analyses confirm that Tyr96 and Asn65 function in PC binding selectivity by the C2-domain and in the regulation of cPLA2α activity. The DHPC-binding mode of the cPLA2α C2-domain, which differs from phosphatidylserine or phosphatidylinositol 4,5-bisphosphate binding by other C2-domains, expands and deepens knowledge of the lipid-binding mechanisms mediated by C2-domains.Ca Mechanic domain . Complexe region . Occupat2-domain .2+, cPLA2α activators include specific lipids. Mutational functional analyses have revealed that ceramide-1-phosphate (C1P), a bioactive sphingolipid generated by ceramide kinase in the trans-Golgi, enhances enzyme translocation to perinuclear regions (2) also activates cPLA2α, but independently of intracellular Ca2+ concentration  that interact with dodecylphosphocholine micelles  substrate  and the known preference of cPLA2 for phosphatidylcholine (PC)-enriched membranes.Structural insights into cPLAmicelles . Hydrogeubstrate . Nonethe2α C2-domain bound to 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). In contrast to the two bound Ca2+ ions reported in the lipid-free structure 3] group. Two of the three bound Ca2+ ions provide bridging interactions between the C2-domain and the DHPC phosphate group, which also interacts directly with Asn65. The DHPC-binding mode of the cPLA2α C2-domain differs substantially from that of the PKCα C2-domain or the Syt1 C2A-domain bound to phosphatidylserine (PS) or PIP2, thereby expanding and deepening our knowledge of the lipid-binding mechanisms that are mediated by the C2-domain.Here, we report the X-ray crystal structure of the cPLAtructure , we obse2α, we initially attempted to generate complexes of the human recombinant protein with various lipids including PC, related phosphoglycerides and C1P analogs. Despite extensive crystallization trials, the resulting C2-domain crystals contained two bound Ca2+ ions, but no bound lipid  as a key specificity determinant for the phosphorylcholine headgroup of PC, whereas Asn65 tethers with the lipid phosphate moiety and facilitates CaPC complexation. Notably, Ca4 also functions to tether the PC phosphate moiety to the protein. Bridging by CaPC to the PC sn-2 chain ester linkage further aids enzyme binding and ‘scooting’ on PC membranes. Further enhancement of C2-domain binding to the membrane, via interaction with Arg59, Arg61 and His 62, is expected when PC-enriched membranes also contain C1P. Taken together, the findings emphasize the unique design features associated with cPLA2α C2-domain structure and function, as well as the versatility of lipid recognition exhibited by different C2–domains. In cPLA2α, the C2-domain is structurally designed to target PC-rich membrane regions in order to increase the enzymatic efficiency of the catalytic domain, which prefers polyunsaturated PCs.Our findings support a function for cPLA2+-bound crystal structure  ligation into a modified pET-28-SUMO vector (kanamycin-resistance). Prior to insertion, the open reading frames were mutated to remove a single BamHI restriction site within the C2-domain ORF without changing the protein sequence. The complete protein sequences for cPLA2α from human, mouse, and chicken are provided in The C2-domain sequence used to achieve solubility during expression and to avoid the need for protein refolding was developed by consideration of the original lipid-free, Catructure  that use1–140 or C2-domain15–140 expression resulted in mostly insoluble protein located in inclusion bodies.C2-domain17–140 expression resulted in soluble protein but with a SUMO tag that was inaccessible to cleavage.C2-domain17–140 with an inserted Gly or Met residue at the N-terminus (C2-domain Gly/Met-17–140) resulted in good expression of soluble protein and cleavage of the SUMO tag.Expression of C2-domain16–140 resulted in good expression of soluble protein and cleavage of the SUMO tag.Expression of C2-domain2 and stored at −80°C until use.Protein expression was performed at 20°C in Luria-Bertani medium containing 0.1 mM isopropyl-β-D-thiogalactopyranoside. Cells expressing the C2-domain were harvested, suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 500 mM NaCl and disrupted by French press. After ultracentrifugation, the supernatant was applied onto a Ni-NTA resin (Qiagen) and treated with Ulp1 to remove the N-terminal His6-SUMO tag. Eluted proteins were further purified by anion exchange  and gel filtration  chromatography. Purified protein was concentrated up to 20 mg/mL in 20 mM MES-NaOH buffer (pH 6.0) containing 100 mM NaCl and 2.5 mM CaClGly-17–140, the only resulting protein crystals contained two bound Ca2+ but no bound lipid. Successful crystallization of C2-domain containing bound DHPC and three bound Ca2+ions was obtained with the chicken C2-domain16–140. The best crystal complexes were obtained from solutions containing 1 mM protein, 5 mM DHPC and reservoir solution containing 100 mM HEPES-NaOH buffer (pH 7.0), 1.4 M MgCl2 and 0.6 M NaCl at 20°C. Crystal complexes were transferred into a cryoprotective solution containing saturated NaCl and flash-cooled at 100 K. X-ray diffraction data were collected at 100 K on 24-ID-C beamline at the Advanced Photon Source. Data were processed and scaled using HKL-2000  with commercial crystallization solution kits, JCSG Core Suite I-IV and PACT Suite (QIAGEN). Despite extensive crystallization trials with human C2-domainHKL-2000 . The cry2α C2-domain (PDB: 1RLW) as a starting model. The built model was refined using alternating cycles of the Coot 3 and phosphate groups of the DHPC head group, as well as extra density corresponding to the ester groups of fatty acid chains rather than to the morpholino and sulfate groups of MES. When we tried to place the MES molecule at this position, the extra strong density corresponding to fatty acid chains stood out. Thus, we identified our structure as DHPC bound to C2–domain.Electron density mapping of the lipid ligand involved consideration of the chemical structures of both DHPC and MES because MES was included in our crystallization buffer. The omit map  clearly α-atom positions by the PDBeFold (http://www.pymol.org/) and UCSF Chimera 1.11.2 (http://www.cgl.ucsf.edu/chimera/).Multiple sequence alignment was performed by CLUSTALW . PairwisPDBeFold  in conjuPDBeFold  and stru2). Unilamellar POPC vesicle preparation by POPC/DHPC bicelle mix dilution is detailed in 2+ was varied. FRET measurements were performed at 25°C in a temperature-controled (±0.1°C) cuvette  using a SPEX FluoroLog-3 spectrofluorimeter (Horiba Scientific). Excitation and emission wavelengths were 284 nm and 520 nm, with band-pass settings of 5 and 10 nm, respectively. Inner filter effects were avoided by using low protein concentration . Relative FRET was calculated as (Iobs – Imin) / (Imax – Imin), where Imin is the dansyl emission in the absence of Ca2+ and Imax is the maximal energy transfer obtained from the binding curve. FRET fluorescence data were plotted as the relative fluorescence signal versus PC concentration and fitted to the equation described by Partitioning of C2-domain and various point mutants to PC model membranes was monitored by FRET and SPR. FRET measurements were performed using Trp/Tyr emission of C2-domain as energy donor and dansyl-PE-POPC-DHPC (5:45:50) bicelle-dilution vesicles as energy acceptors. Bicelle-dilution vesicles were formed by mixing the POPC, dansyl-PE and DHPC in chloroform, drying under a stream of nitrogen and placing under vacuum for ~2 hr, before resuspending in buffer , and Kd values were determined by nonlinear least-squares fitting using the equation: Req = Rmax/(1 + Kd/C). Each data set was repeated three times to calculate a standard deviation value.SPR measurements were performed using a Biacore T200 system  at 25°C under previously described conditions . An uncooccurred  but the 2 as previously reported (2 (10 μM free Ca2+), 10 μM EGTA, 4 mM DTT, and 2.39% H2O. For K0.5 analysis, micelles were created by drying down ten separate concentrations of PAPC by nitrogen and then reconstituting in 8 mM Triton-X100. Micelle concentrations are as follows: 50 μM PC (2.4% mol PC), 100 μM PC (4.7% mol PC), 150 μM PC (6.9% mol PC), 200 μM PC (9.1% mol PC), 300 μM PC (13% mol PC), 400 μM PC (16.6% mol PC), 500 μM PC (20% mol PC), 800 μM PC (28.6% mol PC), 1200 μM PC (37.5% mol PC), and 1600 μM PC (44.4% mol PC). For KsA analysis, all six concentrations of micelles were created by first making a 1600 μM PC micelle by drying down PAPC and reconstituting in Triton-X100. The 1200 μM, 800 μM, 500 μM, 300 μM, and 100 μM PC micelles were made via serial dilution of the 1600 μM micelle with LCMS-grade H2O.A mixed micelle was utilized to measure PAPC hydrolysis by cPLAreported . Briefly2α (250 ng) was mixed with 30% glycerol and 80 mM HEPES. Assay buffer (25 μL) was then combined with micelles (25 μL), followed by protein mix (50 μL) to produce a final reaction volume of 100 μL. A total of five reactions were prepared for each of the ten concentrations. Immediately following enzyme addition to the micelle – assay buffer mixture, a timer was started and mixtures were placed into a 37°C bead bath. At 15, 30, 45, and 60 min, a 100 μL aliquot was pipetted from each concentration into 500 μL of MeOH containing 10 ng of arachidonic acid-d8 (AAd8) to quench the reaction. Samples then were assessed for PAPC hydrolysis via UPLC-LC/MS. Kinetic and statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc).To prepare for enzyme for assays, cPLAhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951269/). AA and AAd8 were separated with an Acentis Express C18 HPLC Column 10 cm x 2.1 mm, 2.7 μm via UPLC using a Shimadzu 2-D UPLC Nexera System in conjunction with a QTRAP 5500 Mass Spectrometer (AbSciex). Mass spectrometry parameters were: Polarity, Negative; Ion Source, Electrospray; Q1 Resolution, Unit; Q3 Resolution, Unit; Curtain Gas, 30; Collision Gas, Medium; IonSpray Voltage, −4500; Temperature, 500; Ion Source Gas 1, 40; Ion Source Gas 2, 60; Entrance Potential, −13. MRM transitions with corresponding declustering potentials (DP), collision energies (CE), and collision cell exit potentials (CXP) were: AA Q1 Mass (da), 303.2; Q3 Mass (da), 259.2; DP, −150 volts; CE, −17 volts; CXP, −14 volts. AAd8 Q1 Mass (da), 311.2; Q3 Mass (da), 267.3; DP, −150 volts; CE, −18 volts; and CXP, −16 volts.AA and AAd8 were purchased from Cayman Chemicals and analyzed using an adapted method from our previous report . The data reveal the positioning of the bound PC headgroup and identify a novel calcium bridge between cPLA2's C2 and PC's phosphoryl group. This study improves our understanding of how calcium and lipid coordination can be mediated in different ways in C2 domains of different