Mitochondria are the powerhouses of eukaryotic cells. Pairs of molecular machines with a rotary action, called ATP synthase, are embedded in their inner membranes and produce adenosine triphosphate, ATP, the fuel of life. These dimers form long rows on cristae tips, helping to endow them with their characteristic and mobile tubular shape. Our structural analyses of bovine dimers show that some structural changes depend on catalysis and others are independent. The monomers pivot and translate at wedge-shaped structures in their membrane domains. The structures suggest how dimeric ATP synthases might interact and fashion themselves to the range of oligomeric arrangements observed in mitochondria, whilst allowing the ATP synthase to produce ATP under a wide range of physiological conditions. The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer–monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae. The membrane extrinsic part of bH3 is supported and largely rigidified by other α-helices in the d and F6 subunits and in the membrane extrinsic region of subunit ATP8, all bound approximately parallel to the membrane extrinsic region of bH3. The upper part of the rod is attached via the C-terminal α-helix of subunit b, bH4, to the C-terminal domain of the OSCP subunit, and the N-terminal domain of this subunit binds to the N-terminal regions of the three α-subunits. The two domains of the OSCP are joined by a flexible linker, which provides the PS with a universal joint. During the rotary catalytic cycle, the α3β3-domain rocks from side to side, as noted before (The ATP synthase complexes embedded in the inner membranes of mitochondria (IMM) are rotary machines that make the ATP required to sustain life . Energy e domain , 3. Three domain . The cate domain , 5, 6. Td before –10, withd before , and thed before . The memd before , with bHd before . Subunitd before –13. How d before , 14. If d before –21. Thusd before . The fusd before , 17, 23,As shown here, the interaction between the two wedges linking the monomers in the dimeric ATP synthase is not a constant feature. It changes to accommodate both the rocking motions of the membrane extrinsic catalytic domain associated with catalysis and other motions that are independent of catalysis observed in the isolated dimeric ATP synthase. These pivoting motions and sliding translations between the monomeric complexes are intrinsic features of the dimeric ATP synthase. They allow it to operate in the ever-changing ultrastructure of the IMM and also to contribute to their ultrastructure.1, the inhibitor has trapped dimeric states where each monomeric catalytic domain contains the same three rotational points in the active catalytic cycle. The positions of these three rotational points relative to the peripheral stalk give rise to three states of the intact monomeric ATP synthase named s1, s2, and s3. Complete structures of the monomeric complex were built , models of dimeric complexes were constructed in a range of combinations of catalytic states and substates . The substates arise from changes that are independent of catalysis in the relative disposition of the two monomers to each other . By hierarchical classification of the earlier dataset  and subsequent refinement, 154,130 particles were resolved into nine discrete classes of the dimeric assembly representing rotational states [s1:s1], [s1:s2], [s1:s3], [s2:s1], [s2:s2], [s2:s3], [s3:s1], [s3:s2], and [s3:s3] at resolutions of 9.2, 11.9, 9.0, 9.4, 8.5, 10.7, 9.7, 11.4, and 13.1 Å, respectively. Composite dimer models were created by rigid body fitting of the maps and associated atomic coordinates  revealed 59 additional substates of the dimer, at resolutions ranging from 13.8 to 23.8 Å. The substates are distinguished from discrete classes of the dimeric assembly in defined rotational states by sequential lettering and are grouped according to catalytic state. For example, the first, second, and third substates of the ATP synthase dimer in which the right and left monomers are in catalytic states 1 and 3, respectively, are referred to as dimer substates [s1:s3a], [s1:s3b], and [s1:s3c].As described before, three datasets of 4,267, 2,238, and 4,096 dose-fractionated exposures of the purified dimeric bovine ATP synthase were collected, and structures of the monomeric enzyme were produced by single-particle analysis of 176,710 particle coordinates . From residues 1–20, subunit j is folded into an amphipathic α-helix jH1, which lies in the lipid head group region on the matrix side of the IMM where it interacts with the C-terminal region of transmembrane α-helix A6LH1 and CDL1. The negatively charged headgroup of CDL1 is bound to jK8, and to residues fQ38 and fY42 of the f subunit in the wedge and residues aT33, and A6LK27 and A6LK30 . Transmembrane α-helix jH2 (with residues 22–39 forming the transmembrane span) lies adjacent to A6LH1 on the opposite side of aH1 to the wedge and is associated with subunit a via interactions between its N-terminal region and residues 107–108, 110–111, and 114–115 of aH4 . The C-terminal region of jH2 (residues 40–49) protrudes into the IMS. Residues j50–60 were not modeled as they were not well-defined in the cryo-electron microscopy reconstructions. They are predicted to have an extended structure  that interacts with the same region in the adjacent j subunit, as indicated by the structures of the dimers in various rotational states and many of their substates, where density beyond the modeled residues can be observed . However, the interactions between the two j subunits are mobile, and the observed angle between the central axes of the central stalk or the PS of the monomers ranges from ca. 75 to 86° in the nine rotational catalytic states (Movies S1 and S2). This range of angles arises by the rigid body of the wedge and the rest of the membrane domain pivoting about contact points between the amphipathic α-helices jH1 (residues 1–20), provided by residues 3, 7, 10, 11, and 14 . The two jH1 α-helices are oriented in the plane of the IMM on the matrix side as the enzyme contorts during the rotational cycle (Movie S3). Residues 3, 7, 10, 11, and 14 in jH1 project toward the interface, possibly interacting via an intervening lipid. This pivoting reduces the net side-to-side displacement of the catalytic domain arising from the rotation of the asymmetrical central stalk and accommodates other changes in the structure of the dynamic mitochondrial cristae.In the dimeric complex, the monomer-monomer interface in the membrane domain consists of contacts between the two j subunits each on the external surface of the wedge (Movie S4) and others where the angle is greater than 90° (Movies S5 and S6). The motions in the former category are similar to those that occur during catalysis in that the relative dispositions of the membrane domains change by a pivoting motion about the interface between j subunits, with the C-terminal IMS protrusions remaining in contact. The flexibility of this pivot is an intrinsic property of the interface, and it has other roles beyond that described above of dissipating the rocking of the catalytic domain in the mitochondrial cristae. Most notably, it provides a mechanism to accommodate the general fluidity of the membrane in the region of curvature associated with the dimeric ATP synthases and the rearrangements that occur. The second category of motions between substates, where the angle between rotatory axes exceeds 90°, arises from sliding and twisting along the monomer-monomer interface presented by the a and j subunits and the membrane wedge, thereby effecting a compound rotation of one monomer relative to the other (Movies S4 and S5). For example, in Movies S5 and S6). In substate [s2:s2c], the C-terminal residues of subunit j are in contact in the IMS as in Movies S5 and S6). In the IMM, this transformation could be part of a mechanism for the dimers and oligomeric rows to adapt to the convolutions of the cristae, with the two contact positions representing a simple steric mechanism that restrains the rotation to upper and lower limits. In this mechanism, the wide angle conformation . The highly dynamic nature of the dimeric complex is demonstrated by the compilation of all the observed conformers in Movie S7.Other variations in motion occurring within groups of particles of a defined catalytic state are substates that are independent of catalysis. They fall into two categories, namely those where the angle between rotatory axes is less than 90° . Although it is not certain whether these very wide rotatory axis angle substates occur in native membranes, as the analysis was performed on isolated dimers, the data strongly suggest that the interfaces between monomers are significantly dynamic and that this dynamism is a general property of the interface, linked to motions of the enzyme that are both dependent and independent of catalysis. Indeed, this dynamism, which manifests as pseudoc2 symmetry about the dimer interface, necessitated specific refinement strategies to prevent the rotation of particle orientations about the pseudoc2-axis in order to isolate and subsequently maintain catalytically homogenous particle subsets during further particle realignment . Therefore, during image processing, each monomer was given the arbitrary designation “left or “right” with respect to a single viewing direction, and, for example, the state [s1:s2] and state [s2:s1] dimers are distinct and do not represent the same molecular state.It cannot be excluded that the wide angle substates with rotatory axis angles greater than ca. 95°, which depart significantly from the consensus structures with rotatory axis angles of ca. 76–86°, arise from the influence of the detergent micelle and the lack of the interactions between dimers that occur in native membranes. In curved membranes, an estimated tension of ca. 10 mN/m is exerted on mechanosensitive channels , 25, andMovies S5 and S6, places adjacent catalytic domains in closer proximity and, therefore, limits the degree of lateral curvature of the oligomeric row. Such a mobile contact also accounts for the positive and negative curvature along the cristae rows , gH2 (residues 42–60), and gH3 (residues 69–93), with transmembranous gH3 augmenting the skeleton of wedge, and amphipathic gH1 and gH2 associated with the top of wedge on the matrix side of the IMM. In the dimeric complex, the two gH2s have the capability to contact each other across the dimer-dimer interface and link the dimers together, as depicted in tae rows C and D atae rows , 17, 23.Movies S5 and S6, narrows the membrane footprint of the dimer, thereby bringing adjacent dimers closer.It is also possible that subunit k could be involved in linking dimers together. During the process of assembly of the closely related human enzyme, this subunit is the last to be incorporated into complex, and in human cells grown under glycolytic conditions, subunit k turned over more