At the same time, it exhibited a good SPE adsorption ability, and the FA extraction was from 1.63 to 3.11 mg kg−1 in grain by-products by SPE coupled with high performance liquid chromatography/mass spectrometry (HPLC/MS). The fluorescence and SPE-HPLC/MS were used for the efficient detection of ferulic acid in real samples with recovery values of 88–114% and 90–97%, respectively. Furthermore, the nanomaterials of MIPs based on QD-grafted COFs were used for FA detection with high sensitivity and selectivity, and it also increased the recycling of waste resources.A fluorescence and solid phase extraction (SPE) adsorption nanomaterials of molecularly imprinted polymers (MIPs) based on quantum dot-grafted covalent organic frameworks (QD-grafted COFs) was prepared by one-pot surface-imprinting synthesis method. Amino groups of silane reagent were at the surface of QDs to coordinate COFs efficiently by Schiff-base reactions, providing thermal and chemical stability to MIPs. It also reacted with the phenolic hydroxyl groups of ferulic acid (FA) through non-covalent interactions. The nanomaterials were used as fluorescence sensing and SPE adsorption toward determination of ferulic acid. The MIPs based on QD-grafted COFs had good fluorescence response ability, and quenching linearly at concentrations of ferulic acid from 0.03 to 60 mg kg Quantum dots are solution-processable semiconductor nanocrystals  with sizIn order to improve the selectivity, sensitivity, and optical performance of fluorescence labeling, porous materials have a strong interest in the detection. The covalent organic frameworks (COFs) are one of the famous porous materials linking molecular building blocks by strong covalent bonds to make crystals of extended structures . RecentlIn this paper, we used molecularly imprinted polymers (MIPs) based on quantum dots-grafted COFs (QD-grafted COFs) as a fluorescent probe for the high selectivity and sensitivity optosensing of ferulic acid. Here, the MIPs are intended to sensitively and selectively sense the bonding interactions between quantum dot-grafted COFs and ferulic acid, and to further transform this chemical information to detectable fluorescence signals. MIPs are a way to overcome the coexisting compounds with similar fluorescence responses to the target, which expanded the applications of optosensing methods based on quantum dots. Ferulic acid (FA), 4 hydroxyl-3-methoxy cinnamic acid, is one of the typically natural antioxidants with the free radical scavenging capability of superoxide anions, peroxyl radicals, hydroxyl and radicals . Based o−1) was obtained from a Water Pro water purification system .The standards of ferulic acid, cinnamic acid, syringic acid, and caffeic acid were purchased from Acros Organics . 1,3,5-triformylphloroglucinol  was purchased from Strem Chemicals, Inc., Newburypor, MA, USA. P-phenylenediamine (Pa) and mesitylene (99%) were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. CdSe/ZnS quantum dots with fluorescence excitation wavelength at 460 nm and emission centered at 605 nm were purchased from Jiayuan . Triton X-100 (99.5%), cyclohexane (99.5%), 3-aminopropyl triethoxysilane , and acetic acid (99.5%) were purchased from Sinopharm Chemical Reagent Co., Ltd., Beijing, China. Tetraethyl orthosilicate  were purchased from J&K Scientific Ltd., Beijing, China. All reagents were at least analytical grade and purchased from Beijing Chemicals . Doubly deionized water  was used to observe the surface morphologies of the MIPs or non-imprinted polymers (NIPs) based on QDs-grafted COFs. The fluorescence spectra were acquired using a multifunctional microplate reader . All fluorescence measurements were performed under the same conditions. The excitation wavelength was 460 nm for emission over the range of 500–700 nm. The high performance liquid chromatography (HPLC) system consisted of two LC-20AB pumps and an RF-10AXL ultraviolet detector . A Visiprep TM-DL solid phase extraction (SPE) vacuum manifold  was used in the preprocessing procedure.COFs were synthesized using modified Schiff-base reactions by our previous paper . The oneThe non-imprinted polymers (NIPs) based on QD-grafted COFs was used as a control. It was similar to the polymerization process with MIPs but without adding FA molecules.For the fluorescence experiment, the MIPs based on QD-grafted COFs (1 mg) were added into a hole of the 96-well enzyme labeling plate containing 200 μL of ethanol. After thorough mixing for a certain time, the fluorescence response signal corresponding to each concentration of target was recorded.The MIPs based on QD-grafted COFs (100 mg) were packed into 3 mL SPE cartridges. After the MIPs based on QD-grafted COFs cartridge was activated and conditioned with methanol and water (10 mL), respectively, a solution of FA in ethanol was loaded. After the retained analytes were eluted with acetic acid/methanol mixture , the eluent was dried under a gentle stream of nitrogen gas at 40 °C. The residue was dissolved in methanol and filtered through a 0.22 μm filter for subsequent HPLC analysis.−1. The detection wavelength for UV detector is 320 nm. All detected solutions were filtered through a 0.45 μm polytetrafluorethylene membrane before use.The HPLC chromatography analysis was achieved on an Inertsil ODS C18 . The mobile phase is a mixture solution of methanol/water  with a flow rate of 1.0 mL min2 surface binding sites, inhibit effectively QDs photooxidation, improve the QDs fluorescent stability, and prevent the diffusion of the toxic heavy metal ions of QDs [The preparation of MIPs based on QD-grafted COFs used a one-pot surface-imprinting synthesis method . Here, ts of QDs . The QDsThe transmission electron microscope (TEM) image of the QDs is shown in The X-ray powder diffraction (PXRD) was used to obtain crystallinity of COFs a, QDs Fb, and thF is the integral of fluorescence intensity, A is the absorbance at the excitation wavelength, and η is the refractive index of the solvent. X and R are the sample and reference, respectively.The photoluminescence quantum yields (QYs) were used to perform fluorescent properties of MIPs based on QD-grafted COFs. Rhodamine B (QY = 89%) was used as a reference. It was measured as 37% at room temperature. The QY was calculated using the equation below:–1 of FA. The adsorption time was investigated from 0 to 30 min, as shown in In order to investigate the binding performance of the MIPs and NIPs based on QD-grafted COFs, an equilibrium binding analysis was carried out using 15 mg kgThe specificity of MIPs based on QD-grafted COFs was characterized with the structural and functional analogs of ferulic acid, cinnamic acid, syringic acid, and caffeic acid. −1 of MIPs based on QD-grafted COFs ethanol solution. The fluorescence quenching was determined after the MIPs based on QD-grafted COFs were added into the FA solution by shaking. After 15 min, the fluorescence spectra and fluorescence intensity of the different concentrations of FA standard solutions were recorded. The linear range of the standard curve was 0.03–60 mg kg−1 calculated by the fluorescence intensity and the change of FA concentration, and the R-square was 0.9305 (−1, calculated by the FA concentration quenched three times the standard deviation of the blank signal to divide the slope of the standard curve. At the same time, it exhibited a good SPE adsorption ability, and the linear range of the SPE coupled with HPLC/MS was from 0.02 to 20 mg kg−1, with a detection limit of 3 μg kg−1.For FA detection, the fluorescence spectra of the MIPs and NIPs based on QD-grafted COFs in the presence of FA were examined in ethanol at room temperature. In a typical experiment, a different amount of FA standard solutions was completely dissolved in ethanol and then 100 μL of the FA standard solution were mixed with 200 μL 1 mg mLs 0.9305 . The det−1. Calculations of the recovery percentages revealed that 88–114% and 90–97% were recovered from the optosensing and SPE-HPLC/MS methods, respectively, indicating good detection results.The potential applications of grain by-products in alleviating or preventing chronic disease have rarely been investigated. Highland barley bran, wheat bran, corn silk, and vinasse were used in the study. The optimal extraction conditions for grain by-products were established as 6 mL 60% acetone heated to 60 °C for 40 min. The FA contents of the grain by-products were shown in The fluorescence method in the study was low detection limit and high sensitivity for FA detection, with the advantages of its cost, simple, fast and efficient properties, and small reagent consumption. The SPE-HPLC/MS method using MIPs based on QD-grafted COFs was a good FA extraction and separation from complex sample matrixes, with the advantages of selectivity and accuracy. The two methods reported in the paper have their respective advantages for FA analysis.−1 and 0.02–20 mg kg−1, and the detection limit was 5 µg kg−1, and 3 µg kg−1, respectively. The system was used for the efficient detection of ferulic acid in real samples with recovery values of 88–114% and 90–97% by fluorescence sensing and SPE-HPLC/MS methods, respectively, indicating a high selectivity and sensitivity.In summary, we propose efficient and rapid fluorescence sensing and SPE-HPLC/MS methods for FA detection in real samples based on a molecularly imprinted polymer based on QD-grafted COFs. The nanomaterial was simple and facile to achieve FA quantitative detection by optosensing and SPE adsorption. It exhibited a high QY as 37% at room temperature. At room temperature, the MIPs based on QD-grafted COFs were stable for nearly 60 days, indicating that the MIPs based on QD-grafted COFs possessed sufficient stability for FA analysis. The high times concentration of analogs to perform the selective experiment showed a good selectivity of the nanomaterial was established through strong FA-APTES interaction. The linear range of fluorescence sensing and SPE-MPLC/MS methods were 0.03–60 mg kg