proteins. The reviewers all agreed that the data were of high quality and that the findings were significant, but also raised some points that need to be addressed.This study reports structural data which explains the selective binding of phosphatidylcholine (PC) to the C2 domain of cytosolic phospholipase AEssential revisions:1) Please address the following questions about PC selectivity:The positively charged choline headgroup of PC appears to lie near the positive calcium charge of Ca1. Is it possibly more favorable to have anionic lipid here (PS due to size or even PE due to size/shape)? The authors discuss this in Figure 4—figure supplement 1 by showing different lipid structures and discussing why they may or may not bind compared to PC.But, could the new structure here actually be revealing something more? Since PC has been studied as the bulk lipid that binds and PC was used to crystallize, perhaps the field has been biased against lipid mixtures ?The authors suggest PS will have some repulsions due to the anionic headgroup but it is difficult to see this. Also, the phosphoryl group of anionic lipid could be stabilized by the calcium bound to the C2 domain.Similarly, the PC headgroup is close to His62, which under some conditions could be partially protonated (~pKa near 6).2) As written, the reduction in binding and/or activity by Y96A or N65D doesn't really prove they are involved in PC selectivity, it just demonstrated mutations reduce affinity and activity. To prove/demonstrate selectivity, one would have to compare the mutants and WT to lipids under these different conditions. The authors may want to consider rephrasing/rewriting these statements based upon their findings and that of the previous literature. For instance, although SM binding is low, what would the effects of mutations be? Would there be selective reduction in PC binding vs. that of SM?2 vs. that of PC or other lipids?Also, the SM vs. PC comparison is good and the authors are right that it hasn't been addressed much before. That being said, is SM relevant for Golgi or ER targeting of cPLA3) Please address the following points regarding previous work in the field:2 activity.The Introduction (paragraph three) points out that the lack of a lipid-bound crystal form of the C2 domain has hampered understanding of the structural basis of lipid activation mechanisms. While this current study does provide the lipid-bound structure to advance the field, it may be worth pointing out how the work of Ed Dennis and colleagues (among others) using molecular dynamics and hydrogen-deuterium exchange mass spectrometry has helped the field in understanding cPLA2 demonstrating a reduction in affinity for DHPC and a reduction in activity, which is confirmed in the current study. Similarly, it was shown that N65A reduced cPLA2 DHPC affinity and enzyme activity.More details should be incorporated on the work done on Y96A and N65A by Bittova et al., 1999, as well as the role of N65A in membrane binding and penetration by Stahelin and Cho, 2001. and work by Burke et al. 2008. Bittova et al. studied the Y96A effects in full-length cPLA2? If not chicken, can the C2 chicken structure and lipid binding be directly compared to its influence on the C2-catalytic role in rat or human? If full-length chicken cPLA2 was used, how does its catalytic domain sequence compare to rat and human, and how does its activity compare to those published previously for the rat or human enzymes?4) Were the studies with full-length protein pursued with rat, human or chicken cPLA2 (rat) needed to be refolded from E. coli expression due to solubility issues. The authors have made great strides here in expressing the C2 domain from chicken cPLA2 in soluble form. This could be of interest to the field so a bit more detail on this solubility in the Materials and methods would be useful.Classically, the C2 domain of cPLA2 of chicken with rat and human species.It would be helpful to have the alignment of the full cPLA5) In Figure 5, where the authors model the crystal structure onto the schematic drawing and compare to EPR results, it would be more informative if distances are compared. For example, distances from x-ray structure, headgroup dimensions, EPR data can be compared. How deep in the membrane does the C2 domain go from experimental studies?6) The binding of DHPC in one site is well appreciated but it is expected that the C2 domain will make contact and interact  with a number of lipid head groups around CBLs 1 and 3. Can this briefly be incorporated into the discussion model?Also, in the crystal lattice, do the hydrophobic side chains of the lipid molecule interact with each other?7) Synaptotagmin 1 C2A domain binds to three calcium ions. Can the authors comment and compare the structures?8) In the Materials and methods, the authors mention C1P purchased from Avanti but it is not clear when or how they were used in the paper? In crystallization attempts? Essential revisions:1) Please address the following questions about PC selectivity:The positively charged choline headgroup of PC appears to lie near the positive calcium charge of Ca1.Admittedly, the viewing angles presented for the C2-domain/DHPC complex are not very clear about the distance between Ca1 and the choline headgroup of PC. To clarify this issue, we have now added the distances between Ca1 and the DHPC choline nitrogen (5.5 Å) and between Ca1 and the DHPC phosphate (5.7 Å) to Table 1. Such distances are not indicative of interactions.Is it possibly more favorable to have anionic lipid here (PS due to size or even PE due to size/shape)? The authors discuss this in Figure 4—figure supplement 1 by showing different lipid structures and discussing why they may or may not bind compared to PC.But, could the new structure here actually be revealing something more? Since PC has been studied as the bulk lipid that binds and PC was used to crystallize, perhaps the field has been biased against lipid mixtures ?The authors suggest PS will have some repulsions due to the anionic headgroup but it is difficult to see this. Also, the phosphoryl group of anionic lipid could be stabilized by the calcium bound to the C2 domain.2α C2-domain for POPS, POPA, POPG, and POPI compared to POPC (subsection “Lipid specificity of cPLA2α C2-domian”). Unfortunately, technical problems prevented acquisition of reliable data for pure POPE as explained in the text. To circumvent the pure POPE problem, this lipid was mixed with POPS or POPC to assess the effect on C2-domain interaction. Whereas no interaction of PE could be detected in the presence of PC, a weak positive response was elicited in the presence of PS. Our findings are supported by earlier studies of refolded cPLA2α C-domain by Nalfeski et al., 1998, who reported strong binding to PC, but much weaker binding to vesicles composed of POPS and various other phosphoglycerides  using FRET and sedimentation assays.To better illustrate the situation, we have revised Figure 2 by replacing panel C with a new space-filling rendition showing the C2-domain contacts that provide the especially ‘good fit’ for the phosphorylcholine headgroup of bound DHPC. The original panel C of Figure 2 has been incorporated into Figure 2—figure supplement 1. Moreover, we carried out additional new SPR experiments using wild-type C2-domain and vesicles composed of various phosphoglycerides including PS. The new data, which are now included in Figure 4, clearly show weak interaction by cPLASimilarly, the PC headgroup is close to His62, which under some conditions could be partially protonated (~pKa near 6).His62 appears to be sufficiently close for van der Waals contacts with the choline methyl groups. We initially suspected that His62 might be able to undergo weak cation-π interaction to the choline moiety (~ 5.0 Å). However, Ward et al., 2013, reported only slight differences in the binding of wild-type and H62A C2-domain to POPC vesicles using SPR. In this same study, changes in pH  reportedly had no effect on binding to C2-domain to POPC vesicles. To clarify the situation, we have modified and added text along with the Ward et al., 2013, reference.2) As written, the reduction in binding and/or activity by Y96A or N65D doesn't really prove they are involved in PC selectivity, it just demonstrated mutations reduce affinity and activity. To prove/demonstrate selectivity, one would have to compare the mutants and WT to lipids under these different conditions. The authors may want to consider rephrasing/rewriting these statements based upon their findings and that of the previous literature. For instance, although SM binding is low, what would the effects of mutations be? Would there be selective reduction in PC binding vs. that of SM?2α C2-domain”). We also have modified text by rephrasing to ‘phosphorylcholine headgroup lipids’ where needed.We agree with the concern raised by the reviewer. We performed the suggested experiment involving testing of the mutants and WT with the sphingomyelin vesicles by SPR and have included the new results in Figure 4 (panel 4D). The data show that the relative changes in binding to POPC and 18:1-SM for WT and mutant C2-domains are similar  and modified the text accordingly .We have added new text providing information about the in vivo location of SM in the Golgi and the expected minimal impact on cPLA3) Please address the following points regarding previous work in the field:2 activity.The Introduction (paragraph three) points out that the lack of a lipid-bound crystal form of the C2 domain has hampered understanding of the structural basis of lipid activation mechanisms. While this current study does provide the lipid-bound structure to advance the field, it may be worth pointing out how the work of Ed Dennis and colleagues (among others) using molecular dynamics and hydrogen-deuterium exchange mass spectrometry has helped the field in understanding cPLAWe agree with the recommendation and have added new text (Introduction paragraph four) as well as the relevant references  to improve the situation.2 demonstrating a reduction in affinity for DHPC and a reduction in activity, which is confirmed in the current study. Similarly, it was shown that N65A reduced cPLA2 DHPC affinity and enzyme activity.More details should be incorporated on the work done on Y96A and N65A by Bittova et al., 1999, as well as the role of N65A in membrane binding and penetration by Stahelin and Cho, 2001, and work by Burke et al. 2008, Bittova et al. studied the Y96A effects in full-length cPLAWe agree with the recommendation and have added new text (Introduction paragraph two) as well as the relevant references  to improve the situation. Also, new text is now modified and added to the beginning of the Discussion paragraph