rapidly than the other subunits of the enzyme, which turned over together at a slower rate . These o1 molecules spanning between the globular F1 domains across the interface between the two dimeric ATP synthases. However, the membrane domains of the porcine tetramer were misinterpreted . Also, the tethering of adjacent catalytic domains by IF1 induces a torsion in the F1 and PS domains, tilting the rotatory axes accompanied by narrowing of the membrane domain in the dimeric unit . This change in the membrane domains could have arisen from the effects of extraction of the complexes from the membrane with detergents and the loss of endogenous lipids . Thus, it is possible that the binding of the two IF1 dimers to form the ATP synthase tetramers occurs during this extraction procedure, and that the formation of tetramers is an artifact. Relaxation of the tilted geometry of adjacent dimers and the dimer-dimer interface in the porcine and ovine tetramers is not possible because of the close proximity of the catalytic domains, and this prevents the formation of planar oligomeric rows. In contrast, the models of oligomerization of active dimers proposed here permit the formation of the variety of oligomeric arrangements needed to produce the geometry of the IMM. Also, they are compatible with the fluidity of the dimer interface observed in the structures of the bovine dimers (Movies S4–S7). These models of oligomerization of active dimers are incompatible with formation of inactive tetramers . The changes to the architecture of the membrane domains in the tetrameric structures produce a rigid curvature along the axis of oligomerization that is incompatible with the ultrastructures of the IMM observed by tomography .Independent of these physiological considerations, the structures of free dimers and equivalent dimers in tetramers differ substantially, and, for example, the angle between the rotatory axes of monomers in each dimer in tetramers is 112–116°, whereas it is ca. 76–95° in free dimers. This wider angle between monomers in dimers within tetramers is accompanied by an outward bending of each rotatory axis and a significant rearrangement of the interface between monomers in the dimers . The membrane domain of the tetramer has undergone a structural rearrangement where the individual monomeric membrane domains appear to have collapsed inwards: See the change in the inclination of the cmography , 23, and1 that has not been considered so far in the context of the possible influence of the inhibitor protein on the topography of the cristae is that one of its two active inhibitory regions could bind to an ATP synthase complex in hydrolytic mode, leaving the second inhibitory region unbound and unable to insert itself into a catalytic site in a second copy of the enzyme. Indeed, such a state might be quite common as the inhibitor could bind to all three of the possible states of the enzyme, s1, s2, and s3, and not be restricted to cross-linking adjacent s1 and s3 states. In contrast to the cross-linking of two dimers discussed above, such a binding mode would be unlikely to have a major impact on the ultrastructure of the cristae.One possible mode of binding of dimeric IFMammalian ATP synthases are highly conserved in the composition and sequences of their subunits, and, therefore, the nature of the association between monomers in the dimeric units will also be conserved. Likewise, the compositions and structures of the ATP synthases in yeasts and fungi resemble the mammalian enzymes in many respects , 31. In 32SI Appendix.The purification of dimeric ATP synthase from bovine heart mitochondria, the preparation of cryo-em grids, data collection, preprocessing of micrographs, automated particle picking, and initial particle selection by two-dimensional classification has been described before . ParticlSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Ants have strict requirements on the building materials of the nest, such as the size, weight, luster and color of soil particles. The soil of underground ant nests is composed of clay particles cemented together to form a hard brick-like material. The ant nest powder shows pozzolanic activity after calcination, which can meet the requirements for active admixture of concrete. Under the standard curing condition, the influence of calcined ant nest clay powder (CANCP) on the durability of concrete is evaluated by chloride penetration resistance, carbonization resistance and freeze–thaw resistance, and the influence of the powder content is investigated. The results show that when the content of CANCP is less than 10%, the chloride penetration resistance of concrete increases with content of CANCP. In the early stage of carbonation, the greater the content of CANCP, the higher the carbonization rate of concrete. In the middle and later stage of carbonation, the carbonation rate of CANCP concrete is significantly lower than that in the early stage, and the carbonation depth is linearly related to the carbonation time. When the content of CANCP is less than 20%, the freeze–thaw resistance of CANCP concrete is better than that of the reference concrete. Ants have strict requirements on the building materials of the nest, such as the size, weight, luster and color of soil particles. The underground ant nest is generally composed of soil and gravel, and is mixed with organic substances such as dead plant bodies and ant secretion, and the clay content is large. Compared with the common clay around the ant nest, its physical and chemical properties have changed greatly. The soil of underground ant nests is composed of clay particles cemented together to form a hard brick-like material. Most of the natural clays have pozzolanic activity after calcination . At presThe calcined ant nest clay powder (CANCP) can partially replace cement and the associated influence on the compressive strength of concrete has been studied . After cPO 42.5 ordinary Portland cement was used , and theA standard mix proportion of design strength of C50 was used as reference concrete (CT for short) in this test, and the calculated mix proportions were shown in CANCP was obtained from the underground nest soil of iridomyrmex anceps after calcination at 800 °C for 2 h and grinding for 60 min by SM-500 ball crusher . The phy2, Al2O3 and Fe2O3 in CANCP reached 88.76% which is greater than the 70% specified in the ASTM  standard for concrete mineral admixtures [The total content of SiOmixtures . The cheD of chloride ion was measured. The vibration forming and test process of the specimens are shown in In this test, the electric flux method of ASTM C1202  was usedIn this test, the method of GB/T 50082-2009  was used2 concentration (20% ± 3%), relative humidity (70% ± 5%), and temperature (20 ± 2 °C).Standard curing was adopted, and the specimens were taken out from the standard curing room two days before the test, and then they were dried for 48 h in the electric air blast drying oven at 60 °C After drying, except one side of the specimen, the other three sides were sealed with heated paraffin. On the exposed side, parallel lines were drawn with a pencil at a distance of 10 mm along the length direction as the measurement point of the predetermined carbonation depth. HTX-12X microcomputer concrete carbonation test chamber was used in the test, and the test was carried out under the conditions of COThe specimens were taken out when the carbonization process reached 3, 7, 14 and 28 d, and the splitting method was used on the pressure testing machine to start the fracture from 50 mm at one end. After the fracture, the cross section was sealed with paraffin again, and the remaining specimen was put into the carbonization test chamber to continue carbonization until the next carbonization age. The residual powder was removed on the section of the specimen obtained by splitting, and 1% phenolphthalein reagent was sprayed immediately on the cross section. After 30 s, the carbonation depth of 10 measuring points was measured respectively. The average value of carbonation depth at each test point was calculated, as the carbonation depth of the specimen, and the calculation accuracy was 0.001 mm. The determination carbonation depth of specimens is shown in First, 100 mm × 100 mm × 400 mm prism specimen was adopted for the quick freeze–thaw test . The speThe mass loss rate ΔW and the relative dynamic elasticity modulus reduction rate ΔD are taken as the indexes to evaluate the freeze–thaw resistance performance of concrete. The mass and transverse fundamental frequency of the specimens were measured every 25 freeze–thaw cycles, as shown in 0 and the chloride ion penetration depth value XD were measured, and the results are shown in According to the electric flux method of ASTM C1202 , the 6 hIt can be seen from the test results that the chloride penetration resistance of concrete increases first and then decreases after adding CANCP. When the content of CANCP is less than 10%, the chloride penetration resistance of concrete increases with the increase of the content of CANCP, and the electric flux of concrete containing 5% and 10% CANCP is 1310.1C and 998.9C, which is 8.8% and 30.4% lower than that of the reference concrete CT. When the content of CANCP is more than 10%, the chloride penetration resistance of concrete decreases with the increase of the content of CANCP, and the electric flux of concrete containing 15% and 20% CANCP is 1639.5C and 2207.1C, which is 14.2% and 53.7% higher than that of the reference concrete CT. On the contrary, the initial current value and chloride penetration depth value of concrete first decreased and then increased after adding CANCP. When the content of CANCP is less than 10%, the initial current value and chloride penetration depth of concrete decrease with the increase of the content of CANCP; when the content of CANCP is more than 10%, the initial current value and chloride penetration depth of concrete increase with the increase of the content of CANCP.2 is 0.964, and there is a good linear correlation between the initial current and 6 h electric flux. Therefore, the initial current can be used to calculate the electric flux to accelerate the test efficiency. The 28 d initial current can be used as the evaluation index to accelerate the detection of the chloride penetration resistance of CANCP concrete.Feldman believes that there is a good linear correlation between the initial current or conductivity and the 6 h electric flux for the same type of concrete. Therefore, it can be considered to provide a fast and convenient evaluation method for the chloride penetration resistance of concrete by testing the initial current or conductivity . The lin2 is 0.943, and there is a good linear correlation between chloride penetration depth and 6 h electric flux. Therefore, the penetration depth XD can be calculated by measuring the electric flux Q, and then the permeability coefficient of chloride ion in the