In this study, a crystal plasticity finite element method (CPFEM) model was used to study the deformation behaviour in an aluminium single crystal (1 1 2)[1 1 -1] processed by accumulative roll-bonding (ARB) up to 9 cycles. The simulation followed the real ARB process based on the developed finite element model. The predicted through-thickness texture matches well with the experimental observations. The deformation behaviours, in terms of crystal rotation, shear strain and slip system activation, in the first and second cycles  were unidirectional, but the deformation was altered after ARB was applied from the third cycle onwards. Such alteration was found to be caused by the thickness position change and deformation discontinuity at interfaces, which were investigated in detail. The role that interfaces play became dominant over thickness position change as increasing ARB cycles. A large strain can be introduced into materials by repeating the standard 4-step procedure 3. Due to large surface friction, a heterogeneous through-thickness deformation  after each cycle has been experimentally revealed in ARB processed metals and alloys4, and the deformation behaviour is altered after the thickness position being changed between cycles (because of cutting-stacking). The through-thickness inhomogeneity in a cycle and deformation alteration between cycles make the deformation behaviour in ARB complicated.Accumulative roll-bonding (ARB), a severe plastic deformation (SPD) method, has been extensively applied to fabricate ultra-fine or even nanocrystalline grained materials5, ALAMEL model6 and viscoplastic self-consistent (VPSC) model12 have been used to simulate texture evolution in ARB processed polycrystals. Only textures at the centre and surface were predicted by the ‘uniform-field’ Taylor model in ref.5, while textures at the centre, surface and quarter were modelled in refs6. Textures of two-phase composites were studied by the so-called ‘mean-field’ VPSC model12. Intrinsic homogenization at different levels is assumed in these CP models, and accordingly, the predicted ARB textures in these studies12 are more in a sense of statistical manner. Through-thickness texture and its transition between cycles have not been explicitly revealed in simulations.Texture modelling has become a powerful tool to study plastic deformation and texture evolution, and various crystal plasticity (CP) models have been developed. The Taylor modelet al.8. The main advantage of the ‘full-field’ CPFEM model over those above mentioned CP models is no homogenization assumption7, in which the crystal plasticity constitutive model is incorporated into the finite element method (FEM) framework. The plastic deformation and texture evolution are fully coupled in the CPFEM model. The equilibrium and compatibility conditions between elements and between grains are reached by basic principles of mechanics13, so the CPFEM model can access both intra- and inter-grain deformation14. Being able to access the former (intra-grain deformation) means the CPFEM model can simulate the deformation texture in (strongly anisotropic) single crystals that is the material used in this study. Non-uniform through-thickness deformation and cutting-stacking in ARB would result in misorientation angles at the bonded interfaces16, as shown later in this study, and the deformation behaviour at the interfacial boundaries can be simulated by the CPFEM model because of its capability to access inter-grain interaction8.The crystal plasticity finite element method (CPFEM) model, a more sophisticated CP model, is more applicable to ARB, as suggested by Knezevic 19, but in these studies ARB was approximated by plain strain compression. Modelling the real ARB process is challenging4, since it is a discontinuous process. The ARB FEM model in ref.20 resulted in a large distortion of FEM mesh, which would cause convergence problems and restricted the ARB simulation to a low cycle number. A modelling method, mapping solution, has been used between ARB cycles to transfer the deformation solution from the deformed mesh to a new mesh7. However, the ARB simulation in ref.2 was based on an elasto-plastic material constitution law without considering texture, while a VPSC model was used in ref.7 but it only simulated up to 2 cycles. Li et al.4 modelled the texture at the surface, centre and quarter up to 5 cycles with the ALAMEL model, but the simulation did not follow the real multi-cycle ARB. In their paper, the deformation history of the first cycle based on the elasto-plastic constitution law was used to model texture in all 5 cycles, i.e., decoupled plastic deformation and texture evolution. According to the authors’ best knowledge, no CPFEM simulation following the real ARB has been conducted up to a large cycle number (≥5).The CPFEM model has been applied to study the plasticity at interfaces of two-phase composites fabricated by ARBIn the present study, a CPFEM model was adopted to predict plastic deformation and texture evolution in an ARB processed aluminium single crystal (1 1 2)[1 1 7, mapping solution, a built-in remeshing technique of the commercial FEM code ABAQUS, was used between cycles. The deformed mesh was replaced by a new mesh after mapping solution, while the deformation solution  in the new mesh was interpolated from the distorted mesh. During the interpolation, the solution at nodes of the deformed mesh was firstly obtained by extrapolating the values of integration points and then the solution at nodes was averaged over all elements around the nodes in the new mesh. The discrepancy caused by the interpolation has been checked in this study by comparing the solution before and after mapping solution, and it was reduced to an acceptable level by refining the FEM mesh.Figure 22, and this layer plays an important role in plastic deformation at a small scale23, but its influence to the global texture is limited. The ‘Wire-brushing’ step was thus omitted in this ARB FEM model. The two stacked sheets in ARB experiments are usually jointed by metal wire or welding before roll-bonding, and accordingly, the relative displacement between the two stacked sheets is prohibited or very small. Moreover, the wire-brushing would cause a high surface roughness of the sheets to be stacked, and the high roughness would greatly reduce the relative slide between the two stacked sheets when the compression stress along the normal direction (ND) increases in the rolling bite. Therefore, rolling was used to replace roll-bonding in this ARB FEM model. The single-layered sheet was rolled at a 50% reduction in the first step ‘Rolling’  was rolled to 2 mm by a 50% reduction in the first cycle and then to 1 mm in the second cycle. The deformed sheet after the first cycle was directly mapped to a new FEM mesh at the beginning of the second cycle, without ‘Cutting’ and ‘Stacking’ steps. ARB was applied from the third cycle onwards, and the two stacked sheets made the starting thickness be 2 mm from 3- to 9-cycle. The element type was CPE4R, which can provide efficient and fast numerical formulation. Enhanced hourglass control was used to increase the resistance to the hourglassing problem and provide more accurate displacement solution. The shape of elements was square, i.e., equal length along the RD and ND. After examination of mesh division, the whole thickness was divided into 40 elements in the first four cycles, and then the element number was doubled after every two cycles. The initial orientation (1 1 2)[1 1 16 was a single crystal (1 1 2)[1 1 The simulation was specifically designed to match the ARB experiment conducted by Kashihara 25. TD-rotation is strongly dominant over RD- and ND-rotation, and thus only TD-rotation among these three components will be presented and discussed in the following text. Figure The crystal rotation angle, i.e., misorientation between final orientation and initial orientation 1 1 2)[1 1 [1 1 \doc26 and high pressure torsion27, and also rolling28, and satisfactory textures have been obtained.The kinematical scheme and hardening model used in the CPFEM model are given in Appendix t/t0, where t is the distance from the upper surface to a thickness