one.2? If not chicken, can the C2 chicken structure and lipid binding be directly compared to its influence on the C2-catalytic role in rat or human? If full-length chicken cPLA2 was used, how does its catalytic domain sequence compare to rat and human, and how does its activity compare to those published previously for the rat or human enzymes?4) Were the studies with full-length protein pursued with rat, human or chicken cPLA2 of chicken, mouse, and human species. The very highly homologous nature of the sequences strongly suggests similar outcomes regardless of the species used.Clarifying text has been added to the Figure 3 legend. We also have included a new figure  showing the sequence alignment of the full-length cPLA2 (rat) needed to be refolded from E. coli expression due to solubility issues. The authors have made great strides here in expressing the C2 domain from chicken cPLA2 in soluble form. This could be of interest to the field so a bit more detail on this solubility in the Materials and methods would be useful.Classically, the C2 domain of cPLAE. coli.We thank the reviewer for acknowledging the technical advance that has accompanied our structural data. We have provided additional new text in the Materials and methods section. This new text describes the technical details associated with the development of C2-domain clones that can be expressed in soluble form in 2 of chicken with rat and human species.It would be helpful to have the alignment of the full cPLA5) In Figure 5, where the authors model the crystal structure onto the schematic drawing and compare to EPR results, it would be more informative if distances are compared. For example, distances from x-ray structure, headgroup dimensions, EPR data can be compared. How deep in the membrane does the C2 domain go from experimental studies?We agree and have now added the information regarding distances from the X-ray structure to the text describing the ad hoc model. We also discuss the C2-domain penetration depth within the context of liquid-crystalline bilayer structural information reported by Wiener and White, 1992. We find agreement with previously published penetration distances into the bilayer for Val97 and Ile39. The new text is now added in paragraph two of the Discussion section.6) The binding of DHPC in one site is well appreciated but it is expected that the C2 domain will make contact and interact  with a number of lipid head groups around CBLs 1 and 3. Can this briefly be incorporated into the discussion model?We have now incorporated this kind of descriptive text at the start of the Discussion section.Also, in the crystal lattice, do the hydrophobic side chains of the lipid molecule interact with each other?2 C2-domain/DHPC structural complex. The new information describes the distances associated between the acyl chains of the bound DHPC in the complexes. For comparison, we also have added the differing crystal packing lattice for lipid free C2-domain (PDB: 1RWL) as a new lower panel and referred to this new information in paragraph three of the Discussion section.We have added information to the legend of Figure 1—figure supplement 2  Synaptotagmin 1 C2A domain binds to three calcium ions. Can the authors comment and compare the structures?Now included as Figure 6—figure supplement 1 is a direct structural comparison2α C-domain. The figure and associated legend, which provide a clear indication of differences and similarities with regards to Ca2+ binding, are pointed out in the Discussion subsection “Comparison of PL recognition with other C2–domains” paragraph three.(superpositioning) of the C2A domain of synaptotagmin-1 and the cPLA8) In the Materials and methods, the authors mention C1P purchased from Avanti but it is not clear when or how they were used in the paper? In crystallization attempts?At the beginning of the Results, we briefly mentioned attempting to generate C2-domain complexes with various lipids including PC, related phosphoglycerides and C1P analogs.


In-situ compression and uniaxial tensile tests are performed for studying the mechanical properties and deformation mechanisms of these two types of 3D chiral metamaterials. Novel deformation mechanisms along different directions are explored and analyzed, such as: uniform spatial rotation deformation, tensile-shearing directed (compression-shearing directed), tensile-expansion directed (compression-shrinkage directed) deformation mechanisms of 3D chiral metamaterials, and competitions between different types of deformation mechanisms are discussed. The proposed 3D chiral metamaterials represents a series of metamaterials with robust microstructures design feasibilities.Rational design of artificial microstructured metamaterials with advanced mechanical and physical properties that are not accessible in nature materials is very important. Making use of node rotation and ligament bending deformation features of chiral materials, two types of innovative 3D chiral metamaterials are proposed, namely chiral- chiral- antichiral and chiral- antichiral- antichiral metamaterials.  Artificially designed metamaterials are usually arranged in repeating patterns, at scales that are smaller than the wavelengths of the phenomena they influence. The smart properties of metamaterials origin from artificially designed structures at sub-wavelength scales, and can be tailored and tuned precisely with their architected shape, geometry, size, orientation and arrangements. The interactions between architected microstructures of metamaterials and electromagnetic, sound and optical waves will result in blocking, absorbing, enhancing, or bending of waves. It has been demonstrated that rationally designed metamaterials have promising multifunctional applications, such as ultralow mass densities and ultrastrong metamaterials16. Auxetic metamaterials exhibit enhanced mechanical properties over conventional materials, such as higher shearing modulus, increased indentation resistance, good absorption properties and higher fracture toughness. Auxetic materials can be applied for designing innovative multifunctional structures, such as: body armor, packing material, knee and elbow pads, robust shock absorbing material and sponge mops. According to the geometrical relations of auxetic unit cell, there are mainly three types of auxetic materials: reentrant materials, rigid square rotation materials and chiral structures17. Chiral structures stands for a series of structures which cannot be mapped onto its mirror image by rotations and translations alone18, and various types of chiral structures exist commonly in nature, such as: DNA, RNA, chiral carbon nanotube, twisting flower petals and stems, plant climbing tendrils and twisted leaves, chiral cellulose21. Besides these chiral materials in nature, various types of multifunctional artificial chiral metamaterials are designed and fabricated as well. Because of their lack of mirror symmetry, chiral metamaterials23 have recently enabled several remarkable phenomena, such as negative refractive index24, superchiral light25, and use as broadband circular polarizers27 or detectors28.As a special type of mechanical metamaterials, auxetic metamaterials with negative Poisson ratio can expand its volume when stretched, and the concept of auxetic materials with negative Poisson ratio was firstly described by Love in 1944 for the first time34. In the classical theory of elasticity, the degrees of freedom are not included for describing the mechanical behaviors of microstructured solid where the microstructure characteristic length is comparable to the solid structure35. Due to the additional degrees of freedom allowed by internal microstructures, chiral Cosserat solids have different mechanical behaviors from solids with a center of symmetry37, The Cosserat  elasticity theory of Eringen37 demonstrated robust efficiency and reliability in the modeling of materials with microstructures, such as: granular or fibrous materials, bone microstructure, or 3D lattice structures, etc. For example, based on the micropolar theory and tensor analysis, Liu et al.38 developed a continuum theory for describing the dilatation–rotation coupling and shear–rotation coupling deformation mechanism of 2D chiral lattice structures. Chen et al.39 proposed a micropolar continuum model for describing the constitutive relation for tetrachiral lattice structure, where 13 independent material constants are employed. Spadoni et al.40 proposed a micropolar continuum model for analyzing the in-plane properties of hexachiral structures, where deformable-ring node model are employed.The mechanical properties of chiral structures can be investigated with different theoretical approaches, such as: strain energy homogenization method, internal external force equilibrium of unit cell, Cosserat (micro-polar) elasticity, etcet al.41 exploited the buckling introduced mechanical instabilities in surface-attached cellular structure, and effects of cellular unit cell geometrical parameters on the formed chiral pattern during the swelling and shrinkage cycle are studied systematically. Shan et al.42 proposed an elastomeric porous metamaterials, where multiple pattern transformations can be induced by buckling. The proposed periodic porous elastic structures can generate mechanical instabilities, and can be used to tune the propagation of elastic waves in phononic crystals, enhancing the tunability of the dynamic response of the system. Ha et al.44 proposed an innovative isotropic 3D tetrachiral metastructure, and studied its mechanical properties via finite element analysis. Fu et al.45 developed the equivalent modulus and Poisson ratio of a novel 3D chiral structures made up of orthogonal assembled 2D chiral honeycomb with four ligaments. Based on the pioneering work on “missing rib” type of chiral structures designed by Smith et al.46, new chiral cellular solids with center cores and softer hinges are designed and fabricated via multi-material 3D printing techniques, and amplified chirality-induced auxetic effect via elevating internal rotation efficiency can be realized47. Making use of the chiral rotation induced unique sequential cell-opening mechanisms, hybrid auxetic chiral mechanical metamaterial are designed, which can be employed for developing new multi-functional smart composites, sensors and/or actuators48. In order to overcome the twist deformation limits of linearly elastic bar, Frenzel et al.49 proposed a microstructured 3D elastic chiral mechanical metamaterials which can realize twist deformation upon compression, the proposed tension/compression induced twist deformation has potential applications in chiral optical metamaterials, such as: optical dynamic cloaking structures. Sha et al.50 