specimen can be obtained according to Nernst–Planck equation.The linear correlation analysis of chloride penetration depth and 6 h electric flux is shown in According to JGJ/T 193-2009 , the carThe carbonation depth histogram and the carbonation depth-time curve of concrete with different content of CANCP are shown in It can be seen from the histogram that the carbonation depth of the concrete with CANCP in each curing age is greater than that of the reference concrete. With the increase of the content of CANCP, the carbonation depth gradually increases. The carbonation time is 28 days, and the carbonation depth of concrete with 20% of CANCP is 9.863 mm, which is more than twice of that of concrete without CANCP. As shown in The mass loss rate and relative dynamic elastic modulus curve of different content of CANCP with the number of freeze–thaw cycles, as shown in It can be seen from The dynamic modulus of elasticity is closely related to the internal structure of materials, that is, the dynamic modulus of elasticity of concrete is more sensitive to the internal structure damage. Under the action of the freeze–thaw cycle, the internal structure of the concrete gradually changes from compact to loose, and the concrete itself inevitably has some original microcracks and defects. With the increase of freeze–thaw cycle times, the microcracks and defects in the concrete continue to expand, the section strength gradually decreases, the internal microstructure of concrete is disturbed, resulting in the decrease of dynamic elastic modulus of the concrete.The decrease of dynamic modulus of elasticity of each content is not obvious in the early stage of the freeze–thaw cycle (before 100 cycles), and the accelerated failure appears in the middle and later stage of the test. The accelerated failure occurred after 150 freeze–thaw cycles for the reference group without CANCP and the group with 20% of CANCP, while the accelerated failure occurred after 225 cycles for the group with 10% of CANCP. The concrete with 10% of CANCP demonstrated the best freeze–thaw resistance.b is used to estimate the freeze–thaw resistance of concrete:The freeze–thaw resistance durability index KIn the formula: P is the relative dynamic elastic modulus of concrete after n freeze–thaw cycles; N is the number of freeze–thaw cycles that the concrete can withstand.The calculated freeze–thaw resistance durability index of CANCP concrete is shown in 2 and Al2O3 in CANCP can generate more hydrated calcium silicate and aluminum silicate with Ca(OH)2, which can increase the binding capacity of concrete to chloride ions and reduce the content of free chloride ions [SiOide ions .Micro filling effect and micro aggregate effect . The fin2 is consumed in the hydrated cement paste, and C-S-H gel is increased during the process of pozzolanic reaction. The pozzolanic effect of CANCP can transform Ca(OH)2 to C-S-H gel, and fill between cement hydration products, which can effectively improve the performance of concrete. Meanwhile, the pozzolanic C-S-H gel with low Ca/Si ratio will produce when CANCP has second pozzolanic reactions with traditional C-S-H gel in the hydrated cement paste, and the pozzolanic C-S-H gel is relatively stable which can improve the performance of the traditional C-S-H gel to improve the performance of the concrete. The addition of CANCP can improve the microstructure of the concrete, reduce the porosity, refine the pore size and make the concrete more dense, and all of these increase the diffusion resistance of the concrete to chloride penetration [2 in concrete containing 10% CANCP, and it shows that concrete containing 10% CANCP has a high degree of hydration; C-S-H gel in concrete with 10% of CANCP is more dense and clustered to a higher degree than ordinary concrete.Pozzolanic activity and pozzolanic effect of CANCP promotes the secondary hydration reaction of cement, resulting in a large number of C-S-H gels. A large amount of Ca(OH)etration . It can When the content of CANCP is not more than 10%, it can effectively improve the chloride penetration resistance of concrete. The reasons are listed below:2, Al2O3 and Fe2O3 in CANCP with the main products of cement hydration is as follows [The secondary hydration reaction of the active components SiO follows :(2)CaO+H2, resulting in C-S-H gel and C-A-H gel with low calcium–silicon ratio. At the same time, the active silicon in CANCP will react directly with the C-S-H gel generated from the cement to form C-S-H gel with low calcium–silicon ratio [2 and Al2O3 in the admixture react with Ca(OH)2 generated by secondary hydration of the cement clinker, which reduces the alkali reserve in the cementitious paste [CANCP has active ingredients, and its secondary hydration requires consumption of Ca(OH)on ratio ,25. Althus paste . When th3 have filling effect. The internal pore structure of cementitious materials is improved, resulting in slow carbonation reaction.The carbonization rate of CANCP concrete at the early stage of carbonization is much larger than that in the middle and later stage of carbonation. This is due to the large number of pores in cementitious material in the early stage. In the middle and later stage, the pozzolanic reaction occurred, and the products of C-S-H gel and carbonization reaction product CaCOThe pozzolanic effect by CANCP can result in the formation of low alkaline calcium silicate hydrate with high strength, increasing the amount of hydration cementitious material in concrete, improving the ability of the traditional C-S-H gel, thereby improving the performance of hardened cement paste and improving the freeze–thaw resistance property of concrete. At the same time, micro filling effect by CANCP can result in denser paste, and improved freeze–thaw resistance property ,28.The content of CANCP should be controlled at a proper proportion, and the best proportion is 10%. When the content is more than 20%, the hydration of CANCP will not be complete. At the same time, non-active components in CANCP remain in the concrete to form more pores which increases the porosity of concrete. Under the action of the freeze–thaw cycle, micro cracks will be formed in the concrete, which lead to the deterioration of the internal structure of the concrete.When the content of CANCP is less than 10%, the initial current value and chloride penetration depth of concrete decrease with the increase of the content of CANCP and it can effectively improve the chloride penetration resistance of concrete.The addition of CANCP can accelerate the carbonation process of concrete. When the CANCP content is not more than 20%, the carbonation resistance grade of concrete is not impacted.When the content of CANCP is less than 10%, the concrete with CANCP can still keep the internal structure compact under the action of freeze–thaw and it can effectively improve the freeze–thaw resistance of concrete.It is proved that CANCP can be used as green concrete admixture, which provides favorable evidence for the feasibility of CANCP as ecological building materials.This paper attempts to prove that CANCP can be used as a mineral admixture to improve the durability of concrete. Based on the experimental tests and the data analyses, the following conclusions can be drawn:


Sulfolobus solfataricus (aDHS) to understand the structural and dynamical features of the GC7 inhibition. The advanced metadynamics (MetaD) classical molecular dynamics simulations show that the GC7 interaction is less stable in the thermophilic enzyme compared to hDHS that could underlie a lower inhibitory capacity of the hypusination process in Sulfolobus solfataricus. To confirm this hypothesis, we have tested GC7 activity on S. solfataricus by measuring cellular growth, and results have shown the lack of inhibition of aIF5A hypusination in contrast to the established effect on eukaryotic cellular growth. These results provide, for the first time, detailed molecular insights into the binding mechanism of GC7 to aDHS generating the basis for the design of new and more specific DHS inhibitors.Translation factor 5A (eIF5A) is one of the most conserved proteins involved in protein synthesis. It plays a key role during the elongation of polypeptide chains, and its activity is critically dependent on hypusination, a post-translational modification of a specific lysine residue through two consecutive enzymatic steps carried out by deoxyhypusine synthase (DHS), with spermidine as substrate, and deoxyhypusine hydroxylase (DOHH). It is well-established that eIF5A is overexpressed in several cancer types, and it is involved in various diseases such as HIV-1 infection, malaria, and diabetes; therefore, the development of inhibitors targeting both steps of the hypusination process is considered a promising and challenging therapeutic strategy. One of the most efficient inhibitors of the hypusination process is the spermidine analog N1-guanyl-1,7-diaminoheptane (GC7). GC7 interacts in a specific binding pocket of the DHS completely blocking its activity; however, its therapeutic use is limited by poor selectivity and restricted bioavailability. Here we have performed a comparative study between human DHS (hDHS) and archaeal DHS from crenarchaeon  Protein synthesis represents the final step of gene expression and, being one of the most energy-consuming process in cells, not surprisingly, it is highly regulated in all domains of life.Regulation can be exerted either on the whole translation process or on specific mRNAs, and it can be mediated by different cis-acting elements present on the mRNAs, as well as by trans-acting factors  into a nonstandard amino acid called hypusine using spermidine as a substrate , that shares with the human homolog 31 and 52% of amino acid identity and similarity, respectively, interacts with aIF5A catalyzing the formation of deoxyhypusine, suggesting that the first step of hypusination is conserved in this organism  , a compoThe use of GC7, alone or in combination with other drugs, was demonstrated to inhibit the growth of various mammalian cells .Here we have used a combination of advanced computational approaches and experimental techniques to analyze the different features of the binding mode of GC7 into the active site in both in vivo experiments on S. solfataricus cells whose growth was unaffected by the presence of GC7.Results from metadynamic (MetaD) simulations highlighted a different stability of the two GC7–DHS complexes, which is due to specific interaction networks established within the binding sites. Interaction of GC7 with aDHS is significantly less stable compared to hDHS, and this result was validated by This comparative and multidisciplinary study provided an in-depth characterization of the molecular mechanism of interaction of GC7 with both human and archaeal DHS paving the way for the design of new, specific, and more sensitive DHS inhibitors.S. solfataricus P2 cultures were grown in liquid Brock's medium  in a shaking