position and t0 is the whole thickness. Therefore, t/t0 = 0 corresponds to the upper surface and 1 to the lower surface.The thickness location is defined as t/t0 = 0 to 0.25) and M2 (t/t0 =  0.25 to 1), with negative and positive rotation, respectively. The TD-rotation angle after 1-cycle is very low, and after 2-cycle it continued to increase greatly in the negative direction in M1, while a small increase in the positive direction developed in M2. The division of M1 and M2 in 1- and 2-cycle is slightly different, which was probably caused by the change of rolling bite geometry29. Shear strain γXY evolved in a cycle alone, not cumulated with ARB cycles, is also shown in Fig. γXY is very high at the upper surface in 1- and 2-cycle, while it is almost zero at the lower surface. The asymmetrical distribution of shear strain indicates a large slide occurred between the sheet and rolls under lubricated rolling conditions.It is conventional rolling in the first and second cycles. Figure 16. The slip system a1 and a2 are co-planner, and c3 and d3 are co-directional, where the distribution of the four slip systems before processing is shown in Fig. γa1) is close to γa2, since non-TD-rotation is negligible. It is the same for c3 and d3. Therefore, only a1 from the a1-a2 set and c3 from the c3-d3 set are used as representatives in the following text. The imbalance of shear strain on slip systems , i.e., difference between cumulative γa1 and γc3 (=γa1 − γc3), after 1- and 2-cycle is plotted in Fig. t/t0 = 0 to 0.72, γc3 is higher than γa1, while γa1 is dominant from t/t0 = 0.72 to 1. The alternation between the primary and secondary slip systems divides the M2 band into M2a and M2b  in 2-cycle is similar to that in 1-cycle, since it was conventional rolling (not ARB) in 2-cycle.In this study, the activated slip systems are a1 (1 1 1)[0 M2b Fig. . The disγa1 and γc3) before and after 3-cycle, which clearly shows the feature of cutting-stacking process, and this feature was preserved after this cycle though the slip system activation changed in some regions marked as ‘Rev’. In Rev1 and Rev5, the shear strain imbalance completely reversed, while it just decreased in Rev2, Rev3 and Rev4, like TD-rotation in Fig. ARB was applied from the third cycle. Figure one Fig.  is quiteone Fig.  though t16 and simulation after various ARB cycles, respectively. The difference in orientation, or misorientaiton, between Cu and Dn is only 8°, which is smaller than the tolerance of 10°. The classified Cu in Fig. The TD-rotation caused texture transition between Cu, Dn, roCube and Dp Fig. . Figure t/t0 = 0.5), while its direction is unidirectional at the other side, which has also been observed in 3-cycle  and 0.5 (centre), where negative TD-rotation means the crystal orientation rotated toward roCube and Dp  and previously evolved textures (transferred to this cycle by mapping solution), and accordingly, the plastic deformation and texture evolution were continuously coupled for all ARB cycles. In contrast, the plastic deformation and texture are not fully coupled in other CP models used for ARB simulations  due to the homogenization assumption. In this ARB FEM model, the mesh distortion did not cumulate with ARB cycles, since the mesh distortion evolved in a cycle was eliminated by the mapping solution at the beginning of the next cycle, so the convergence problems caused by the mesh distortion was avoided. Through-thickness texture at the meso-scale is the main objective of the current study, so the realistic topology and microstructure of interfaces were not explicitly modelled, but misorientation angles and slip system activation were used to characterize and represent the difference in deformation at the two sides of interfaces. This simplification is similar to that extensively used and widely accepted in polycrystal modelling34, where different initial orientations are assigned to neighbouring grains, but not considering the realistic microstructure of grain boundaries. The good agreement between the simulated textures and experimental observations  in Fig. ΩTD). The direction of shear stress SXY and normal stress SXX altered in the succeeding period  was obviously lower than that on the other set , so the total plastic spin (γXY = −0.038). In M2b , is negatively low.At C1, the activation of a1-a2 caused a positive plastic spin  Fig.  was requiod Fig. , from 0.M2b Fig. , in whicγa1,a2−γc3,d3), slightly larger than that at C1 (=−0.31), which is supposed to cause higher plastic spin at C2, but this is actually not the case, since the plastic spin is not only determined by the shear strain imbalance, but also crystal orientation (Eq. (5) in Appendix γXY is positive, although the TD-rotation at C2 (3.7°) is lower than at C1 (5.2°) and imbalance between γa1,a2 and γc3,d3 at C2 is higher than at C1.The deformation at C2 is similar to that at C1 , although both A1 and C1 (in 1-cycle) have the same initial orientation. The difference in slip system activation was caused by the different stress, since stress is the only reason for slip system activation (Eq. (6) in Appendix The deformation at another two points, A1 in M1 and B1 in M2a, in 1-cycle is also listed in Table 4, and it is claimed31 that the change between shear deformation (at the surface) and compression (at the centre), or simply called thickness position change here, is the reason for the texture transition. The surface being moved to the centre results in a large change in thickness position, so the transition from shear texture (at the surface of last cycle) to rolling texture (at the centre) is obvious and thus has been widely observed4. In contrast, the thickness position change at the surface is extremely low, so the shear texture, e.g., roCube in this study, is stable at the surface  and lower surface (t/t0 = 0.875 to 1), while the other part is Dp or Dn. The selected orientation (Dn or roCube) was placed at only one position in each simulation case, as shown in Fig. 35.To study the effect of interfaces, the stability of Dn and roCube, as the main components of rolling and shear texture, respectively, under different interfaces are investigated here during conventional rolling with a 50% reduction. Figure 875 Fig. , while tThe distribution of crystal rotation and deformed FEM mesh at the four different thickness positions are compared in Fig. t/t0 = 0.375 and 0.625) dropped greatly, which suggests the high stability of the neighbouring Dn. The shear deformation in the central roCube layer is in the same direction as its neighbouring Dn , while negative TD-rotation evolved near the interface (t/t0 = 0.875). In this case (with neighbouring Dn), the misorientation at t/t0 = 0.875 is significantly higher than that at the other two interfaces , while grain-interaction is strong near grain boundaries and becomes weak as approaches the inner region, because the grains in coarse-grained materials are large enough to accommodate the deformation gradient. These two factors are crystal orientations and interfaces in the multi-layer sheet of ARB, where the former is strongly influenced by the thickness position change , while the interfaces can be simply treated as grain boundaries. Textures rotate into stable positions during deformation, and after being moved to a new position (by mapping solution), the previously developed stable textures become unstable at the new positon that has different external loading. In 3-cycle  is large, so the effect of thickness position change  is dominant. Therefore, the regions with reversed rotation mainly concentrate near the centre (having large changes in thickness position) though with a small exception (Uni2 in Fig. 37 and Cu/Nb9. This increasing effect of interfaces can be seen from the dominant role of grain-interaction in polycrystals with small grains. As the grain size decreases34, the area fraction of grain boundaries increases and inner region decreases, and thus the crystal rotation has to be large to accommodate the orientation gradient over a short distance.The multi-layer sheet in ARB can be simply analogized by an aggregation of grains in polycrystals. The in-grain deformation of polycrystal is mainly determined by two factors. The first factor is orientations of grainscle Fig. , there i2. The slip system activation is critical to microstructural characteristics and the change of slip system activation would alter microstructure evolution38. The influence of rolling direction to slip system activation and microstructure in ARB has not been well researched. However, it has been found in multi-pass unidirectional rolling that the microstructure continued to refine in the way that developed in previous passes29, while the previously formed microstructure was destroyed by the next pass in multi-pass reverse rolling. The continuous refinement in the unidirectional rolling and destroyed microstructure in the reverse rolling were due to the same and different activated slip systems, respectively29. The alternation of slip system activation could be a potential reason for that ARB is faster than conventional