proposed the design of large-scale chiral metallic glasses with extensive hardening and large ductility properties, the mechanical behaviors of the metallic glass chiral nanolattice (MGCN) can be significantly altered through changing the thickness and length of the ligaments in the nanolattices.Recently, Kang in-situ compression tests are performed for studying the mechanical properties and deformation mechanisms of these two types of 3D chiral metamaterials. Novel deformation mechanisms such as: compression-shearing, compression-shrinking auxetic deformation of chiral unit cell, antichiral unit cell and chiral-antichiral hybrid unit cell along different directions are explored and analyzed. Secondly, in-situ uniaxial tensile tests are carried out, and competitions between different types of deformation mechanisms are discussed. With the progress of micro- and nano- manufacturing techniques, the proposed 3D chiral metamaterials show promising performances for future industrial applications, such as: sound absorption and vibration metamaterials, morphing structures, chiral optical metamaterials, shape memory actuators and biomechanical devices.In this paper, depending on the geometrical relations between nodes and ligaments, two types of innovative 3D chiral metamaterials are proposed, namely chiral- chiral- antichiral, and chiral- antichiral- antichiral metamaterials. Firstly, two series of these two types of 3D chiral metamaterials with different geometrical parameters are designed, and L along x, y and z directions, cubic node side length d, ligaments thickness t, the numbers of unit cells along x, y and z directions are Nx, Ny and Nz respectively.Chiral structures are architected with circular, polygonal, elliptical, sphere or cubic nodes and ligaments connecting neighboring nodes in 2D or 3D spaces, the deformation modes of chiral metamaterials are featured by node rotation and ligaments bending deformation under external loading conditions. Depending on the geometrical relation between ligaments and nodes of 3D chiral metastructures, there are chiral and antichiral topological configurations along x, y and z directions respectively. As shown in Fig. In this subsection, two series of 3D chiral metamaterials with different geometrical parameters are designed, namely Type A and Type B, respectively. The geometrical parameters of these designed two types of 3D chiral metamaterials are shown in Table s = 1021.00 MPa, where the deviation of modulus is: ±0.75 MPa, and the average ultimate strain of the material is εmax = 0.16. After finishing the material properties tests, compression tests of two series of 3D chiral metamaterials are performed, where the loading force, displacement and deformation images during the loading process are recorded, the samples are loaded until compression strain level 10%, and deformation process of these two types of 3D chiral metastructures are shown in Fig. Before performing compression tests of these two types of 3D chiral metamaterials, the mechanical properties of the SLS nylon material are tested. Totally, 5 uniaxial tensile samples are fabricated, and uniaxial tensile experiments are performed on an Instron®5985 machine at a displacement rate of 1 mm/min. Finally, the average elastic modulus of the 5 as-fabricated tensile samples is:EDuring the linearly compression process, elastic bending deformation of the ligaments and rotation of circular nodes are dominant, the chiral ligaments form full-wave deformation mode, while the antichiral ligaments form half-wave deformation mode. As shown in Figs in-situ tensile tests of 3D chiral metamaterials are performed, where the loading force, displacement and deformation images during the loading process are recorded, and deformation process of these two types of 3D chiral metastructures are shown in Fig. In this subsection, uniaxial tensile tests are performed for exploring the deformation mechanisms of these two types of 3D chiral metamaterials. The geometrical parameters of designed two types of 3D chiral metamaterials are shown in Table 52. In the compression-shearing experiments shown in Fig. in-situ uniaxial tensile experimental tests shown in Fig. As to the deformation mechanisms of these two types of 3D chiral metamaterials, rotation of the cubic chiral nodes and bending of ligaments are predominant. (1) As to the 3D chiral- antichiral- antichiral metamaterials, compression-shrinkage and tensile-expanding deformation mechanisms are dominant, where the rotation and bending of chiral-chiral and chiral-antichiral ligament-node pairs will results in auxetic deformation behaviors; (2) As to 3D chiral-chiral-antichiral metamaterials, which can be viewed as cubic solid medium cut out with four-pointed planar star pore systems from two perpendicular directions, where the horizontally inclined ligaments undergo a higher degree of flexural deformation than their vertically inclined counterparts, meaning the rotational symmetry of order four is not being preserved. As shown in Fig. With the progress of micro- and nano- manufacturing techniques, the proposed 3D chiral metamaterials show promising performances for future industrial applications, such as: nano chiral metatllic glass with extensive hardening and large ductility, sound absorption and vibration attenuation metamaterials, morphing structures, optical chiral metamaterials, shape memory actuators and biomechanical devices.in-situ compression test are performed for studying the mechanical properties and deformation mechanisms of 3D chiral metamaterials. Secondly, in-situ uniaxial tensile tests are performed for exploring the competitions between two main types of deformation mechanisms: shearing deformation and auxetic rotating deformation of chiral structures.In this paper, two types of innovative 3D chiral metamaterials are proposed, namely chiral- chiral- antichiral and chiral- antichiral- antichiral metamaterials. Firstly, two series of 3D chiral metamaterials with different geometrical parameters are designed, and the 3D chiral metamaterials samples are fabricated with Selective Laser Sintering (SLS) nylon sintering techniques, and The proposed 3D chiral metamaterials represents a series of metamaterials with robust microstructures design feasibilities. With the progress of micro- and nano- manufacturing techniques, the proposed 3D chiral metamaterials show promising performances for future industrial applications, such as: nano chiral metatllic glass with extensive hardening and large ductility, sound absorption and vibration attenuation metamaterials, morphing structures, optical chiral metamaterials, shape memory actuators and biomechanical devices.


BMC Biology characterises biophysically oligomeric and filamentous structures formed spontaneously by the Toll-like receptor signalling adaptor MyD88. Naturally occurring mutants of MyD88 that cause immunodeficiency are unable to form these structures. By contrast a somatic mutant that promotes the survival of tumour cells forms oligomers much more readily than the wild-type protein. These findings suggest that assembly of oligomeric MyD88 is critical for the regulation of inflammatory signalling.A paper published in  Innate immune and inflammatory signalling underlie a large burden of human disease ranging from sepsis to rheumatoid arthritis and neuronal degeneration. Thirty years ago Charles Janeway correctly predicted a central role in these signalling processes for what he termed pattern recognition receptors (PRRs), germline-encoded molecules that recognise conserved structures associated with microbial pathogens (PAMPS). One important group of PRRs is the Toll-like receptors (TLRs). These molecules respond, broadly speaking, to non-self lipids and nucleic acids presented at the cell surface or in endosomes, respectively Fig. . BecauseBMC Biology by Gambin and colleagues [PAMPs, such as lipopolysaccharide (LPS) from Gram-negative bacteria, bind directly to TLRs to induce formation of an active dimeric complex. Dimerization of the cytosolic TIR domains provides a transient nucleation signal for the assembly of an oligomeric scaffold, the myddosome, that is required for downstream signal transduction , 3. The lleagues  providesMyD88 consists of two modules, a death domain (DD) and a TIR domain. Dimerisation of the TLR TIR domains creates a new binding surface that can interact with the corresponding TIR domains of MyD88, nucleating the assembly of a closed myddosome complex. In resting cells it is thought that MyD88 is auto-inhibited, perhaps by interactions between the TIR and death domains. Transient interaction with the activated receptor is viewed as relieving this inhibition, allowing the formation of the helical myddosome assembly .In the present study the authors have used an in vitro system to produce full-length MyD88 and in addition the isolated DD and TIR domain. This allows protein to be produced in a controlled manner and single molecule fluorescence methods were then used to assay the aggregation propensity of DDs, TIR domains and full-length MyD88. This analysis reveals that, at nanomolar concentrations, full-length MyD88 is able to form large polymers or filaments whereas only short oligomers of TIR domains are detected. At this low concentration the DD is also able to form oligomers, although these are much smaller than the polymers formed by the full-length protein.Two mutations in MyD88 that cause immune deficiency in humans were then tested for their effect on the propensity of full-length MyD88 to oligomerise. Both of these, one in the DD and one in the TIR domain, abrogated polymer formation. A third variant, L252P in the TIR domain, is of particular interest because it is found as a dominant somatic mutation in many B-cell lymphomas . The preA key question unanswered by this study is the stoichiometry of endogenous MyD88 in unstimulated cells. Overexpression of MyD88 leads to the appearance of speckles in the cytosol, likely caused by oligomerisation, but endogenous MyD88 appears to be uniformly distributed. This might indicate that it forms only short oligomers rather than filaments or perhaps that it is monomeric. One published study provides evidence that MyD88 is monomeric or possibly dimeric in lysates prepared from resting cells, but this conclusion may be unreliable as preparation of the lysates could cause significant dilution of the MyD88 . A more The role played by oligomers and filaments in innate immune signal transduction has been an emerging theme over the last few years. These structures have been described as supra-molecular organising centres and are formed during activation of the TLR-TRIF and inflammasome NLRP3-ASC-Caspase1 pathways as well as in pro-inflammatory signalling through MyD88 . A recen