water bath at a speed of 150 rpm at the temperature of 348 K (75°C). Growth was monitored by measuring the optical density at 600 nm (OD600). For a typical experiment, an S. solfataricus P2 culture with a starting OD600 of 0.05 was split into six different flasks of 10 ml each, and GC7 (Sigma-Aldrich) was added in the following concentrations: 0 (control), 10, 50, 100, 250, and 500 μM respectively. OD600 measurements were taken at 0 hour (h), 15, 24, 48, 72, and 96 h. At a time point of 48 h, a 1-ml aliquot from each sample was harvested and centrifuged. The resulting cell pellets were washed three times with fresh Brock's medium and then resuspended with a resuspension buffer . Cells were lysed by six freeze and thaw cycles, and total protein concentration of each sample was determined using the Bradford reagent (Sigma-Aldrich). The levels of hypusinated aIF5A in each lysate sample were analyzed by Western blot.Western blot analysis was performed as previously described  in its homodimeric state  . The overall quality of the model was assessed with PROCHECK  on backbone and ligand atoms. Subsequently, MD simulation in an NVT ensemble was used to heat the system from 0 to 100 K in 1 ns with restraints lowered to 500 kJ mol−1 nM−2. Temperature was controlled by the Berendsen thermostat  for both systems. The number of contacts in the native pose was around 650 for both the H. Sapiens and S. solfataricus systems, and we considered the ligand as exited from the binding site when the number of contacts reaches the number of 200. This number is small enough to guarantee the overcoming of the energetic barrier splitting the bound and the unbound states. The residence time in MetaD calculation was computed as the average time required to observe the unbinding (number of contacts <200) in the different replicas. A change in this cutoff value does not appear to affect the result of the analysis (data not shown).Metadynamics is an enhanced sampling method based on the introduction of a history-dependent bias on a small number of suitably chosen collective variables  with the residues composing the protein binding pocket. Results of these calculations are shown in for hDHS . Path A for hDHS . Notablyfor hDHS .Although the two DHS enzymes share a significant sequence identity and similarity  as reported in the previous paragraph, the results described so far prompted us to investigate more thoroughly the inhibition mechanism of GC7 in hDHS and aDHS.H. sapiens and S. solfataricus. To experimentally confirm these theoretical results, we have tested the effect of GC7 on S. solfataricus cellular growth. S. solfataricus cells were grown in the absence and in the presence of different concentrations of GC7 . Results presented in S. solfataricus even at the highest concentration (500 μM) of the inhibitor. These results are in contrast with those obtained on eukaryotic cells  by Western blot. Two primary antibodies were used: one able to recognize both modified and unmodified aIF5A . Chromatograms showed the profile of native polyamines, visible following fluorescent derivatization, relative to Proline, Spermidine, and Spermine. Analysis of GC7 treated cells showed two additional peaks identical in retention time to those obtained following standard injection, suggesting that, despite the physical barrier represented by the cell wall, GC7 is efficiently incorporated into the cells . The dimeric forms of DHS were simulated containing two binding pockets each bound to a GC7 molecule. We will refer to each binding pocket as binding site 1 and binding site 2. hDHS was simulated at 300 K (27°C), while aDHS was simulated at 348 K (75°C) to better reproduce the biological condition in which the two enzymes are active. The simulations show that GC7 is strongly bound to the human enzyme maintaining the original binding mode for the entire simulation time . This analysis highlighted a higher fluctuation of the S. solfataricus binding site due to the numerous changes in the position of the ligand.Finally, to get insights on the reduced stability of GC7 within S. solfataricus, lying respectively at the two ends of GC7. This is in line with the movement of the ligand that rotate within the binding site, losing contacts at the two ends , successfully and widely used in the study of protein–protein interaction and mechanisms of inhibition exerted by small molecules or peptides  occurs more frequently , and it implies a protein conformational rearrangement to allow the ligand exit   . On the 165–170) . In this165–170) . Our Metl growth  and aIF5l growth  in contrl growth . Considel growth .S. solfataricus show a higher mobility of the ligands in both binding sites, a further evidence of a formation of an unstable complex in this cell   . Interestaricus) . RemarkaH. sapiens enzyme.Therefore, ineffectiveness of GC7 on the archaeal model may be due not only to the different growth conditions but also to the aminoacidic composition of the binding site compared to the The identification of the unbinding path of GC7, together with the comparative description of the structural and dynamical differences between aDHS and hDHS, provides a broad set of crucial information for the design of a new class of inhibitors, not only able to block the hypusination process but also the formation of the DHS–eIF5A complex. In the first instance, these information can be used to filter out the most promising potential inhibitory compounds during a virtual screening routine and, second, to drive the design of peptides/peptidomimetics, molecules highly suitable to inhibit protein–protein interactions . We have found that, different from hDHS, GC7 does not inhibit the activity of aDHS.S. solfataricus). Furthermore, we have confirmed the ineffectiveness of GC7 in inhibition of both cell growth and hypusination by experimental approaches. Moreover, MetaD simulations confirm the instability of the GC7 inhibitor within the aDHS binding pocket. These data provide a detailed knowledge of the DHS-GC7 interaction, laying the foundation for the design of new specific and more efficient DHS inhibitors.Comparative analysis has shown that aDHS lacks key residues for the GC7 interaction into the active site (Glu 136, Asp243, and Asn292 that are substituted by Asp95, Thr200, and Trp248 in The original contributions presented in the study are included in the article/This research article was conceived by DD and AL with contributions from all authors, under the supervision of DD and AL. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


On the 4th of August 2020, a massive explosion occurred in the harbor area of Beirut, Lebanon, killing more than 100 people and damaging numerous buildings in its proximity. The current article aims to showcase how open access and freely distributed satellite data, such as those of the Copernicus radar and optical sensors, can deliver a damage proxy map of this devastating event. Sentinel-1 radar images acquired just prior (the 24th of July 2020) and after the event (5th of August 2020) were processed and analyzed, indicating areas with significant changes of the VV  and VH  backscattering signal. In addition, an Interferometric Synthetic Aperture Radar (InSAR) analysis was performed for both descending (31st of July 2020 and 6th of August 2020) and ascending (29th of July 2020 and 10th of August 2020) orbits of Sentinel-1 images, indicating relative small ground displacements in the area near the harbor. Moreover, low coherence for these images is mapped around the blast zone. The current study uses the Hybrid Pluggable Processing Pipeline (HyP3) cloud-based system provided by the Alaska Satellite Facility (ASF) for the processing of the radar datasets. In addition, medium-resolution Sentinel-2 optical data were used to support thorough visual inspection and Principal Component Analysis (PCA) the damage in the area. While the overall findings are well aligned with other official reports found on the World Wide Web, which were mainly delivered by international space agencies, those reports were generated after the processing of either optical or radar datasets. In contrast, the current communication showcases how both optical and radar satellite data can be parallel used to map other devastating events. The use of open access and freely distributed Sentinel mission data was found very promising for delivering damage proxies maps after devastating events worldwide. In emerging situations such as industrial and technological accidences, earth observation sensors can provide near-real-time information to local stakeholders and international organizations. This has been already demonstrated in the past in several examples such as the case study of the blast at Cyprus Naval Base , at the Fukushima Daiichi nuclear disaster , etc. High-resolution satellite sensors are mainly used for rapid mapping and recording of the damage over large extents. However, the use of freely distributed sensors such as those of the Copernicus Programme did not thoroughly investigate in the past. This is in contrast with their wide use in monitoring other hazards all over the world, which has been demonstrated in several articles ,4. This The event took place in the evening of the 4th of August 2020 in the area near the main harbor of Beirut, the capital of Lebanon. According to the media, a fire near the port ignited a large nearby store of ammonium nitrate, which is a highly explosive chemical often used in fertilizer. More than 100 people died, while more than 5000 were wounded. It was estimated that at least 300,000 people were left homeless from this event, while the destruction was estimated to cost between 10 and 15 billion USA dollars .From the early beginning, several agencies have tried to support ground rescue investigations. Examples of this effort are by the Center for Satellite Based Crisis Information (ZKI) of the German Aerospace Centre (DLR)  that useWhile the above-mentioned results are very important for local stakeholders as they provide damage proxy maps in a short time, the detection of damaged areas is usually based on the processing on either optical or radar products. In this paper, we aim to investigate the Beirut explosion that took place on 4th of August 2020 by using in parallel both optical and radar Copernicus Sentinel data. The detection of damage areas in different wavelengths of the spectrum can further strengthen the final damage proxy maps.The paper is organized as follow: initially, the methodology and the data used are presented, while the results from the change detection and InSAR analysis from the Sentinel-1 datasets are shown. These results are also compared with optical Sentinel-2 images as well as other published—in the media—high-resolution results. Finally, a general discussion for the potential of space-based applications for monitoring areas under threat is presented.The study area was focused on the harbor area of Beirut in Lebanon, where the