rolling in microstructure refinement39.Similar to TD-rotation, both unidirectional and reversed slip system activation have been observed Figs  and 6b, A CPFEM model was used to study the deformation behaviour in an aluminium single crystal. The CPFEM simulation followed the real multi-cycle ARB process up to 9 cycles.The predicted through-thickness texture has been validated by the experimental observations. A heterogeneous through-thickness deformation was revealed, and alternation of deformation between ARB cycles was also observed. The dynamic balance between the increased and decreased crystal rotation resulted in the stable distribution of textures.The textures developed in previous cycles became unstable after being moved to a new thickness position in ARB, which is manifested by partial rotation reversal and alteration of slip system activation. By contrast, the deformation in conventional rolling is unidirectional due to no change of thickness position.The inhomogeneous through-thickness deformation and cutting-stacking process resulted in large misorientation at the interfaces. The interfaces, like grain boundaries, affected the deformation on the two sides of them, which was investigated by studying the stability of Dn and roCube with different neighbourhood.With increasing ARB cycles, texture evolution in aluminium becomes less sensitive to thickness position change, but it is affected more by interfaces.Appendix: CPFEM theory and hardening model


In bistatic forward-looking synthetic aperture radar (BFSAR) ground moving target detection (GMTD), the suppression of the strong and heterogeneous ground clutter is one of the most crucial and challenging issues. Due to the bistatic forward-looking mode and long observation time, Doppler ambiguity, range and Doppler cells migration and non-stationary characteristics will exist in clutter receives, which leads to severe performance degradation of the traditional method. Hence, this paper proposes a GMTD method based on joint clutter cancellation in echo-image domain for BFSAR to achieve effective GMTD in heterogeneous BFSAR clutter. First, the pre-filtering and keystone transform are applied to suppress Doppler ambiguity and correct range cell migration, respectively. Then, time-division space-time adaptive clutter cancellation is adopted to suppress clutter at the first time in the echo domain, which can eliminate the effect of the migration of Doppler cells. However, its performance will be severely degraded due to the strong non-stationary characteristic of BFSAR clutter. Finally, adaptive displaced phase center antenna is exploited to suppress the residual non-stationary BFSAR clutter in image domain. Experimental results have shown that the strong non-stationary clutter of BFSAR has been sufficiently suppressed by the proposed method and the SCNR provided is enough to detect a moving target well. Synthetic aperture radar (SAR) is unique in disaster monitoring, resources exploration, military reconnaissance and security-related fields, due to its high-resolution day and night observation ability for areas of interest, independent of weather conditions. In general, a traditional SAR system is employed for stationary scene imaging ,2,3. HowIn practice, when SAR system is working at downward-looking mode, receiver will generally receive massive strong ground clutter whose Doppler spectrum is broadened due to the platform motion. In this condition, ground moving targets are often overwhelmed by the strong clutter received, leading to a low signal to clutter and noise ratio (SCNR). As a consequence, clutter suppression is one of the crucial issues in SAR GMTD.Current research and publications on SAR clutter suppression method are mainly divided into two types according to the system channel numbers: Single channel methods and multichannel methods. Clutter suppression methods based on single channel ,19,20, sIn addition, due to the forward-looking mode, the ground clutter of observed area will have a large radial velocity. And with the long observation time of BFSAR, three problems will be introduced into the received signal of BFSAR: Doppler ambiguity, range cell migration (RCM) and Doppler cell migration. The assumption that there is no range and Doppler migration during one coherent processing interval (CPI) will be invalid, which leads to the poor performance on traditional GMTD methods. In BFSAR, the azimuth Doppler ambiguity of the received signal will be caused by the large radial velocity of clutter. Range cell migration will lead to target energy dispersing over multiple range cells. Target signal will be aliasing with the superposition of the clutter from different range cells, which decreases the SCNR in detection. Also, the target signal cannot be directly distinguished from the clutter in the space-time domain due to its Doppler cell migration, which leads to severe clutter suppression performance deterioration. Consequently, the SCNR for GMTD in BFSAR cannot be improved enough from the raw SAR data by the traditional methods. Therefore, the traditional SAR GMTD methods are not suitable for BFSAR ground moving target detection.This paper proposes a BFSAR moving target detection method based on joint clutter cancellation in echo-image domain with three receiving channels. The proposed method not only can eliminate the influence of Doppler ambiguity and range and Doppler cell migration on clutter suppression, but can also reduce the effect of non-stationary characteristic of BFSAR clutter and improve the SCNR for target detection. First, we use the pre-filtering and first-order keystone transform to suppress the Doppler ambiguity and correct range cell migration for the received signal, respectively. Then, time-division space-time adaptive clutter cancellation (STACC) is adopted between two adjacent receiving channels to suppress clutter at the first time in echo domain, which can eliminate the effect of the migration of Doppler cells. However, since the space-time distribution of BFSAR clutter is closely related to bistatic configurations and platform parameters, BFSAR clutter will present strong non-stationary peculiarity, which severely degrades the clutter suppression performance. As a result, massive non-stationary residual clutter will be retained and moving targets may be still buried by the residual clutter. Some compensation methods for the clutter non-stationary peculiarity, such as angle-Doppler compensation method and derivative-based updating method, are carried out. But these methods need the prior information of the angle and Doppler frequency of the clutter spectrum and high computational complexity, respectively. In order to suppress the strong non-stationary BFSAR clutter and lighten the computational burden of the system, adaptive DPCA (ADPCA) is exploited as the second clutter cancellation to suppress the residue clutter in image domain. With ADPCA processing, the clutter will be coarse focused and the difference of the phase between different channels can be accurately compensated in image domain. Thus, the residue non-stationary clutter of BFSAR will be sufficiently cancelled in the image domain. Experimental results of traditional STAP, direct time-division STACC after the correction of the cell migration and joint clutter cancellation in echo-image domain have shown that the strong and non-stationary ground clutter of BFSAR can be suppressed sufficiently by the proposed method and the SCNR can be improved greater, where a moving target can be well detected. The remainder of this paper is consisted by the following sections: The geometric configuration of bistatic forward-looking SAR is shown in k-th channel, and it can be obtained bySupposing the transmitted signal is a linear frequency modulated (LFM) pulse, the received signal of the The range histories with respect to moving target k-th channel in range frequency domain azimuth time domain can be expressed by [After range Fourier transform, the signal received by the essed by (5)S by the filter function (10), we can haveFrom the Equations (6) and (7), we can know that the absolute values of After the bulk-deramp pre-filtering, the Doppler ambiguity caused by the large Doppler centroid has been suppressed and the coupling effect between range and azimuth directions has been weaken to some extent. However, we can find that the residual first-order coupling term is remained in the filtered signal In Equation (13), the first-order polynomial of The third term in Equation (14) represents the range modulation term, and the range compression can be carried out with the new range frequency modulated rateAfter the first-order keystone transform, the linear range migration has been completely corrected. Need to note that the quadratic range cell migration (the second term of the Equation (14)) is still retained. The linear range migration is more obvious compared with higher-order range cell migration. Thus, the effect of quadratic range cell migration can be neglected in BFSAR.After the two steps presented above, the effect of Doppler ambiguity and the