GYY-4137 is widely used in these studies because it slowly releases H2S, but there is disagreement as to whether it requires enzymes to release H2S. In this article we describe the release of H2S in water without enzymes and that it releases H2S faster in organic solvents than in water or when mixed in topsoil. Furthermore, we describe the long-term effect of dosing pea, radish, and lettuce plants with GYY-4137 for up to six weeks. The effect of GYY-4137 on plant growth for six weeks was either positive or negative depending on the loading of GYY-4137 and how it was applied to plants. The addition of GYY-4137 to lettuce plants via potting mix resulted in reduced growth and death of the plants. In contrast, application of GYY-4137 to the leaves of lettuce plants increased the harvest weight of the leaves by up to 86%. Our results demonstrate that GYY-4137 can have a positive, important effect on the growth of plants but that this effect is dependent on several factors.Hydrogen sulfide (H The worldwide population is expected to grow from its current level of 7.2B to 9.6B people by 2050.–3 To mee2S) to increase the growth, survival, and yields of crops. H2S is a gasotransmitter that is synthesized enzymatically in plants and used as a signaling molecule. Research in the last dozen years has repeatedly demonstrated that therapeutic amounts of H2S have dramatic effects, including increased growth of roots, protection against heat stress and drought conditions, increased overall size and mass, alleviation from freezing stress on leaves, protection from high water salinity, and prolonged fruit shelf life.[2S in plants is a new field–much of the key work has been completed since 2007 –yet it has already been shown to have positive effects on corn, soybeans, wheat, sweet potatoes, cucumbers, strawberries, rice, spinach, tomatoes, broccoli, and kiwi.[2S by hydrolysis, increased the growth of radish, peas, and lettuce plants. We report that the harvest yield of radishes doubled when milligram loadings of GYY-4137 were applied.One partial solution to this problem is the application of hydrogen sulfide (Helf life.–13 The iand kiwi.–39 In th2S is that it is a low boiling point gas (boiling point = -60°C), and it is highly toxic. Exposure to levels of 2 ppm of H2S in the air can lead to negative health effects such as headaches or breathing problems for people who suffer from asthma, and exposure to 100 ppm is “immediately dangerous to life and health”.[2S is challenging to handle, most scientists who study it add solid NaSH to water to yield an aqueous solution of H2S. These aqueous solutions can be used to deliver H2S, but they also lead to a rapid release of H2S into the atmosphere. Much prior work to investigate the effect of H2S in crops involved dosing plants or seeds daily with aqueous solutions of H2S at levels of 10 μM to several mM, which led to rapid evaporation of H2S and the potential release of >100 ppm of H2S into the atmosphere. The low boiling point of H2S and its high toxicity places obvious limitations on how aqueous H2S can be used to grow crops. To address this challenge, chemicals such as GYY-4137 that slowly release H2S were used to investigate the effect of exogenous H2S on plants  or onto the leaves (lettuce) to investigate the effect of how different loadings of GYY-4137 and different locations of applications had on their growth. Furthermore, we report on the hydrolysis of GYY-4137 in water and organic solvents and how soil can affect its hydrolysis.In this article we report on the effect of H2S or GYY-4137 on pea, radish, or lettuce plants. Previously mentioned articles in 1978 and 1979 used gaseous H2S and reported surprisingly large effects on the growth of lettuce plants, but this work was not further explored for over three decades. A paper in 2015 reported the effect of growing lettuce seeds for 5 days in water with different concentrations of H2S.[2S had a 40% increase in elongation compared to control plants grown without H2S. This effect lessened at concentrations of 0.1 and 0.5 mM H2S, and plants grown in 1.0 mM H2S had over 50% reduction in elongation compared to the control plants. A paper in 2014 reported that low levels of aqueous H2S slowed senescence of harvested lettuce for 48 h.[2S on pea plants.[2S in the water led to an increase in size of 68% for pea plants grown in 100 μM H2S for 5 days compared to control plants grown in the absence of H2S. Young pea plants exposed to H2S and either arsenate or flooding had higher survival rates compared to plants that were not exposed to H2S. Finally, the only reports of the effect of H2S on radish plants were published in the 1990s and report that all loadings of H2S had a negative effect on these plants.[2S for longer than a week or any effect on yield.Little work has been reported on the effect of aqueous Hs of H2S. Seeds grfor 48 h. Two papea plants., 63 The e plants.–66 None 3 potting mix) or 2.5” square pots (232 cm3 potting mix). Plants were grown on a rooftop with watering as needed.Plants were grown in a 1:1 mixture of #1 Professional Germination Mix and #4 General Purpose Growing Mix, Beautiful Land Products of Iowa LLC in West Branch, Iowa, prewet before loading pots to maximize consistency. Lawesson’s reagent, morpholine, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-TRIS), and all solvents used were obtained from Sigma Aldrich. GYY-4137 was synthesized from a published method. Lettuce GYY-4137 was administered to the seeds using four different methods. In method 1, enough potting mix to fill 12 pots was added to a larger bin and watered to give a consistent wetness. The GYY-4137 was added to the potting mix and blended thoroughly by hand until it was evenly distributed. Finally, 12 pots were filled with the potting mix and packed finger tight. In method 2 GYY-4137 was added to freshly planted seeds as a solution. In this method seeds were sown approximately 2 cm deep and covered with potting mix. Enough GYY-4137 for 12 plants was added to 60 mL of tap water and vigorously stirred. Five mL of this aqueous solution of GYY-4137 was pipetted into the soil directly above the seeds.In methods 3 and 4 the plants were dosed with GYY-4137 at planting (day 0) and subsequently every 7 days. In method 3 the seeds were initially planted according to method 2, in which aqueous GYY-4137 was added to the potting mix above the seed immediately after planting. Every 7 days enough GYY-4137 for 12 plants was added to 60 mL of water and vigorously stirred. The aqueous GYY-4137 was added to the potting mix immediately adjacent to the leaves of the plant. In this method plants were dosed with the same amount of GYY-4137 every 7 days as they were dosed at day zero. For instance, if a plant was originally dosed with 10 mg of GYY-4137 at day zero, it was dosed with 10 mg of GYY-4137 every 7 days. In method 4, used for lettuce only, after planting according to method 2, leaves were treated weekly with an application of dosing of GYY-4137. Every 7 days enough GYY-4137 for 12 plants was added to 60 mL of tap water and vigorously stirred. Five mL of aqueous GYY-4137 was added to the shoot tip of each plant in the amount added to the soil above the seeds at day zero.For each set of plants and each method of delivering GYY-4137, twelve seeds were planted at each loading of GYY-4137. For instance, radish plants were grown using methods 1 and 2. For each method radish seeds were exposed to eight different loadings of GYY-4137, and twelve radish seeds were planted at each loading for a total of 96 radish seeds sown for method 1 and 96 seeds for method 2.Lettuce seeds were sown in 2.5” pots about 2 cm deep. The lettuce plants were grown in a greenhouse located at the University of Iowa Biology building. Lettuce seeds were sown on June 15, 2017 and harvested on July 6 for the 3-week harvest and July 27–28 for the 6-week harvest. Pea seeds were planted about 2 cm deep in 4” pots. The pea plants were grown outside from June 23, 2017 and harvested on July 17 for 3-week harvest. The 6-week harvest was done on August 3 and 4. Radish seeds were planted 2 cm deep in 2.5” pots. The radish plants were grown outside from June 30, 2017 to July 31, 2017. All the plants were watered once daily or as needed.Radishes were harvested at 4.5 weeks. After cutting off the shoot and brushing off the potting mix, roots were weighed on a laboratory balance. Pea plants were harvested at both 3 and 6 weeks by cutting the plant at the root/shoot boundary and removing the leaves of the plant. The height of the shoot was measured from the root/shoot boundary to the base of the uppermost expanding leaf. The shoot (stem plus detached leaves) was then weighed on a laboratory balance. Lettuce plants were harvested at 6 weeks by cutting the stem at the root/shoot boundary. Soil was gently brushed from the plants, and the total shoot weight was obtained by weighing the leaves and the stem. The leaf weight was determined by cutting the leaves from the stem and weighing the leaves separately.2S release from aqueous solutions of GYY-4137 at 0.5 M was assayed using lead acetate strips that turn from white to brown and then black when exposed to H2S. GYY-4137 was added to BIS-TRIS buffered to pH 7 to ensure a constant pH. In each of the experiments either 0.6 mL (subscript 1) or 1.2 mL (subscript 2) of buffered GYY-4137 was used. Aqueous GYY-4137 was added to a 20 mL scintillation vial and a separate, smaller vial which contained a lead acetate strip was placed into the 20 mL vial. The lead strip did not come in contact with the aqueous GYY-4137 but H2S released by GYY-4137 partitioned into the atmosphere and contacted the strip. The 20 mL vial was capped tightly and further sealed with parafilm. The vials were kept at room temperature for 10 days.H1 and A2 the lead strips were exposed to aqueous GYY-4137. In samples labelled B1 and B2 the GYY-4137 was replaced with NaSH  as a positive control for H2S release. In samples labelled C1 and C2 potting mix used here was added to the 20 mL vial with aqueous GYY-4137. Samples labelled D1 and D2 were identical to C1 and C2 except that NaSH was used in place of GYY-4137. For samples labelled E1 and E2 locally collected topsoil was added to the aqueous solution of GYY-4137. Samples labelled F1 and F2 were identical to E1 and E2 except that NaSH was used rather than GYY-4137.In samples labelled A2O/D2O at room temperature by 31P NMR spectroscopy. Two studies were performed at 0.12 M and 0.50 M GYY-4137 to investigate the rate of hydrolysis above and below the solubility limit of GYY-4137 in water (0.13 M). GYY-4137 was dissolved in 1.5 mL of 90% H2O/D2O buffered with BIS-TRIS (1 M) at pH 7. The 31P NMR spectra were collected at day 0 and at day 35. The