explosion occurred, and its surrounding area . While mFor the needs of the study, Copernicus radar freely distributed datasets were used. Once the available images were selected from the Alaska Satellite Facility (ASF) services , these wThe radar processing at the HyP3 cloud platform included two products as follows. A change detection map based on a pair of Radiometrically Terrain-Correct (RTC) Sentinel-1 data and an Interferometric Synthetic Aperture Radar (InSAR) analysis map, using both ascending and descending Sentinel-1 images, was used to map changes in the area. For the first product, the log difference for both VV  and VH  backscattering gamma 0 amplitude polarizations of the RTC Sentinel-1 images was estimated based on the following equation for each image pixel:1 and S2 [In addition, the coherence values were also mapped over the area. The degree of coherence is defined as the normalized complex correlation coefficient of the complex backscatter intensities S1 and S2 . CoherenCoherence values may range from 0 to 1; the larger the number, the higher the coherence. A low coherence value may indicate areas that have changed between the two overpasses of the Sentinel-1 radar sensors.The InSAR analysis of an ascending and a descending pair of Sentinel-1 images was executed—as mentioned before in the HyP3 platform—using the Gamma software. In short, the InSAR Gamma algorithm for the Sentinel-1 images comprises of eleven 11) steps as follows. (1) determine the overlapping area using the two Sentinel-1 images. (2) Download the European Union Digital Elevation Model (EU-DEM) v1.1, a hybrid Shuttle Radar Topography Mission digital elevation model (SRTM), and ASTER Global Digital Elevation Map (ASTER GDEM) data fused by a weighted averaging approach over the area of interest . (3) Cre steps asThen, the final products from the change detection and the InSAR analysis were imported into a Geographical Information System (GIS), namely the ArcGIS v.10 software, whereas zonal statistics per each zone, namely Zone A to Zone D, were estimated. The overall results generated a damage detection proxy map that was also compared with available high-resolution RGB WorldView-2 images as well as other reports from international agencies, namely the German Aerospace Center (DLR) and the ARIA teams. In addition, Sentinel-2 optical images, taken before and after the explosion, were downloaded from the Sentinel Data Hub  in orderAs mentioned earlier, two pairs of Sentinel-1 images were used for the change detection and the InSAR analysis. In addition, two pairs of Sentinel-1B images in descending and ascending orbits were processed for the InSAR analysis. The images were taken on the 31st of July 2020 and the 6th of August 2020 with a descending orbit see , while aFinally, Sentinel-2 images of Level 2A (Bottom of Atmosphere (BOA) reflectance images) with a spatial resolution of 10/20 m acquired before (24th of July 2020) and after (8th of August 2020) the explosion, with limited cloud coverage over the area of interest were downloaded from the Sentinel Data Hub. The RGB optical composite of the Sentinel-2 image taken after the explosion and the NIR-R-G pseudo color composite of the same image were used for comparison purposes through interpretation with the Sentinel-1 image analysis.In addition, Principal Component Analysis (PCA) was applied to the integrated Sentinel-2 dataset (images of 24th of July 2020 and 8th of August 2020 together) to detect any significant spectral changes near the harbor area. The PCA is a well-known statistical analysis process that takes into account the spectral variations within the image . While tThe HyP3 big data cloud platform provided by the ASF was used to process the radar Sentinel-1 datasets. The platform is designed on Amazon cloud services, and upon submitting the request form, the end users may select several products and processing algorithms. Further details for the potentials of this platform can be found in . The finThis section provides an overview of the results generated for this analysis of the radar images. These results are also compared with optical Sentinel-2 data as well as other high-resolution images such as those of WorldView-2 and the public products from the DLR and ARIA teams.Below, the results from the Sentinel-1 radar images is provided, following the change detection image analysis in Integrating both these polarization results, the majority of the differences of the backscattered signal (VV and VH polarizations) are around the blast zone mainly in Zone A and Zone B . This is2). As shown, the majority of the damaged area is found in Zone A with an area of 17.9 hectares, which equals 37.2% of the total damaged area, while another 24.5% of the total damage area is reported in Zone B that extends up to 1000 m away from the blast site. Zonal statistics analysis indicate a similar pattern for Zone C and Zone D with an approximately average damage area of 9 hectares (18.5%); however, the majority from these areas are in the medium log difference threshold units (−0.25 to −0.15 and 0.15 to 0.25). Zone A also reports the highest percentage in terms of areas of having a log difference more than >−0.25 and >0.25 .The next table  shows thSynthesizing the results from The majority of the damaged areas are found between a medium threshold value (from −0.25 to −0.15 and 0.15 to 0.25 log difference of the VV and VH polarization) with a total area of 84.4 hectares  that equals 72.1% of the total damaged area. The remaining 27.9% of the damaged area (32.5 hectares) is found at the higher threshold values (>−0.25 and >0.25).Following the change detection analysis, the InSAR analysis for mapping the micromovements of the area because of the generated seismic waves from the explosion was performed. As mentioned earlier for the needs of the InSAR analysis, a pair of Sentinel-1B Single Look Complex (SLC) images was used, with acquisition dates of 31st July 2020 and the 6th of August 2020.Based on this wrapped interferogram, which is mainly useful for visualization purposes, the unwrapped interferogram was calculated for the descending orbit Sentinel-1 images a, which The line of sight (LOS) displacement map was estimated for both the ascending and the descending pairs of SLC Sentinel-1  into meaIn the area of interest, and based on the descending orbit Sentinel-1 images see , we can Of course, these examples should be taken with great caution, as a low coherence is reported at this site due to the VV and VH changes observed from the explosion, as well as the sensitivity of the Sentinel-1 sensors (in relation to their wavelength that corresponds to ≈5.54 cm). However, the findings here are quite noteworthy, as even with low coherence, the InSAR analysis was able to detect the blast site and locate significant changes, such as those reported from the change detection analysis.The coherence map for both descending and ascending orbits are shown in The results from the Sentinel-1 images have also been compared with the Sentinel-2 optical image taken after the 4th of August 2020. The RGB composite of the image is shown in Visual interpretation of the optical image can depict some of the most apparent damages over the area  a change detection approach and (b) an InSAR analysis. The overall results were also confirmed through other studies that have been reported only some hours after the event, such as the case of the ARIA and the DLR teams. In addition, the availability of a high-resolution WorldView-2 image taken over the area of interest has confirmed the results generated from the analysis of the Sentinel-1 sensors.To evaluate the overall performance of this study, we have compared the results presented above with other published material (maps) and available high-resolution WorldView-2 images. Initially, we have compared our change detection results (Damage Proxy Map) with the one of the ARIA team , as thesWithin the area around the harbor of Beirut, digitization of the buildings that have been damaged has been reported by the MapAction platform . SimilarA direct comparison of the high-resolution WorldView-2 image that was taken just after the explosion over the harbor of Beirut was carried out. This image is shown in A damage proxy map based on the processing of Sentinel-1 images was carried out in this study over the harbor area of Beirut, Lebanon after the high explosion occurred in the area on the 4th of August 2020. The analysis of this study was achieved through a change detection analysis of Sentinel-1 images, while the seismic waves results were also mapped using an InSAR Sentinel-1 image analysis.The analysis was able to be carried out due to the systematic observations of the Copernicus Sentinel-1 sensors operated by the European Space Agency (ESA), while the processing chain was carried out by the big-data cloud HyP3 platform operated by ASF. This combination allows end users to process in a short time  a series of radar processing chains in almost global coverage. Of course, this is feasible due to the free and fully open policy (FFO) of the Sentinel datasets.The findings of this study are well aligned with other products delivered by specialized space centers such as the DLR and the ARIA teams, while a comparison was also performed using a high-resolution WorldView-2 image. While the medium-resolution Sentinel-1 images and their products do not allow us to detect individual destruction in a building level, the 10-m resolution is enough for estimating with high accuracy the damaged area. The use of Sentinel-2 images could support the detection analysis through visual interpretation of significant changes in the landscape as well as through a PCA temporal analysis. This was more obvious using the NIR-R-G pseudo color composite; however, due to the spatial resolution of the image (20 m), the detection of smaller areas was quite challenging.The use of both sensors, namely the Sentinel-1 and Sentinel-2, can be further utilized in the future to support emergency situations, with an almost global coverage. In this case, other events not so well known can be monitored by the scientific local community to support specific emergency needs. Of course, these maps can be only indicative of the destruction, as ground verifications are needed. In addition, the analysis of the results from this series of analyses should be seen with great caution, as the images might suffer from other factors that influence the final outcomes. For instance, the relative micromovements can be due to other sources, such as the atmospheric component and even DEM accuracy, while the change detection of the VV and the VH polarizations need to be linked also with their coherence.Overall, the use of Sentinel data was found very promising for supporting ground investigations after an event . Future steps can include considering the automation of the whole procedure, minimizing the time between the data processing and the event itself.