large-scale range cell migration have been eliminated. Although the energy of the target has been gathered into one range cell, the signal of the moving target is still submerged in the superposition of the strong clutter at target range cell from different azimuth. In order to improve the SCNR and detect the moving target from the strong clutter background, the two-channel time-division STACC is operated to achieve the first clutter cancellation in the SAR echo domain. For BFSAR, the Doppler spectrum of both ground moving target and clutter will span into multiple Doppler cell, because of their Doppler migration. Thus, the Doppler frequency of one scattering point corresponds to a certain range during a CPI, which makes the no Doppler migration assumption for traditional method invalid ,36. UndeIn order to eliminate the strong ground clutter of BFSAR, the effect of the Doppler cell migration must be removed. In this section, a clutter suppression method based on STAP for BFSAR is introduced to deal with the ground clutter with the Doppler range cell migration. For the long integration time of BFSAR, we can divide it into a huge quantity of periods, which are much shorter than a CPI. The length of one period can be decided by the azimuth resolution During each period, the Doppler frequency of the moving target and clutter scattering point can be approximated as an unchangeable constant. Thus, the signal processed by STACC in BFSAR can be regarded as a narrowband one in a period and the effect of Doppler cell migration is eliminated through the time division.Then, the first clutter cancellation in SAR data domain can be carried out. For the range cell under detection, we can construct an optimal filter for space-time clutter cancellation in each period, the processing of space-time clutter cancellation in range time azimuth time domain is given byThen, the weight vector of the filter can be obtained byIn practice, due to the complex unknown environment, the covariance matrix i-th range cell can be obtained byThe training data We call this method time-division space-time adaptive clutter cancellation and its construction can be regarded as a sliding time-variant FIR filter, whose optimal weight vectors are updating during each period. The procedure of the first clutter cancellation in SAR echo domain is shown in With the time division processing, the first clutter cancellation can be achieved in SAR echo domain. However, the spectral distribution of the BFSAR clutter is closely related to the bistatic configuration, platform velocities and flight directions. That means the Doppler frequency of BFSAR clutter is range-dependent, namely BFSAR clutter is non-stationary, especially for the close distance range situations ,33. Due In In this section, we consider the problem of the residual non-stationary clutter cancellation. As mentioned above, the performance on clutter suppression will be heavily impacted due to the non-stationary characteristic of BFSAR clutter. Thus, ADPCA is introduced to deal with the second clutter cancellation in image domain. After the two-channel time-division STACC, the data of the channel The deramp function can be constructed as follows:By multiplying with the deramp function mentioned above, we can haveDue to Then, the coarse focused image of two channels can be obtained by taking the Fourier transform respect with In order to align the equivalent phase center of the different receive channels, the compensation function for the image of the The spatial distribution of the phase in Equation (31) is shown in Finally, the second cancellation in image domain can be expressed asFor the residual clutter, we haveFor the ground moving target with the slow velocity, we haveAs can be seen from Equations (33) and (34), after the second clutter cancellation in image domain, the residual non-stationary clutter can be cancelled sufficiently in the image domain and moving target is retained and coarse focused. The energy of moving target has been almost gathered in image. Then, the goal of the ground moving target detection for BFSAR can be achieved.Heretofore, the ground moving target detection method for BFSAR with three receiving channels has been detailed. The procedure of the proposed method is given in Experiments and results are given in this section to verify the effectiveness of the proposed method. In the experiments, the proposed method will be compared with the traditional STAP and direct three-channel time-division STACC method after Doppler ambiguity suppression and correction of cell migration. The performance on SCNR improvement and detection of these methods are analyzed. The simulation parameters are listed in The receiver is equipped with a 3-channel linear array along the flight direction, and the spacing  2000 Hz ,23. With the close detection range situation, namely the strong non-stationary ground clutter environment, we consider that the original coordinates of transmitter and receiver are  m and  m, respectively. In this paper, an effective BFSAR ground moving target detection method based on joint clutter cancellation with three receiving channels is proposed. Considering the Doppler ambiguity and cell migration of BFSAR, the proposed method mainly concludes four steps. First, the bulk-deramp pre-filtering is applied to suppress the Doppler ambiguity. Then, the first-order keystone transform is the second step to correct the large-scale range cell migration. The energy of the moving target will be gathered into one range cell. After the steps mentioned above, two-channel time-division STACC will be conducted between two adjacent channels as the third steps to suppress the BFSAR clutter at the first time in echo domain without the effect of the migration of Doppler cells. Due to the bad performance of the non-stationary BFSAR clutter suppression, ADPCA is exploited as the fourth step to cancel the residual non-stationary clutter in image domain. Experimental results have shown that the non-stationary clutter of BFSAR can be suppressed sufficiently after the joint clutter cancellation and the SCNR improvement of the propose method is greater than other methods. The GMTD performance of the proposed method makes it easy to achieve the goal of ground moving target detection.


The direct electron transfer (DET) of enzymes has been utilized to develop biosensors and enzymatic biofuel cells on micro‐ and nanostructured electrodes. Whereas some enzymes exhibit direct electron transfer between their active‐site cofactor and an electrode, other oxidoreductases depend on acquired cytochrome domains or cytochrome subunits as built‐in redox mediators. The physiological function of these cytochromes is to transfer electrons between the active‐site cofactor and a redox partner protein. The exchange of the natural electron acceptor/donor by an electrode has been demonstrated for several cytochrome carrying oxidoreductases. These multi‐cofactor enzymes have been applied in third generation biosensors to detect glucose, lactate, and other analytes. This review investigates and classifies oxidoreductases with a cytochrome domain, enzyme complexes with a cytochrome subunit, and covers designed cytochrome fusion enzymes. The structurally and electrochemically best characterized proponents from each enzyme class carrying a cytochrome, that is, flavoenzymes, quinoenzymes, molybdenum‐cofactor enzymes, iron‐sulfur cluster enzymes, and multi‐haem enzymes, are featured, and their biochemical, kinetic, and electrochemical properties are compared. The cytochromes molecular and functional properties as well as their contribution to the interdomain electron transfer  and DET (between cytochrome and electrode) with regard to the achieved current density is discussed. Protein design strategies for cytochrome‐fused enzymes are reviewed and the limiting factors as well as strategies to overcome them are outlined. DET is therefore the prior condition for catalytic turnover and direct bioelectrocatalysis. In direct bioelectrocatalysis an anode substitutes soluble or membrane‐bound electron acceptors for dehydrogenases and oxidases, whereas a cathode replaces the electron donor for reductases. DET‐capable oxidoreductases are applied as anode biocatalysts (e. g. cellobiose dehydrogenase or sulfite dehydrogenase) or cathode biocatalysts (e. g. laccase or bilirubin oxidase) in third generation biosensors, mediator‐free biofuel cells and bioelectrosynthesis. Since DET was discovered for cytochrome c peroxidase, horseradish peroxidase, lignin peroxidase and manganese peroxidase)c can bridge the reaction of oxidoreductases to electrodes, which was demonstrated for cellobiose dehydrogenase, bilirubin oxidase and sulfite oxidase.6 years ago.Multi‐cofactor enzymes carry a cytochrome domain, or enzyme complexes with a cytochrome subunit, but also oxidoreductases with a copper cofactor ,The performance of third generation biosensors and biofuel cells critically depends on the DET rate, which can become the rate‐limiting step for catalysis. An increased DET goes hand‐in‐hand with higher turnover