NMR spectra were obtained using a Bruker Avance-300 at 300-MHz, Bruker DRX-400 at 400-MHz, and Bruker DPX-500 at 500-MHz. The hydrolysis of GYY-4137 was also measured in DMSO-d6 at a concentration of 0.13 M. The 31P NMR spectra were collected at day 0 and periodically after that time.The hydrolysis of GYY-4137 was monitored in 90% H2S detection with H2S electrode was measured with the Analysenmesstechnik GmbH Amperometric H2S Micro-sensor. The buffer was 1 M BIS-TRIS titrated to pH 6.7 with HCl. In each experiment 70 mL of this buffer was used. A baseline of the concentration of H2S was first measured and then enough solid GYY-4137 to yield concentrations of 0.12 and 0.50 M were added. The aqueous GYY-4137 was capped with a rubber stopper that had holes to fit the sensors of the electrode. The H2S electrode was inserted through the holes and the cap was sealed tightly with parafilm.HStatistical analysis was performed using IBM SPSS Statistics 25. Levene statistical analysis was performed to ensure homogeneity of variances. If the variances were homogenous then one-way ANOVA tests were used to show statistical relevance. Post-hoc test used to show statistical relevance with the control was the Dunnett’s multiple comparison test. If the variances were found to not be homogenous then the non-parametric Kruskal-Wallis test was performed. Data represented are mean±standard error with ** indicating α<0.05 and * indicating α<0.1.6 using 31P NMR spectroscopy for 109 days  released from GYY-4137 was in equilibrium with H2S(g) and was not allowed to escape from the capped vial. The H2S(g) diffused to the lead acetate strips and reacted to produce highly insoluble, black PbS. Lead strips enclosed in vials with aqueous GYY-4137 at a concentration of 0.5 M for 10 days had a slight browning on their surface (A1 and A2) which is consistent with the release of a small amount of H2S. Two sets of experiments were completed where the potting mix used in this work was added to the aqueous GYY-4137 (C1 and C2) and topsoil from a nearby field was added to aqueous GYY-4137 (E1 and E2). Similar browning of the lead strips was observed for samples with and without soil used in this work, but little to no browning was observed when Iowa topsoil was added to aqueous GYY-4137. Samples B1, B2, D1, D2, F1, and F2 were positive controls in which NaSH was added to the vials rather than GYY-4137 to demonstrate the dark colors of lead strips.The effect of soil on the degradation of GYY-4137 was investigated using lead acetate strips that turn from white to black when exposed to HRadishes were grown using methods 1, 2, and 3 for delivering GYY-4137 for 40 days. Results for method 1 (blending GYY-4137 powder into the potting mix) is shown in Pea plants were grown for 3 and 6 weeks with different loadings of GYY-4137 provided by method 1 . At 3 weTwo other sets of pea plants were grown using methods 2 and 3 to deliver GYY-4137. In one set of experiments, peas were grown for 6 weeks after method 2 delivery (a one-time application of aqueous GYY-4137). Shoot height and fresh weight was determined . A seconFour different experiments were completed with lettuce plants to investigate the effect of GYY-4137 on growth after 6 weeks, as assayed by shoot fresh weight. In two sets of experiments GYY-4137 was added to the potting mix prior to planting. In the first set, GYY-4137 was thoroughly mixed with the soil prior to the addition of seeds (method 1) . In a seIn a fourth set of experiments GYY-4137 was applied by method 4 to lettuce seeds. It was added adjacent to the seeds at planting and at days 7, 14, 21, 28, and 35 aqueous solutions of GYY-4137 were applied to the leaves. Shoots were harvested and weighed at week 6 .2S in water, its rates of hydrolysis in water and other solvents is uneven. In organic solvents it hydrolyzes with residual water over days to weeks. In experiments reported here, it hydrolyzed by 74% in 7 days in DMSO-d6 and in another paper it hydrolyzed by 50% in 13 days when dissolved in chloroform or acetone.[31P NMR spectroscopy. Similar results were found when the release of H2S from GYY-4137 was monitored with an H2S electrode. At concentrations of 0.12 M and 0.50 M at a pH of 6.7 the steady state concentration of H2S was approximately 3 and 5 μM which were approximately 105 lower concentrations than GYY-4137. The concentration of H2S did not scale linearly with the concentration of GYY-4137 because the solubility limit of GYY-4137 was only 0.13 M. At a concentration of 0.50 M GYY-4137 much of the GYY-4137 was an insoluble solid at the bottom of the vial. These experiments demonstrate that GYY-4137 that is swollen in water, but not dissolved, will release low levels of H2S. These results demonstrate that the local solvent environment for the hydrolysis of GYY-4137 is important for consideration of its rate of degradation. The presence of organic chemicals near GYY-4137 can strongly influence the rate of release of H2S. Furthermore, GYY-4137 that is not fully dissolved in H2O will also release H2S which is an important consideration for its applications in agriculture when GYY-4137 is added to soil for extended periods of time.Although it is regularly reported that GYY-4137 hydrolyzes to release H acetone. In contr2S from GYY-4137 in the presence of two different growth media was investigated. In a bark-based potting mix used here, H2S was detected by darkening of a lead acetate strip after 10 days. When Iowa topsoil dug locally was used, no release of H2S from GYY-4137 was observed using lead acetate strips. Although qualitative, these results further demonstrate the importance of considering the local environment of GYY-4137.The release of H2S when watered or concentrated aqueous solutions of GYY-4137 that slowly release H2S. The loading of organic material in the soil may affect the release of H2S from GYY-4137 by providing a nonpolar, organic environment that will accelerate the release of H2S. It is also clear that it is not necessary to have enzymes to release H2S from GYY-4137, although the presence of enzymes may speed the release of H2S.These experiments describe the importance of considering the soil composition as well as concentrations of aqueous GYY-4137 in measurements of plant growth responses. Water from dilute aqueous solutions of GYY-4137 added to soil may evaporate leaving behind solid GYY-4137 that releases H2S on radishes demonstrated reduced growth rates, our experiments demonstrated that GYY-4137 applied on radish plants could increase their root harvest weight .[The increased growth of lettuce plants exposed to GYY-4137 on the leaves was consistent with the results from 1978 and 1979 where lettuce plants were grown in greenhouses continuously exposed to gaseous H season)., 62 The 6 GYY-4137 was over 74% hydrolyzed within 7 days. This demonstrated the importance of considering the local environment of GYY-4137 when used in agricultural studies and also further demonstrated that GYY-4137 does release H2S by hydrolysis in water. The rate of release of H2S from GYY-4137 is not straightforward and may involve its uptake into plants through the roots or leaves.The hydrolysis of GYY-4137 is complex and proceeds significantly faster in organic solvents than in water. Less than 3% of GYY-4137 hydrolyzed in water after 5 weeks, but in DMSO-d2S exhibited increased growth but when GYY-4137 was added to the pots of lettuce plants only negative effects were observed. In contrast, when GYY-4137 was applied to lettuce leaves the weight of the leaves increased by up to 82%. The location of application of GYY-4137 was clearly an important aspect of how it affected lettuce plant growth and should be considered in future work with other plants. The importance of the duration of release of H2S was shown by the different results for pea plants harvested at 3 and 6 weeks. The loading of GYY-4137 that had the most positive effect shifted to higher doses for plants harvested at 6 weeks compared to 3 weeks. This result was unexpected and unreported. The origin for the change in effect of plants to different doses of GYY-4137 at different times is important to consider and should be studied in future work.This research demonstrated the dual importance of the location and duration of application of GYY-4137. Prior work with lettuce exposed to gaseous H2S has the potential to increase the harvest yields of radishes and lettuce. Importantly, only milligram quantities of H2S were needed to have a large effect on the growth of these crops, but the location and number of times GYY-4137 needed to be applied differed for the crops. Although this work was completed in a greenhouse, it may have implications for the growth of crops in agricultural fields.This work demonstrated that HS1 Fig31P peaks for each chemical is shown to illustrate its splitting pattern. No internal standard was used to collect the 31P NMR spectra. The usual internal standard is phosphoric acid, but this was not used due to concerns that it would affect the degradation rate of the GYY-4137. The lack of an internal standard resulted in a small drift for the 31P peaks in the spectra shown below. To ensure that the drift of the NMR spectrometer was small, the chemical shift of phosophoric acid in a separate NMR tube was regularly checked and used to calibrate the instrument.The (TIF)Click here for additional data file.S2 FigNo change in the spectra was observed from day 22 to day 104.(TIF)Click here for additional data file.S3 FigC and D was observed by 31P NMR spectroscopy.At day 104 water  was added to the NMR tube. At day 109 no change in ratio of chemicals (TIF)Click here for additional data file.S4 Fig31P NMR spectroscopy.On day 109 morpholine  was added to the NMR tube and no change of the integration of the peaks was observed on day 114 by (TIF)Click here for additional data file.S5 FigC and D. The NMR tube was heated in 85°C oil bath for 24 hours. The 31P NMR spectrum on day 21 showed complete consumption of D leaving only compound C. This result indicated that D could convert to C but that the reaction was slow at room temperature.GYY-4137 was heated in an NMR tube for 20 days to yield a mixture of chemicals (TIF)Click here for additional data file.S6 Fig2O/D2O. The 31P NMR spectrum after 35 days showed less than 3% of the GYY-4137 hydrolyzed.GYY-4137 was completely dissolved at a concentration of 0.12 M in 90% H(TIF)Click here for additional data file.S7 Fig2O/D2O at a concentration of 0.50M. The solubility limit of GYY-4137 in water is approximately 0.13M so much of the GYY-4137 was a solid at the bottom of the NMR tube. The 31P NMR spectrum after 35 days showed less than 3% of the GYY-4137 hydrolyzed.GYY-4137 was added to 90% H(TIF)Click here for additional data file.