The objective of this work is to study the delamination of bismuth ferrite prepared by atomic layer deposition on highly oriented pyrolytic graphite (HOPG) substrate. The samples’ structures and compositions are provided by XPS, secondary ion mass spectrometry (SIMS) and Raman spectroscopy. The resulting films demonstrate buckling and delamination from the substrates. The composition inside the resulting bubbles is in a gaseous state. It contains the reaction products captured on the surface during the deposition of the film. The topography of Bi-Fe-O thin films was studied in vacuum and under atmospheric conditions using simultaneous SEM and atomic force microscopy (AFM). Besides complementary advanced imaging, a correlative SEM-AFM analysis provides the possibility of testing the mechanical properties by using a variation of pressure. In this work, the possibility of studying the surface tension of the thin films using a joint SEM-AFM analysis is shown. The scursors) ,8, on zicursors) , siliconcursors)  and strocursors) . Besidescursors) ,13, posscursors) , differecursors)  and postcursors) .Swelling and bubble formation can be both an undesirable result of the film production process and an intentional surface modification, for example, by laser irradiation . The forThe focus of this work is on using simultaneous scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the description of delamination areas. SEM and AFM are the most popular methods in material science for the study of surface appearance. They allow the diagnostics and investigation of the surface and near-surface area in high resolution. In the case of correlative SEM-AFM microscopy, SEM provides both imaging and navigation through the region of interest. The same sample can be subjected to AFM measurement for nanoscale data acquisition on the surface height. Installing AFM in SEM has several advantages, protecting the device from acoustic vibrations, thermal drift and other noise exposure in the chamber of an electron microscope.The investigation of delaminations is of interest to both practical applications and the detection of rare physical effects in this structure. In the near-surface region of the substrate, significant residual stresses arise due to the formation of highly nonequilibrium nanostructured and nano-phase states. The patterns of formation of such states are not well studied yet.−7 Pa.A commercial sample of highly ordered pyrolytic graphite (HOPG), 7 mm × 7 mm, ZYA grade was used. The samples were purchased from NT-MDT Spectrum Instruments . The processing of the substrate material is important for the preparation of heterostructures . The upp3  was used as the source of Bi. Ferrocene (Fe(C5H5)2, Sigma-Aldrich spol. s r.o., Prague, Czech Republic) realized the Fe component. In the time interval between letting the precursor into the chamber, ozone was supplied for 4 s. A purge was carried out with N2 carrier gas of 99.999% purity.An ALD set-up ALDCERAM ML-200  was used for the preparation of Bi-Fe-O films. To obtain the Bi-Fe-O composition, Bi(mmp)3 was in the interval of 408–418 K. It provided sufficiently reproducible impulses of precursors. The optimum temperature for the evaporation of the ferrocene precursor was 364 K [3 provided a higher growth rate than Fe(C5H5)2. The growth rate was ~0.12 nm/super cycle. For the homogeneous mixing of the resulting Bi-Fe-O layer, the layer growth of BiOx and FeOx compounds was carried out in a single technological cycle. The initial layer was 150 cycles of BiOx, with a super cycle of Bi(mmp)3 input at 1.2 s. Then, a layer of FeOx was deposited during 150 cycles. The super cycles of the intake of Fe(C5H5)2 took 2 s each. The inlet gas pipelines transporting the precursors were at 423 K [The evaporation temperature of Bi(mmp)as 364 K . The reaat 423 K . The sub5 set-up . Depth profiling was performed using a Bi+ 30 kV analysis beam of 200 μm2 area and O2+ 500 V for sputtering. The O2+ beam also had a signal intensity enhancing effect. Raman spectroscopy was carried out on a confocal Raman imaging system Alpha 300 R  using a 5 mW and 532 nm irradiation laser. The additional study of the surface composition was carried out by an AXIS SupraTM X-ray photoelectron spectrometer (XPS) . A cluster source of argon ions was used for depth profiling with the following characteristics: 5 keV Ar500+, 2 mm × 2 mm etching area, 5 s of pre-etching and post etching time, 1800 s etching time of one circle. The reason for the low energy and long time of etching was the prevention of the preferential etching of oxygen. The data were processed by CasaXPS v.2.3.23 software .A qualitative analysis of the samples in depth was carried out by secondary ion mass spectrometry (SIMS) using a TOF-SIMSTM  integrated to SEM Lyra 3 . Topography detection was, in this case, performed by self-sensing tuning fork probes which detected the change in their resonance properties due to the change in distance to the sample. The chamber pressure during measurement was 10−2 Pa. When measured in air, the pressure was normal and equal to 101,325 Pa. The scanning of the topography at atmospheric pressure was carried out immediately after scanning in vacuum. This allowed avoiding excessive pollution from outside and accidental jumps to another area, which could be the result of mechanical manipulations.A correlative SEM-AFM analysis was carried out using a scanning probe microscope. LiteScope3 = O2 + O*). According to the literature [2H+ and NO+) are present in the film but at a very low concentration in comparison with the main components. We suppose that there are two possible reasons for the appearance of nitrogen in the film. The first reason is the impurities that can enter the reaction chamber through the ozonizer. Oxygen of 99.2% purity which contains residual nitrogen was used. Thus, at the stage of ozonation, the oxidized nitrogen could get into the chamber according to Equation (1):Oxygen is distributed evenly over the thickness of the film; it is a part of iron and bismuth oxides and is also in the combination of gaseous products. The bismuth concentration is higher on the surface and near the substrate, i.e., on parts of the film with the greatest number of defects and dangling bonds. In addition, the presence of a small amount of OH groups was observed. This is because ozone decomposes at a temperature of 250 °C, with a half-life of 1.5 s  and (3) are possible inside the chamber:+ and C2O+ appear in the depth below Fe and Bi and can be associated with the oxidation of carbon by the O2+ ionizing beam.These reaction products can be captured by surface states or defects, causing even more stratification of the surface. The second reason is connected with the fact that the nitrogen-containing components, as can be seen from the SIMS profile, are mainly concentrated at the same depth as the iron. This allows suggesting that iron can catalyze a nitrogen fixation reaction, similar to reports in literature ,23. The −1 Pa) as well as small amount of impurities in the reaction gases and precursors can be the reason for the rest of the latter in the growing film.The bismuth surfactant properties contributed to the retention of gas molecules under the surface of the bubbles and the initial adhesion of the reaction products on the surface of the substrates. It can be assumed that the initial vacuum of the ALD process  153 cm−1, E(LO2) 172 cm−1, A1(LO2) 216 cm−1, E(TO5) 280 cm−1, E(TO6) 314 cm−1 and A1(LO4) 489 cm−1 phonons (Raman spectroscopy was performed to determine the molecular bonds in the Bi-Fe-O system. The Raman spectrum contains A1(TO1) 133 cm phonons .These LO and TO phonons indicate the presence of self-organized bismuth ferrite ,25.XPS survey spectra a and val3/2 peak shows the Fe3+ state at 710.8–710.6 eV at the surface and near-surface area (3/2 and Fe 2p1/2 peak of about 14 eV is evidence of the presence of the Fe3+ oxidation state [2+ appears after 39.5 h of etching at 709.4 eV [3+ peak could be observed at the 719 eV and 720 eV satellite peaks. The surface before profiling is converted by Bi3+ and shows the XPS peaks of Bi 4f7/2 and Bi 4f5/2 at 158.9 eV and 164.3 eV [The Fe 2pace area a. The dion state . The che709.4 eV . The pea709.4 eV . They oc164.3 eV . The shi164.3 eV . The etc164.3 eV .−) [According to Jaiswar Sh. and Mandal K.D. , O1s XPS−) . The inc−)  and hydr−)  groups cCorrelative SEM-AFM allows the fast and easy comparison of images from both techniques. The localization of the region of interest using SEM allows one to quickly focus on the surface of the sample by selecting the desired scan area a. The heAt certain values of the electron beam parameters, a thin film of the material can be transparent . In the HOPG can be considered as graphene layers connected by van der Waals forces. Graphene, in turn, is known as a material capable of retaining substances in a gaseous state. The problem of the adsorption of ferrocene compounds on the HOPG surface was theoretically and practically considered in , where tThe combination of SEM and AFM is useful in many ways. SEM allows for the precise navigation of the AFM tip on the surface and a constant tip quality control as well. Because the same areas are measured by two techniques, the overlapping of the results provides better material and topographical information than just one method on its own. This makes the AFM and SEM combination an ideal measurement tool for the evaluation of nanomaterials, especially carbon-based nanostructures .When in proximity to the measured sample, the probe can be affected by a number of forces—mechanical contact force, van der Waals forces, capillary forces, electrostatic and magnetic forces, Casimir forces, chemical bonding, solvation forces, etc.