numbers, higher specific currents, higher sensitivities and lower detection limits. It also helps to lower the polarization potential of third generation biosensors, which decreases the rate of interfering oxidation processes at the working electrode and thereby increases the selectivity of the biosensor. The optimization of biocatalysts and their molecular, catalytic or electron transferring properties becomes simpler due to the rapid development of protein engineering techniques.Recent protein engineering efforts mimic naturally evolved electron transfer proteins and enzymes by fusing a cytochrome domain to glucose dehydrogenase. A good interaction of the cytochrome with the active‐site cofactor is important to obtain high IET and DET rates and consequently a high current density for glucose biosensors. To evaluate the potential of enzymes featuring an electron transferring cytochrome for third generation biosensors and biofuel cells, this review presents and classifies multi‐cofactor enzymes, evaluates the molecular requisites for DET and investigates the potential of engineered enzymes with a fused cytochrome domain for DET.2The name cytochrome was introduced in 1925 by David Keilina, b and c, which still forms the basis of the current classification into six classes: a, b, c, d, f and o. These cytochromes differ in the chemical substituents of the haem tetrapyrrole ring, its linkage to the protein, or both. Two different nomenclatures are used to denote subclasses following either function (e. g. cytochrome 1c) or giving the α‐band absorption maximum (cytochrome 550c).c into four subclasses (I–IV).The detection and identification of cytochromes is supported by their distinctive, redox‐sensitive spectra. Individual cytochromes are resolved on the basis of their lowest energy absorption peak, the α‐band.b and c. The difference lies in the covalent attachment of the protoporphyrin IX ring in c‐type cytochromes via two thioether bonds between its vinyl groups and the two cysteine residues from the typical cytochrome c pentapeptide CXXCH motif, whereas haem b is not covalently bound. The protein's binding pocket also determines the haem's function including electron transfer, oxygen transport, catalysis and others. The protein environment modifies the haem reactivity by the axial iron ligands, the haem burial in the protein and its solvent accessibility, and the arrangement of polar or charged amino acid side chains around the haem.In multi‐cofactor enzymes two cytochrome types are found: cytochrome b, the proximal and distal ligands are usually histidine imidazole groups (His‐His), whereas in cytochrome c, one His imidazole is typically replaced by a Met sulfur group (His‐Met). It is a convention, that the His of the CXXCH motif is referred to as the proximal ligand and the other side of the haem as the distal side. A strong ligation of the iron results in a low‐spin state and typically the iron center in cytochromes alternates between an oxidized Fe(III) low‐spin state with a single unpaired electron and a formal charge of +1 and a reduced Fe(II) low‐spin form with no unpaired electrons and a net charge of zero. The change between the reduced and oxidized state is facilitated by the iron remaining in the low‐spin state.c’ are known. A weak or no axial coordination of the iron results in the high spin‐state and correspondingly a low redox potential.In cytochromes two axial iron ligands prevent the access of small molecule ligands or substrates and determine its function as an electron storage and electron transfer protein. In cytochrome c‐type cytochromes over b‐type cytochromes mainly arise from the π‐electron‐acceptor character of the thioether sulfur atom of the distal Met, which stabilizes the ferrous over the ferric state.The cofactors of metalloproteins exhibit potentials between −700 to +780 mV vs. SHE. For cytochromes redox potentials between −400 and +400 mv vs. SHE are reported , which define their biochemical functions. The redox potential of the haem cofactor is modulated by the stabilization of the buried haem by the protein matrix, modulation by polar and ionized side chains and backbone dipoles; the axial ligands, ligand geometry and first‐ and second coordination sphere of the iron; haem type and covalent bond to the haem, solvent accessibility, ionic composition and pH of the solvent, electrostatic interactions with the environment, and temperature.551c, cytochrome b from flavohemoglobin), orthogonal bundle , αβ‐roll , β‐sandwich  up‐down bundle . Therefore, cytochromes vary in molecular mass, surface geometry, isoelectric point and surface charge distribution, but also show variations in the haem environment, ligation geometry and therefore the redox potential.Cytochromes are electron storing and transferring haemoproteins, which were evolved from a variety of existing haemoprotein folds. This results in the structural diversity of cytochrome protein‐fold families Figure  such as:c. It is also the model protein for DET studies in electrochemistry. Many electrode materials and surface modifications have been applied to promote DET of cytochrome c. Also, the immobilization method shows an influence on DET, especially the orientation of the cytochrome on the electrode. Oriented immobilization has been employed to optimize the haem‐to‐electrode distance and thereby the electronic coupling of the haem to the electrode.The best‐studied cytochrome is cytochrome 3Searching the EC classification of enzymes, we found 39 sub‐sub classes of an enzyme featuring a cytochrome domain or a cytochrome subunit that is responsible for the intermolecular electron transfer to or from an electron donor or acceptor protein (Table S1). They belong to EC class 1 (oxidoreductases) and are multi‐cofactor enzymes containing flavins, porolinoquinoline quinone, different haem‐types, iron‐sulfur clusters or molybdenum as prosthetic groups and a cytochrome domain or cytochrome subunit as electron transferring moiety. Here, the differentiation into cytochrome domains and subunits distinguishes single chain multi‐cofactor enzymes featuring the cytochrome as a domain of the enzyme and multi‐cofactor enzyme complexes with a co‐assembled cytochrome subunit. About half 20) of the EC sub‐sub classes feature membrane‐associated multi‐cofactor enzymes that participate in electron transport chains. The majority of membrane‐associated enzymes are complexes (17) consisting of at least two subunits, whereas only 3 single‐chain membrane‐associated enzymes were found. The other half of sub‐sub classes (19) feature soluble enzymes, which interact with soluble or membrane‐associated electron transfer proteins. Most of the soluble enzymes (16) are single polypeptide chains and only 3 are enzyme complexes. An overview on the different active‐site cofactors in cytochrome carrying multi‐cofactor enzymes is presented in Figure 0 of the The found multi‐cofactor oxidoreductases can be classified by their active‐site cofactor, by the type and number of the cytochrome, or by their catalytic activity or physiological function. Since the 39 found examples have very diverse activities and are located in various intra‐ and extracellular places, the enzymatic activity and physiological function is not a suitable descriptor for the classification of the enzymes in this review. A systematic approach, justified by their different functions and redox properties, is the classification according to their (1) active‐site cofactor or cofactor closest to the cytochrome and (2) the type and number of cytochromes. The catalytic activity and physiological function of enzymes in the same class and even subclass can differ strongly. By sorting enzymes from the 39 EC sub‐sub classes according to these criteria following classes are obtained: (1) flavoenzymes carrying a cytochrome (flavocytochromes), (2) PQQ‐enzymes carrying a cytochrome, (3) molybdenum cofactor enzymes carrying a cytochrome, (4) Iron‐sulfur enzymes carrying a cytochrome, and (5) multihaem enzymes carrying a cytochrome. Prominent and electrochemically characterized enzymes of each class are listed and classified in the following five subsections.3.1b‐type cytochrome: L‐lactate dehydrogenase (flavocytochrome 2b), EC 1.1.2.3; cellobiose dehydrogenase, EC 1.1.99.18; and nitric oxide dioxygenase (flavohemoglobin), EC 1.14.12.17. The second subclass contains membrane‐associated enzyme complexes with a c‐type cytochrome: gluconate 2‐dehydrogenase, EC 1.1.99.3; p‐cresol methylhydroxylase, EC 1.17.99.1; and spermidine dehydrogenase, EC 1.5.99.6. The third subclass contains flavocytochromes with multiple c‐type haems: sulfide‐cytochrome‐c reductase (flavocytochrome c sulfide dehydrogenase), EC 1.8.2.3; NADPH oxidase, EC 1.6.3.1; fructose dehydrogenase, EC 1.1.99.11; and fumarate reductase (flavocytochrome 3c), EC 1.3.1.6. Available structures of these multi‐cofactor flavoenzymes are given in Figure Flavoenzymes with an attached cytochrome domain catalyze the dehydrogenation, mono‐oxygenation or deoxygenation of substrates. By sorting them according to their cytochrome type, three subclasses