Phase transformations greatly affect physical properties of rocks and impose a first-order control on geodynamic processes. Under high deformation rates, rheological heterogeneities cause large spatial variations of stress in materials. Until now, the impact of higher deformation rates, rock heterogeneity and stress build up on phase transformations and material properties is not well understood. Here we show, that phase transitions are controlled by the stress build-up during fast deformation. In a deformation experiment , rock heterogeneity was simulated by a strong elliptical alumina inclusion in a weak calcite matrix. Under deformation rates comparable to slow earthquakes, calcite transformed locally to aragonite matching the distribution of maximum principal stresses and pressure (mean stress) from mechanical models. This first systematic investigation documents that phase transformations occur in a dynamic system during deformation. The ability of rocks to react during fast deformation rates may have serious consequences on rock rheology and thus provide unique information on the processes leading to giant ruptures in subduction zones. Rock microstructures are a result of coupled chemical and mechanical processes3. Previous studies have demonstrated that material heterogeneity can lead to the development of heterogeneous stress and pressure (mean stress) distributions5. On the long term, large scale geological processes are commonly assumed to be slow and stress variations on geological timescales negligible. However, as recently documented by geophysical methods, transient periods with higher deformation rates (10−9–10−10s−1) and stress build-up are more frequent than previously thought7. These fast deformation events can result in large earthquakes with direct societal consequences. For example, slow earthquakes may play an important role in the more damaging earthquake cycles in subduction zones. These processes can take place along the megathrust and other planes of weakness in response to loading, releasing low frequency seismic waves6. Slow-slip events during subduction were inferred from accurately measured crustal movements and it was shown that these occur over periods of hours to weeks9. Understanding processes during fast deformation events depends on identifying material behavior and its rheology. While the effects of high deformation rates (10−5–10−6s−1) on phase transformations were previously studied in nominally homogeneous experiments12, the processes in heterogeneous materials remain unclear. Interestingly, the time scale for these slow earthquakes (~10−9s−1) approaches experimental strain rates (~10−6s−1) which opens a new horizon for designing deformation experiments on reactions in rheologically heterogeneous materials.In reacting rocks, phase transformations have a great influence on rheology through variations in mineral assemblages, mechanical properties, the presence of fluid or a change in grain size16. The effect of elliptical heterogeneities on the stress field of a deforming viscous material has been investigated by analytical solutions17. These models show that material heterogeneity, expressed as viscosity heterogeneity, can lead to the development of stress and pressure spatial variations around a strong inclusion , on mineral transformations. We took an inspiration from and validated the results against the analytical solutions of a strong ellipse within a weak matrix17. In addition, we performed numerical models that corroborate the first-order predictions of the stress field from analytical solutions  phase transition in a Griggs-Type deformation apparatus. The error of confining pressure and differential stress measurements is ca. 0.03–0.05 GPa (see Methods for details and references). The sample material was dried cold-pressed natural calcite-powder (63–125 μm grain size) with added 0.1% of H2O. We introduced a viscosity heterogeneity by embedding a strong (i.e. more viscous) and non-reactive elliptical alumina (Al2O3) inclusion within the calcite powder. The inclusion was initially tilted by 45° relative to the axial-shortening direction. The experiment was performed at 600 °C to avoid kinetic limitations, and a confining pressure of 1.47 GPa, i.e. 0.08–0.1 GPa below the phase transition of calcite to aragonite  and produced a barrelled cylinder of 7.4 mm in length.We conducted a pure shear viscous deformation experiment close to the calcite-aragonite  finite strain rate of 10opy Fig.  to deteropy Fig.  and S7. opy Fig.  and S9.F18. This model is a geometrical 1:1 representation of a cross section through the experiment and resolves the stress distribution between deformation pistons, sample and ellipse . Deformation was achieved by imposing a constant displacement rate in the upper alumina piston  the bulk stress values that were extracted from the model  and the experimentally produced phase transition distribution shows a direct correlation between the modelled local pressure (mean stress) and the phase transformation pattern all over the sample Fig. . Aragoni4 Fig. . This ag20.The development of significant stress variations in the model and the experiment is caused by high deformation rates. The results document that such deformation rates can lead to an increase of mean stress in the sample and local stress perturbations (positive and negative) in the order of 0.1–0.3 GPa for the case of calcite. These variations in stress and pressure were responsible for the fast transformation of calcite to aragonite, a higher-pressure polymorph that has different physical and mechanical properties. Interestingly, when composite materials are considered, the effective mechanical behaviour is grossly influenced by the individual phase properties as well as their spatial distributionOur results show that the phase transformation was not completed, as evidenced by the partial retainment of calcite grains in association with aragonite. However, the locally elevated pressure could be responsible for the higher degree of overstepping above the calcite/aragonite transition as documented by higher amounts of aragonite in those regions see Fig. . This in8 require deformation rates that are approaching our experimental conditions. Therefore, high stress that develops during subduction processes may be accompanied by mineral reactions and mineral transformations that affect the effective mechanical behaviour of heterogeneous rocks.The effect of fast deformation rates on mineral transformation is not limited to experimental conditions only. In fact, recently discovered fast deformation processes that occur in subduction zones21. Our results show that these values of differential stress can trigger mineral reactions and phase transformations locally. Such mineral transformations and the accompanying change in material properties during the deformation thus have a dramatic impact on the stability and style of deformation, as well as on the degree of coupling of the subduction interface.During the periods of inter-seismic loading of the lithosphere, differential stress can be in the order of 0.1 GPa23. The vertical temperature gradient along this sample is assumed to be ca. 20–30 °C and no horizontal temperature gradient is assumed across the sample25. In order to have a quantitative estimate of the temperature gradients, we calculated the steady-state temperature distribution in two dimensions following the experimental geometry  averaged strain rate of 10−6s−1.The deformation experiment was carried out in a Griggs-Type deformation apparatus at 600 °C and 1.47 GPa confining pressure. For our experimental set-up, we estimate the error in confining pressure to be +0.01/−0.05 GPa. For differential stresses the error is estimated to be ±0.03 GPa due to the weak, yet solid confining medium (Potassium-Iodide)2O3) ellipsoid had a 3.75 mm long axis and 1.65 mm short axis and was initially tilted by 45° relative to the displacement direction. To obtain this configuration, the powder was pre-compressed at a 45° angle; a small groove was introduced into this surface, so that the ellipsoid could be positioned and then further covered with calcite powder. For 1000 mg of calcite, 0.8 μl of H2O were added. The capsule was then cold-pressed at 0.05 GPa before closing. The assembly was hot pressed for 14 h at 1.47 GPa and 600 °C before deformation. After the experiment, the sample was quenched from 600 °C to 200 °C at a rate of 20 °C/s to assure a good preservation of reacted phases.The sample material was calcite-powder  which we obtained from crushing a high purity icelandspar single crystal . The mineralogy of the single crystal was verified by Raman spectroscopy. The powder was placed in a 0.15 mm thick, welded Pt jacket. The alumina  is 10.4 mm.3 was in the calcite field. Mechanical data (deformation stress and displacement) for the experiment was corrected for machine compliance, machine friction during run-in and increasing sample area. The raw data and resulting corrected stress-time and stress-strain curves (two correction procedures are discussed) are portrayed in the Supplementary Figs. The pressure of the phase transition was previously calibrated on this machine in hydrostatic experiments – to ensure that σ18. Following ref. 18, the marker in cell method is used for model advection. Temperature is assumed to be constant during deformation.To model the stress and pressure distribution in the deformed sample, we solved the mechanical problem numerically. We solve the Stokes equations for slow viscous, incompressible flow with a nodal resolution of 301 × 501 grid points. The code uses the MATLAB® direct solver and employs a staggered grid for discretisationBoundary conditions were set to free slip at the sides and top of the model, while the bottom was set to no-slip. An internal zone of constant displacement rate was set in the upper alumina piston  in order to control the bulk deformation rate of the sample region. Time steps were set to a maximum of 450 s. After 700 calculation steps a strain of ε = 0.33 was reached.19 , and recalculated to strain-rate dependent effective viscosity ) Pa−n s−1); all in SI units.The rheology of the deforming material  was modelled using a power-law viscosity16 Pa s  and for salt 3 × 1011 Pa s.The viscosity for the other materials was assumed to be constant: for alumina 10−1 27), aragonite (peak at 205 cm−1 28) or a mix with colours accordingly ranging from blue to yellow.Raman mapping was carried out using WITec Alpha 300R microscope and UHTS 300 Spectrometer VIS-NIR at Heidelberg University. The x-y stage operated at a step-size of 12 μm and a 50x objective was used. The grating was set to 1200 grooves/mm. The excitation laser wavelength was 531.98 nm. The laser intensity at the sample was 50 mW, which yields clearly distinguishable spectra with 0.25 s acquisition time (Supplementary Fig. Supplementary information