—depending on the setup of the microscope and the probe ,43,44. TThe AFM measurements show dome-like structures and holes on the sample. When the topography imaging by AFM is merged with an SEM material contrast, a distinct difference between the holes and the surrounding material can be seen, as well as the height and thickness of the film. The formation of these bubbles can be explained as a local delamination caused by the compensation of surface tension created by the differing crystal conformation of the substrate and the thin film . The obsIn this work, we assume only spherical elements of a swollen film. The pressure value in the chamber was controlled by the electronics and software of the SEM microscope during the measurements in vacuum. Incertitude in the calculations could occur because the atmospheric pressure inside the clean laboratory room could be higher and add positive pressure from 2.5 up to 12.5 Pa. Even the pressure inside the one room could have a differential range from 0.025 to 0.175 Pa . We did 0 in our case is the volume under normal pressure and V is the volume in vacuum.According to , swellinThe swollen element has the shape of half an ellipsoid, the volume of which can be calculated as half the volume of an ellipsoid by Equation (5):Using data on the height of the tension (swelling) in vacuum, we can calculate the surface tension. Inside the bubbles there is excess pressure, which is compensated by the action of surface tension forces.The excess pressure under the surface of half an ellipsoid can be described as in Equation (6):The total pressure exerted on the film consists of the pressure of the medium and the excess pressure of the gases inside. Comparing the total pressure in vacuum and at normal pressure we get Equation (7):Substituting location a Based on the available data, one can also calculate other parameters, for example, the change in potential energy of the surface molecules by Equation (8):2 and the calculated excess of potential energy ΔE is 4,2 fJ. The observed phenomena were reversible when the “bubbles” maintained their ellipsoid shape. However, some blisters were broken after the evacuation of the chamber (In Equation (8), the area of the layer ΔS is 0.65 µm chamber . Upon thThe presented SEM-AFM measurements should be performed after careful adjustment of AFM and tests on various shape calibration samples, because the height differences can be caused by other possible contributions. Among them are ambient conditions as temperature, humidity and the gas composition of the atmosphere. AFM measurements in dry atmospheres  could also bring additional information about the chemistry and physics behind the blisters’ formation. The influence of atmosphere conditions could be complex. So, for example, capillary adhesion and friction which depend on humidity will have a different influence on hydrophobic and hydrophilic surfaces . The intIn this work, the results of studying the exfoliation of Bi-Fe-O film from the HOPG surface are presented. An increased interest in multiferroics led the researchers to investigate their combination with other prospective materials. The results aim to fill the space in the field of bismuth ferrite preparation on graphite substrates. The observed film delamination effect can be used to further obtain 2D nano-dimensional materials by the mechanical  collection of exfoliated flakes.The results show that simultaneous SEM-AFM contributes to obtaining structural information from local regions as morphological, geometric, physical and chemical characteristics. This is important for a better understanding of the many aspects of material science, such as defects, phase transformations, etc., and the correct interpretation of surface topographic features, as well as its functional role in the testing of the surface in relation to material performance and lifetime.


In this paper, a novel optical approach to double the sensitivity to angular rate of interferometric fiber optic gyroscope (IFOG) is proposed. Two fiber polarization combiner/splitters (FPCSs), as the key components, are added in the traditional IFOG light path. The FPCSs are able to either combine two orthogonal polarizations transmitting at two different polarization-maintaining fibers (PMFs) into the two orthogonal axes of one PMF, respectively, or split two polarizations transmitting at the two orthogonal axes of one PMF into two polarizations to transmit at two different PMFs, respectively. Through the specific placement and coupling of these two FPCSs, the incident light can transmit twice along the polarization-maintaining fiber coil (PMFC). The novel approach is verified experimentally and the experimental results show consistency with the theoretical analysis. The proposed approach is able to double the sensitivity of IFOGs and can increase the signal-to-noise ratio (SNR) without increasing the length of PMFC, which is very susceptible to environmental influences and is of great significance in the technical improvement of IFOGs, as well as the miniaturization of IFOGs. The inertial navigation system (INS), as an autonomous navigation device that does not depend on external information, is able to provide continuous and real-time information, such as the position, attitude, and speed of the carrier. It has the advantages of good concealment and high precision, and is not affected by meteorological conditions and human disturbances as well ,2,3,4,5.Numerous achievements have been made out to improve the sensitivity of IFOGs, including software compensation and hardware optimization. Software compensation ,15,16,17In this paper, we propose a novel approach, which is able to effectively double the sensitivity by doubling the light paths without increasing the length of polarization-maintaining fiber coil (PMFC), which is the sensing coil in IFOGs. The corresponding demonstration was setup and the effectiveness of the novel approach was verified. The experimental results show consistency with the design analysis.The diagram of the proposed method is shown in As shown in The two polarizations transmit at two orthogonal axes, or split two polarizations, which transmit at two orthogonal axes in a single PMF, into its orthogonal linear polarizations through two fiber ports. Light only travels along a single axis, such as the slow-axis, of the PMF attached to Port1 and Port2, while it can travel along both the slow-axis and the fast-axis of the PMF attached to Port3, as shown in The light signal from the light source is converted into two linearly polarized light beams transmitted clockwise (CW) and counter-clockwise (CCW) by the Y-branch coupler, respectively. The CW light beam travels along the slow-axis of PMF attached to Port1 of FPCS1 and exits from its Port3 along the slow-axis of PMF attached to it. After one turn around the PMFC, the CW light beam enters the FPCS2 through its Port3 and get out from its Port1. The coupling angle of the PMFs attached to Port1 of FPCS2 and Port2 of FPCS1, is 0°. Therefore, the light beam enters FPCS1 again through Port2 and then exits from its Port3 along the fast-axis of PMF. After another turns around the PMFC, the CW light beam enters FPCS2 for the second time and exits from its Port2 along the slow-axis of the PMF. As for the CCW light beam, it travels along the slow-axis of PMF attached to Port2 of FPCS2 and exits from its Port3 along the fast-axis of PMF attached to it. After one turn around the PMFC, the CCW light beam enters FPCS2 through its Port3 and exits from its Port2. The coupling angle of the PMFs attached to Port1 of FPCS2 and Port2 of FPCS1 is 0°. Therefore, the light beam enters FPCS2 again through Port1 and then outputs through its Port3 along the slow-axis of PMF. After another turn around the PMFC, the CCW light beam enters FPCS1 for the second time and exits from its Port1 along the slow-axis of the PMF. Thus, because of the specific placement and coupling of these two FPCSs, the incident light can transmit twice along the PMFC, doubling the sensitivity of IFOG indeed.1, with the input regular rate of 7.5°/h and D2 with the input regular rate of −7.5°/h of PM-IFOG at the two conditions, were collected in real-time and then saved to the PC. The scale factor can be calculated as K = (D1 − D2)/15°/h. The experimental result data divided by K on the two conditions are shown in In order to verify the effectiveness of the proposed method, the intrinsic frequency of the novel polarization maintaining interference fiber optical gyroscope (PM-IFOG) was measured. A square-wave voltage was applied to the phase modulator of the IOC. Tuning the frequency of the square wave, the delay time of the sensing coil for light wave after doubling is ~6.21 μs, as shown in Based on the noise analysis theory for gyroscopes, angle random walk (ARW) appears where the slope of Allan error curve is −0.5 and bias stability is thought to be the minimum value of the curve where the slope is 0 divided by 0.664 . The val8/(°/s), but after doubling, the scaling factor k2 was 7.65452 × 108/(°/s). The doubling factor was k2/k1 = 2.018. Compared to the expected value 2, the experimental doubling factor was 1% larger. This may lead by the pigtails of the two FPCSs, each of whose length is ~1.5 m.The demonstrations before and after doubling were placed on a motor-driven turntable with the rotation rates of −3°/s and 3°/s, respectively. The scaling factor experimental results are shown in IFOGs are widely used due to their excellent performance and are the core component of many devices including high-precision INS. Improvement of the sensitivity of IFOGs is extremely required in some specific applications. A novel approach for doubling the sensitivity of PM-IFOG is proposed. Two FPCSs were added in a conventional PM-IFOG system in a specific way of placement and coupling. In the process of detecting the earth’s rotation angular velocity, experimental results showed that the sensitivity of PM-IFOG with the novel approach indeed was basically twice that of the normal PM-IFOG, which was consistent with the theoretical analysis, and thus verified the validity of the proposed method. Meanwhile, the SNR of the PM-IFOG with novel approach also obtains a promotion. In conclusion, the PM-IFOG with the proposed doubling method has the obvious advantages of higher sensitivity and constant environmental adaptability, which is quite meaningful in engineering applications, especially in the field requiring long fiber coils.