can be separated. The first subclass consists of soluble (intra‐ or extracellular) flavoenzymes with a Cellobiose dehydrogenase and L‐lactate dehydrogenase are intensively studied biocatalysts for biosensors and biofuel cells.3.2b‐type cytochrome: pyranose dehydrogenase, EC 1.1.2.B5; (2) carrying a c‐type cytochrome: lupanine hydroxylase (EC 1.17.2.2), lactate dehydrogenase (EC 1.1.5.B3), polyvinyl alcohol dehydrogenase (EC 1.1.2.6), 1‐butanol dehydrogenase (EC 1.1.2.9), type II alcohol dehydrogenases (EC 1.1.9.1) and a novel pyruvate dehydrogenase from Gluconobacter sp.; (3) carrying multiple haem b/c moieties: type III alcohol dehydrogenase, EC 1.1.5.5 and aldehyde dehydrogenase, EC 1.2.5.2. Subclasses 1 and 2 are composed of monomeric enzymes with a distinct domain for each cofactor and soluble. In subclass 3, membrane‐associated type III alcohol dehydrogenase and aldehyde dehydrogenase heterodimers feature an additional 50 kDa subunit containing three haem c moieties.c‐type cytochrome has been elucidated so far (1YIQ), it is presented together with a structural model for a promising enzyme from subclass 1: pyranose dehydrogenase  carrying a e Figure .Soluble PQQ‐dependent pyranose dehydrogenase is capable of DET on glassy carbon electrodes.3.3b‐type cytochrome domain and is found in vertebrates, while sulfite dehydrogenase (EC 1.8.2.1) carries a fixed c‐type cytochrome subunit and is found in bacteria. The proposed catalytic mechanism of sulfite oxidase and sulfite dehydrogenase involves two intramolecular one‐electron transfer steps from the molybdenum cofactor to a b‐ or c‐type cytochrome, respectively.Two enzyme classes contain a molybdenum atom complexed by a molybdopterin molecule in combination with a cytochrome. This molybdenum cofactor is often abbreviated as Moco. Sulfite oxidase (EC 1.8.3.1) carrying a mobile −2 were achieved at different polarization potentials. A third‐generation sulfite biosensor was developed based on human sulfite oxidase on indium tin oxide, which exhibited a sensitivity of 35 nA mM−1 and a linear range between 0.5–20 μM.c as mediator and a self‐assembled monolayer of 11‐mercaptoundecanol cast on a gold electrode.Direct electrochemistry using different electrode materials was observed for sulfite dehydrogenase3.4b‐type cytochrome(s): succinate dehydrogenase, EC 1.3.5.1; fumarate reductase, EC 1.3.5.4; molybdenum: nitrate reductases, EC 1.7.5.1; dimethyl sulfide/cytochrome 2c reductase, EC 1.8.2.4; ethylbenzene hydroxylase, EC 1.17.99.2; chlorate reductase, EC 1.97.1.1; selenite reductase, EC 1.97.1.9; CoB‐CoM heterodisulfide reductase (coenzyme), EC 1.8.98.1; hydrogen/quinone oxidoreductase (Ni), EC 1.12.5.1; (2) two c‐type cytochromes: nitrate reductase, EC 1.9.6.1 and (3) a sirohaem/nitrite reductase, EC 1.7.1.15; assimilatory sulfite reductase, EC 1.8.1.2  a single or two E. coli or denitrifying bacteria. Direct electrochemistry of NarGHI from E. coli was reported using a pyrolytic graphite “edge” rotating electrode.Over the last two decades, significant efforts to develop nitrate biosensors have been directed to immobilization within or on conducting and non‐conducting polymers.3.5c‐type haems), or (2) different haem‐types, e. g. cytochrome cd1 EC 1.7.2.1 with a haem c and a haem 1d, Figure Several multihaem oxidoreductases that act on nitrogenous compounds are listed in enzyme sub‐sub class E.C. 1.7.2. carry a cytochrome as acceptor. These contain either: (1) the same haem‐type, e. g. hydroxylamine dehydrogenase EC 1.7.2.6 featuring seven 1cd has been used for the determination of nitrites using cytochrome 552c as a redox mediator.−1 cm2 μM−1.Cytochrome 4b‐ or c‐type cytochromes in various protein folds.Multi‐cofactor enzymes with a known protein structure are listed in Table b folds are: 8‐helix orthogonal bundle (globin), up‐down bundle, β‐sandwich, and the αβ‐barrel and αβ‐complex (αβ‐roll). The observed cytochrome c folds are: 3‐helix orthogonal bundle, 4‐helix orthogonal bundle and αβ‐complex. The cytochromes vary in size, with the 3‐helix orthogonal bundle (40–74 aa) being the smallest, the 4‐helix orthogonal (80–94 aa), αβ‐roll (82–86 aa) and 8‐helix orthogonal bundle (144 amino acids) in the median range, and the β‐sandwich (208–213 aa) and the up‐down bundle (214–255 aa) being about twice as big. The volume of the cytochrome domains vary also according to the number of accommodated amino acids and is typically smaller for the single‐haem cytochromes (10–12 nm3) and bigger for the multi‐haem cytochromes (22–32 nm3). Exceptions are the big single‐haem β‐sandwich fold (∼26 nm3) and the structurally very compact cytochrome domain of flavocytochrome 3c (12 nm3), which contains 4 haem cofactors.The observed cytochrome b and His/Met ligation for cytochrome c. Exceptions from this rule are found in b‐type cytochromes: cellobiose dehydrogenase with a His/Met ligation, flavohaemoglobin with only a proximal His ligand and ethylbenzene dehydrogenase with a Lys/Met; and c‐type cytochrome: fumarate reductase with a His/His.The ligation of the haem iron in the proximal and distal position is most often typical and shows His/His ligation for cytochrome 2 (the interface area is the combined area of both interacting surfaces) with a tendency of mobile cytochrome domains having a smaller interface and fixed subunits having a bigger. Typical protein‐protein interfaces show an interface size distribution between 2 and 28 nm2, with 8 nm2 being the median size. The strength of the interaction is enhanced by steric surface complementarity, opposite charge pairs and hydrophobic pairs of amino acids.The observed edge‐to‐edge distances between the haem and the closest cofactor in the closed conformation/in the enzyme complex are between 0.47 and 1.22 nm, the median distance being 0.76 nm. The interface between the catalytic domain or subunit and the cytochrome domain or subunit has an area between 8 and 24 nm5The cytochrome domain acts as an electron acceptor in dehydrogenases/oxidases and as an electron donor in reductases. Correspondingly, the redox potential of the cytochrome in the investigated dehydrogenases and oxidases is 34 to 494 mV vs. SHE to function as an electron acceptor of the catalytic cofactor, whereas in reductases the redox potential is between −238 to 20 mV vs. SHE. Details of multi‐cofactor enzymes with a demonstrated DET  are listed in Table P. chrysosporium CDH with 84 or 262 mV  of the active‐site. An IET faster than the TN makes the catalytic reaction rate limiting and is unlikely to be a criterion for further evolutionary selection. A slow IET rate reduces the catalytic turnover and would be subjected to evolution. The found IET rates  are therefore in a similar range as the TN. For example the IET rate in L‐lactate dehydrogenase is 90 s−1, whereas the catalytic TN is 214 s−1 (measured with ferricyanide as one‐electron acceptor of the flavin). So the cytochrome‐flavin IET is very efficient and only little rate limiting. In cellobiose dehydrogenase a broad range of IET rates can be found 0.13–45.8 s−1), which is due to the different enzyme producers and possible slightly different physiological functions and conditions, but also pH is an important factor and restricts IET in most CDHs to pH values below 7 with maxima between pH 3–5.The redox potential of the cytochromes and the other cofactors depends on pH, temperature, buffer species and concentration and therefore the values from literature are different to compare. However, the redox potential difference between the cofactors measured at the same conditions is interesting to compare. Especially the flavoenzymes have a wide spread redox potential difference, e. g. Whereas the IET between the two protein redox partners has been evolved and optimized over time, the DET to electrode surfaces likely depends less on the protein, but more on the complementarity of the electrode surface. A suitable environment in regard to hydrophobicity, hydrophilicity, charges, ion species and concentration, etc. has to be provided to optimize DET. Nevertheless, for some enzyme/electrode combinations high DET rates, comparable to the TN and IET rates are reported, e. g. for fructose dehydrogenase, gluconate 2‐dehydrogenase and cellobiose dehydrogenase. For enzymes with no reported DET rates the minimum DET rate based on the enzyme loading and the obtained current was recalculated in Table −2 are reported. Especially for enzymes/electrode combinations with a low current density a suboptimal DET can be assumed. However, it has to be taken into account that the actual surface area in contact with the enzyme and the geometric surface area used for the calculation differ greatly. Nanomaterials and chemical modifications of electrode surfaces have been used to increase the available surface for enzyme immobilization and to optimize the DET by improved binding and orientation of the enzyme. However, also simple, unmodified electrodes based on carbon work very well. When used and calibrated as biosensors, the enzyme‐modified electrodes reach sensitivities between 31 and 275 nA mm−2 mM−1.The efficiency of the