Chickpea flour was found to be nutritionally superior compared to refined wheat flour, and conventional processing of the flour was found not to affect its content of protein, fats, carbohydrates, and phosphorus, while total dietary and crude fibers were significantly increased. The fatty acid profile was minimally affected, while magnesium and potassium were reduced. The sensory test conducted among panelists (n = 60) showed that the incorporation of processed chickpea flour into the dough of mankoushe zaatar with ratios of 30% and 50% provided an end-product with better taste and overall acceptability compared to the regular mankoushe. Hence, conventionally processed chickpea flour can be used as a fortifier to improve the nutritional quality of bakery products without negatively affecting their sensory properties.Chickpea flour is known to have good nutritional values. Nevertheless, it is commonly made from ground grains, and characterized by an “off-flavor”. Processing of chickpea grains before flour formation reduces the intensity of the off-flavor. Therefore, two experiments were conducted: first to examine the effect of conventional processing  on the nutritional composition of the chickpea flour; and second, to investigate the impact of processed chickpea flour incorporation with different ratios on the sensory properties of  Pulses are important crops cultivated around the world because of their nutritional value. They play an essential role in human nutrition, particularly in the dietary pattern of low-income individuals in developing and underdeveloped countries. Pulses are good sources of protein, carbohydrates, minerals, and vitamins. These grain legumes are characterized by high dietary fiber content, oligosaccharides, slowly digestible starch, and resistant starch ; thus, tWorldwide, chickpeas received attention because of their physiological benefits. Extensive efforts have been conducted to increase the intake of these grain legumes by integrating them as flours into food products: bread ,8, spaghmankoushe zaatar. It is a widely consumed pastry similar to pizza, in which the dough base is topped with mixed herbs, known as zaatar. In this study, chickpea grains were conventionally processed , and the impact of processing on the chemical composition of the flour was also examined.Thus, the aim of this study was to examine the effect of processed chickpea flour incorporation with different ratios on the sensory properties of the traditional Lebanese pastry Cicer arietinum L.), Kabuli type, were purchased from the local Lebanese market. Chickpea grains were hand-sorted to remove foreign materials. Some of the grain was kept raw, and some was soaked in distilled water in the ratio of 1:10 (w/v) for 17 hours, after which the water was discarded and fresh tap water was added to the chickpeas in the ratio of 1:10 (w/v). Grains were boiled at 100 °C for 1 hour in a double-jacketed kettle, drained, then dried in an industrial air drier  for 8 hours at 38–40 °C. The two types of chickpea, raw and treated grains, were ground into powder form with an electrical, multifunction swing type portable grinder , sifted through a 60-mesh stainless steel sieve (250 µm particle size) to obtain a fine flour. Each kind of flour was packed in a vacuum form into nylon bags for later use. Refined wheat flour (as control) was obtained from Crown Flour Mills, Beirut, Lebanon. Chickpea grains  Offshore, Beirut, Lebanon. The additional ingredients  were all purchased from the local market. Three samples were prepared for the proximate fatty acids and macrominerals analysis. Sample 1 contained refined wheat flour as a control, sample 2 contained raw chickpea flour, and sample 3 contained processed chickpea flour.For the topping, The proximate composition analysis was carried out on the control flour, the raw chickpea flour, and the processed chickpea flour, according to the standard methods of the Association of Official Analytical Chemists  . MoisturFatty acid (FA) composition was measured in the three flour samples. Palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic FA were determined according to the International Organization for Standardization (ISO) (ISO 12966-4:2015) . The com3 and 2 mL of H2O2. Then 0.5 g of the sample was diluted in 50 mL. Some tubes were spiked with 1 ppm of P, Mg, and K each. One blank was spiked with 5 ppm of P, Mg, and K. Minerals were determined via inductively coupled plasma-atomic emission spectrometry (ICP-AES) [The macromineral content of phosphorus (P), magnesium (Mg), and potassium (K) was determined in the three different samples. All samples were digested with 4 mL of NHOICP-AES) . All theMankoushe is a dough-based pastry topped with mixed herbs, known as zaatar. zaatar is composed of ground oregano leaves, sesame seeds, sumac spices, and salt, mixed in the following percentages, respectively: 35.86%, 43.04%, 17.93%, and 3.15%. In the present study, three types of mankoushe were made in which the base was modified and the topping was maintained: regular mankoushe with 100% refined wheat flour, as a control; “chickpea-30” mankoushe, containing dough made up of a mixture of refined wheat/processed chickpea flour (70/30); and “chickpea-50” mankoushe, in which the dough was made up with a mixture of refined wheat/processed chickpea flour (50/50). Afterward and upon use, sunflower oil was added to the topping mixture based on the following percentages: 31.70% topping mixture and 68.30% oil. As for the dough, the three types were prepared under controlled conditions, according to the formulation given in mankoushe were baked in an electric oven  for 6 min at 300 °C. Food samples were prepared just before the sensory test.The base is usually made of refined wheat flour, and n = 60), aged between 20 and 40 years (mean ± standard deviation (SD) of 24.15 ± 3.8). Subjects were completely blinded to the three mankoushe samples. The test samples, assigned with three number codes, were presented randomly to the panelists and served at room temperature. Four sensory characteristics: texture, color, taste, and overall acceptability were evaluated using a nine-point hedonic scale, with a score of 1 (Dislike Extremely), 5 (Neither Like nor Dislike), or 9 (Like Extremely) [The study was approved by the Ethics Committee at the Doctoral School of Science and Technology (DSST), and all subjects were asked to sign a consent form before participation. Participants were recruited through an advertisement posted at the DSST, Lebanese University. A sensory analysis (Preference Test) was performed under optimal conditions . For the purpose of maximizing participant homogeneity, the study was conducted among untrained university females (tremely) . p < 0.05 level.Data were analyzed using Statistical Package for Social Sciences . Food composition results are expressed as the mean value ± standard deviation of three separate determinations, and data were evaluated using two-way ANOVA and Tukey’s post-hoc test. Sensory parameters were analyzed by comparing the means using one-way ANOVA followed by Tukey’s post-hoc test. In all analysis, significant differences were determined at the p < 0.001) in both chickpea flours compared to the refined wheat flour. However, moisture and carbohydrate contents were detected to be considerably lower. Chickpea flours appeared to be of higher nutritional value compared to refined wheat flour in terms of proteins, fat, ash, and fibers. These findings were in line with those reported by Hefnawy et al. [The proximate compositions of flours are presented in y et al. .p < 0.001) in processed (1.85%) compared to the raw (2.9%) chickpea flour. Total dietary fibers (15.75%), and crude fibers (3.7%) were modestly, but significantly (p < 0.001) increased in processed chickpea flour compared to raw chickpea flour, in which total dietary fibers and crude fibers were 14.70% and 1.63%, respectively. It appears that conventional processing of chickpea grains ultimately increased the fiber content of the flour. Fibers are the edible parts of pulses that are resistant to digestion and absorption in the human small intestine, with complete or partial fermentation in the large intestine. Fibers are of high importance since they have beneficial physiological effects, including laxation, blood glucose, and cholesterol attenuation [Between the two types of chickpea flour, proteins, fat, and total carbohydrates appeared to be similar. However, ash content was significantly lower  in palmitic acid between refined wheat (21.96%) and both chickpea flours (about 12%). While the amount of saturated FA stearic acid (2.72%) was lower in wheat compared to raw and processed chickpea flours (about 4%), the linolenic, linoleic, oleic, and palmitoleic acid contents were similar between flours . The fatty acids profile  shows a In the current study, conventional processing had minimal effects on the FAs, except for stearic. Nevertheless, the impact of processing on the FA profile of chickpea flour is not well documented, and varied affects have been reported .Furthermore, findings show that the main FAs found in raw and processed chickpea flours is the linoleic acid (about 58%) followed by the oleic acid (23%). Linoleic acid has numerous beneficial impacts, including improved blood lipid profile, improved insulin sensitivity, lower incidence of type 2 diabetes, and anti-arrhythmic effects . Oleic ap < 0.001), while P was minimally affected (9.26%). Mineral losses are likely to be attributed to their leakage into the cooking water or to the conventional processing steps. Soaking, cooking, drying, and milling are critical processes in flour production. Nonetheless, these steps are known to affect the concentrations of inorganic elements in grains [The phosphorus (P), magnesium (Mg), and potassium (K) content of chickpea flour  was highn grains .P, Mg, and K are essential minerals to human health. They have been suggested to be potentially protective against obesity and metabolic disorders ,16,17. PChickpea flours (raw and processed) in the present study differed in their mineral content from the ones reported earlier ,19. The mankoushe—regular, chickpea-30, and chickpea-50—are presented in mankoushe with regard to texture and color. As for the taste, chickpea-50 mankoushe was significantly accepted (p = 0.026) compared to regular. Regarding the overall acceptability, a significantly higher score (p = 0.005) was observed between the regular and the two types of chickpea mankoushe. However, the chickpea-30 and chickpea-50 varieties were similar in all sensory attributes.The sensory acceptability results of the three types of mankoushe significantly. The sensory enhancement seen in this study could also be attributed to Maillard reactions [Processing is well-known to enhance the flavor of pulses . In the eactions . It has eactions .The use of raw chickpea flour as an ingredient in bread generated inconsistent data. Chickpea bread with an incorporation rate of 30% showed either similar  or lowermankoushe at different ratios enhances the sensory qualities of the baked product. Therefore, it is a promising fortifier to improve the nutritional properties of wheat-based products. Raw and processed chickpea flours appear to be of a high nutritional value compared to refined wheat flour, even though conventional processing of chickpea grains affects the chemical composition of the flour. Nevertheless, processed chickpea flour is still considered to be a good source of protein, fibers, minerals, and healthy fats. Processed chickpea flour is suitable as an ingredient for bakery products, since its incorporation into the dough of