High heeled shoes have long been worn in society and they are known to cause biomechanical imbalances to not only the foot, but the whole musculoskeletal system. This study aims to show the detailed changes that happen to the shape of the transverse arch of the foot in high heels, using two different inclination degrees.68 women participated in this study. Two custom-made high heels were made with inclinations of 15 degrees and 30 degrees (cm). A weight-bearing ultrasound was used to assess the coronal view of the transverse arch in standing. ANOVA and Tuckey tests were used to compare the results between 0 degrees, 15 degrees and 30 degrees inclinations.The transverse arch height was slightly increased as the heel height increased (0DI-15DI: p = 0.5852 / 15DI-30DI: p = 0.395 / 0DI-30DI: p = 0.0593). The transverse arch length (0DI-15DI: p = 0.0486 / 15DI-30DI: p = 0.0004 / 0DI-30DI: p = 0.1105) and the area under the metatarsal heads (0DI-15DI: p = 0.0422 / 15DI-30DI: p = 0.0180 / 0DI-30DI: p = 0.9463) significantly decreased as the heel height increased.The main changes were viewed in the 30 degrees inclinations compared to 0 degrees inclination. When the toes are dorsiflexed in high heels, it stimulates the Windlass mechanism which in turn stiffens the plantar fascia and adducts the metatarsal heads, while the soft tissues shrink in response to loads.High heels affected the shape of the transverse arch even in short term standing, and these effects increased as the height of the heel increased. High heeled footwear (HH) are shoes that are higher under the heels than under the other parts of the shoe , 2 and tWearing HH for a long term is said to lead to chronic foot pain and deformities .In the literature, HH are said to cause calluses , posturaThe transverse arch is one of the three arches of the foot , 11 whicThis study aims to describe the changes that happen to the shape of the transverse arch when standing with HH on. We used an ultrasound machine that allows to view the coronal plane of the transverse arch and custom-made high heels. We measured the transverse arch height (TAH), heights of the metatarsal heads, sesamoid rotation angle (SRA), transverse arch length (TAL), length between the metatarsal heads, total area under the metatarsal heads and the areas between the metatarsal heads; in three inclination angles: flat (0°), moderately high heels (15°), and high heels (30°). We hypothesize that the arch will drop and widen while the area underneath the metatarsal heads will decrease, and that this change increases as the height of the heel increases.During a health care event for women in Aichi prefecture–Japan, in 2016, 90 adult women voluntarily participated in taking their foot measurements. The purpose of the study was explained to them, and signed consents were obtained from each participant before the measurements. Exclusion criteria were: history of lower leg surgery or fracture of the foot and ankle, disc herniations, neurological or congenital disorders, diabetes and pregnancy. The current study was performed in accordance with the current local guidelines and the Declaration of Helsinki and approved by the Ethical Committee of Kyoto University Graduate School and Faculty of Medicine .Two custom-made HH were prepared for this study. These two HH were made with flat hard mattress board and slip resistant sheets were attached on the inclined plane and basal plane of each HH . These HA weight-bearing plantar ultrasound imaging device (WPUID)  was madeFirst, participants were seated and set their dominant foot on the solid gel of the WPUID and the other foot on the digital weight scale; then, the examiner fixed the position of the participants’ sesamoid bones and 5th metatarsal head (5MTH) on the solid gel area. Second, the participants stood on the WPUID, holding their foot on the solid gel area and the other foot on the digital scale until the digital scale showed almost half of their weight. Then, 0 degrees inclination (0DI) condition’s ultrasound images were taken. Third, the examiner placed the 15 degrees inclined heels under their midfoot to rearfoot holding both feet stable and took ultrasound images in the 15 degrees inclination (15DI) condition when the digital scale of the other side of the foot indicated half of their weight. Then, ultrasound images in the 30 degrees inclination (30DI) condition were taken in the same manner. B-mode ultrasound images were taken at a frequency of 9.0 MHz once by one researcher for each condition, considering the participants’ fatigue. A total of 3 ultrasound images were taken per foot.After transferring all ultrasound images to a computer, they were analyzed using ImageJ software  , Fig 2B.Statistical analysis was done using JMP Pro 14 . One-way ANOVA was used to compare the parameters between the three conditions  and Tukey test was used as a post-hoc test. The p-value was set at 0.05. All results are displayed as mean ± standard deviation.The total number of the participants was 90 adult women and after applying the exclusion criteria and excluding the ultrasound images that were not clear for analysis, 68 women (68 dominant feet) were left for the analysis of this study. The demographic data of the participants are represented in The results of TAH, TAH ratio, SRA, TAL and total area in the three measurement conditions are represented in The results of the metatarsal head heights in the three measurement conditions are represented in The results of the length between the metatarsal heads in the three measurement conditions are represented in The results of the area between the metatarsal heads and the plantar skin in the three measurement conditions are represented in In this study, we aim to describe the changes that happen to the shape of the transverse arch of Japanese young women when standing at 0, 15 and 30 degrees of heel inclination; using a weight-bearing ultrasound device. The significant changes that our results showed are 1) significantly decreased TAL and area under the metatarsal heads as heel inclination increased, 2) significantly decreased heights of the 2MTH, 3MTH and 4MTH in 30DI compared to 0DI, 3) significantly decreased area between LS and 2MTH, 2MTH and 3MTH, 3MTH and 4MTH, 4MTH and 5MTH in 30DI compared to 0DI. The main significantly different results were between 0DI and 30DI. However even in 15DI, the TAL significantly shortened and the areas between LS and 2MTH and 4MTH and 5MTH significantly decreased, compared to 0DI, indicating that even lower HH height affects the shape of the transverse arch. In both heights (15DI and 30DI), the space between LS and 2MTH and the one between 4MTH and 5MTH seem to be the most affected.The TAH was not significantly changed from 0DI to 15DI and 30DI, but the TAH ratio had significantly increased in 30DI compared to 0DI. When loaded, the arch normally collapses as a response to loads  and reboAs for the heights of the metatarsal heads, the heights of 2MTH, 3MTH and 4MTH had significantly lowered in 30DI compared to 0DI. Lowered height of the metatarsal heads is caused by the increased pressure on them, and pressure is increased on the metatarsal heads in HH . DecreasAs for the SRA, it slightly lessened as HH increased, without significance. This may be due to the decreased length of the MS and the LS (as per our results) in response to loading. Previous reports are in agreement with the fact that the heights of the sesamoids decrease when loaded, noting that the MS bears more loads than the LS .On the other hand, we had hypothesized that the transverse arch would widen. However, the TAL had shortened with significance in 15DI and in 30DI compared to 0DI, as well as the space between 4MTH and 5MTH in 30DI compared to 0DI. This may be due to the tightening of the deep transverse metatarsal ligaments, plantar ligaments, and dorsal interossei muscles that connect the metatarsal heads in the forefoot, and the dorsal interossei muscles and adductor halluces muscles; and the adduction of the metatarsals secondary to the Windlass mechanism . In pastFurthermore, the total area under the metatarsal heads had significantly decreased in 15DI and in 30DI compared to 0DI. In details, the area between LS and 2MTH and the area between 4MTH and 5MTH has significantly decreased in 15DI compared to 0DI. This significant decrease in the area continues in 30DI between LS and 2MTH, 2MTH and 3MTH, 3MTH and 4MTH, and 4MTH and 5MTH compared to 0DI condition. This decrease in area is due to decreased height and length between the metatarsal heads, which are used to calculate the area in our study. Decreased area between the metatarsal heads and the plantar skin can cause tightening on the nerves, tendons and vessels located between the toes. This pattern is similar to Morton’s neuroma; a condition caused by repeated loading on the metatarsal heads causing trauma to the plantar intermetatarsal nerve and its impingement by the adjacent metatarsal heads and metatarsophalangeal joints , 21. It Women have more elastic joints by nature , 26 whicAlthough this study describes the changes that happen at the level of the transverse arch in HH, it has several limitations. First, the duration of the measurements is short, and the shape of the arch may change when the muscles are fatigued after longer durations of wearing HH. Second, the custom-made HH are standardized to the average foot size in Asian women which may not have fit all our participants. Third, these custom-made heels had a wide heel and no toe box, and the effects may be different had it been a stiletto-heel type or a closed narrow toe box. Nonetheless, we have shown the detailed changes that happen to the transverse arch in HH. These results can be used for a better customization of HH or the paddings used with the HH.Short term standing in HH shortens the transverse arch and tightens the area between the metatarsal heads and the plantar skin of the foot. In this study, we showed that HH affected the shape of the transverse arch and these effects increased as the height of the heel increased.