whole electron transfer (IET+DET) can be assessed from the reported current densities of the enzyme‐modified electrodes. Specific current densities between 1.3 and 1045 nA mm6DET‐capable enzymes from the classes of flavocytochromes and PQQ‐dependent multi‐cofactor enzymes will be discussed in detail in the following sections, by using the above derived framework of molecular and functional properties to evaluate their performance on electrodes.6.1Cellobiose dehydrogenase (CDH) is one of the best‐documented DET‐enzymes for application in biosensor and biofuel cells. It is an extracellular flavocytochrome secreted by fungi to obtain and transfer electrons from the oxidation of its natural substrate cellobiose to its physiological electron acceptor lytic polysaccharide monooxygenase.Myriococcum thermophilum CDH the closed‐state with the β‐sandwich cytochrome b domain bound to the glucose‐methanol‐choline (GMC)‐oxidoreductase flavodehydrogenase domain was observed (4QI6), whereas for Neurospora crassa CDH the open‐state with a swung‐out cytochrome domain was found (4QI7). The structures demonstrate that the cytochrome‐dehydrogenase interaction is not strong and therefore the cytochrome domain is mobile, with a distance of 3.87 nm between both domains in the crystal structure of the open‐state N. crassa CDH.The crystal structure of the full‐length CDH was reported in 2015.P. chrysosporium CDH.Site‐directed mutagenesis confirmed the interaction site of both domains. The biggest contribution to binding comes from the electrostatic interaction of a haem propionate‐A group interacts with an Arg residue on the dehydrogenase domain on the side of the substrate channel.M. thermophilum CDH the IET rate is especially low, about ∼150 times lower than the TN ..5 Table . DivalenPichia pastoris (which is known to overglycosylate and O‐glycosylate recombinant proteins), CDH also can be produced in alternative expression systems.Trichoderma reesei turned out to be the best host to produce low‐glycosylated CDH of high uniformity of the glycoform.Based on its ability to perform DET, CDH has been studied to develop third generation biosensors and enzymatic biofuel cells.6.22b (Fcb2) is an L‐lactate:cytochrome c oxidoreductase found in the intermembrane space of yeast mitochondria. Fcb2 is homotetrameric enzyme that catalyzes the oxidation of L‐lactate to pyruvate with a subsequent reduction of cytochrome c. The first crystal structure of Fcb2 was published in 1990.2 contains one flavin mononucleotide (FMN) and one haem b in two structurally and functionally distinct domains. The two domains are linked by an interdomain “hinge” peptide and there is a C‐terminal “tail” which is involved in the tetramer integrity. Mutational studies of the functional residues in active‐site together with molecular dynamics studies uncovered the substrate oxidation mechanism.2 has been successfully changed toward larger (S)‐2‐hydroxy acids by enzyme engineering,2.Flavocytochrome 2 was also studied in detail by stopped‐flow spectroscopy, laser flash photolysis and temperature jump experiments. These confirmed the IET from the fully reduced FMN to haem b is several times faster than the catalytic turnover.b was shown to be much slower and to be the overall rate‐limiting step in the catalytic cycle.2 crystal structure, the cytochrome domain is moved relative to the tetrameric flavodehydrogenase domain core in one out of two subunits.c as electron acceptor.c overlap, which demonstrates the cytochrome domain's mobility, which has to move away from the flavodehydrogenase domain for the interaction with cytochrome c.The IET in Fcb2 were developed for L‐lactate determination in the 1980s.2 overproducing yeast, Hansenula polymorpha, which was used as biological recognition element in amperometric L‐lactate biosensors and DET was achieved on different electrode materials.2, cytochrome c and Complex‐IV in a cascade. The sensitivity of the best obtained graphite electrode is 250 nA mM−1. Also different redox mediators were tested in combination with the yeast cells to improve the performance.Saccharomyces cerevisiae, was employed to create a yeast‐modified, mediated amperometric biosensor for lactic acid detection.Both DET and MET sensors based on Fcb77.1Coprinopsis cinerea carrying a cytochrome b domain.b containing cytochrome domain has a β‐sandwich fold and is homologous to the cytochrome domain of cellobiose dehydrogenase. Like CDH, PDH can also function as electron donor of LPMO.The first PQQ‐dependent enzymes in an eukaryote was reported in 2014 by Nakamura. It is a PQQ‐dependent pyranose dehydrogenase (PDH) from the basidiomycete b in the cytochrome domain was determined by cyclic voltammetry to be 130 mV vs. SHE,c or electrodes as terminal electron acceptors. Bioelectrocatalysis of PDH demonstrated that the measured pH profile of the biocatalytic current was similar to the pH profile of cytochrome c. The optimal pH values is 8.5.DET from PDH to glassy carbon electrodes has been observed using L‐fucose as a substrate.7.2Gluconobacter and Acetobacter, DET has been demonstrated almost 2 decades ago.b/c‐containing subunit and additionally a multihaem c‐containing subunit as well as a PQQ subunit. The intramolecular electron transfer route was proposed after determining the redox potentials of all four haem c cofactors.For PQQ‐dependent alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (AldDH) from acetic acid bacteria, mainly −2, respectively.−2 was achieved based on the DET of both enzymes.6‐tags. The most favorable orientation was found for the variant with the haem c in closest proximity to the electrode.The DET of ADH and AldDH was first observed on screen printed carbon electrode. The achieved current densities with 30 mM ethanol/acetaldehyde was 320 or 1.3 nA mm−2 at a 50 mM lactate concentration (pH 7.15).Gluconobacter showed a low substrate specificity, which is advantageous for the deep oxidation of the pyruvate (completely oxidized to carbon dioxide) in biofuel cell anodes,−2 at a 25 mM pyruvate concentration (pH 7.15).Other isolated PQQ‐dependent enzymes from acetic acid bacteria are a lactate dehydrogenase (LDH), which showed DET on both gold and carbon electrode surfaces with maximum current density of 360 nA mm8The search for specific biosensor catalysts with DET properties has driven research to generate DET enzymes based on FAD‐dependent glucose dehydrogenase (GDH) for glucose biosensors. GDH is a thermostable, oxygen insensitive redox enzyme widely used in second generation biosensors. Since the FAD cofactor of the enzyme is deeply buried within the protein, it is almost impossible to achieve efficient DET. Fusion of GDH with a cytochrome domain was successfully introduced as a way of creating new multi‐cofactor enzymes capable of DET.b domain of CDH from P. chrysosporium N‐terminally to glucose dehydrogenase from Aspergillus flavus  by the presence of the divalent Ca2+ ions.M value of the electrode immobilized enzyme for glucose was determined. The KM of the heterogenized enzyme is 8.6 mM and 6‐times lower than the KM obtained in solution (53 mM), which indicates that the catalytic reaction is limited by either IET or DET. Since the cytochrome domain of P. chrysosporium CDH exhibits a high DET, the IET between the cofactors is rate limiting. The measured specific current density at a 5 mM glucose concentration and 605 mV vs. SHE was ∼4.5 nA mm−2.To that purpose, the group of K. Sode fused the β‐sandwich cytochrome s Figure .69c The Burkholderia cepacia glucose dehydrogenase by a much smaller, cytochrome c domain from the magnetochrome‐containing protein MamP2) than the wild‐type GDH (4 nA mm2) at a lower onset potential (∼50 mV vs. SHE), which improves the glucose sensing properties of the enzyme. The use of a mobile heptapeptide linker (GSGYGSG) and the choice of a small cytochrome domain improved the overall electron transfer to the electrode, but it was not elucidated if IET or DET was improved.Another study by the group of L. Alfonta replaced the large, 43 kDa multi‐haem cytochrome c subunit of B. cepacia GDH's multi‐haem cytochrome c subunit was investigated by a truncation experiment.2 at a 5 mM glucose concentration) on a thioalkane modified gold electrode.The function of A. flavus has a much higher catalytic TN for glucose (780 s−1) than GDH from B. cepacia (1.7 s−1) or CDH from C. thermophilus (39 s−1). This high glucose activity is a good basis for a DET biocatalyst and keeps the FAD cofactor in its reduced form which enhances the probability of an IET event. On the other hand, reduced FADH2 is susceptible to the futile generation of H2O2, if the electrons are not removed quickly from the cofactor, which would compromise the stability of the biosensor catalyst. The abstraction of the first electron from FADH2, leading to the FAD semiquinone suppresses the generation of H2O2. Besides efficient electron transfer to the electrode, this is another argument for a fast IET by selecting the most suitable cytochrome domain.The GDH from A. flavus fusion enzyme is limited by the IET, which was reported to be 0.172 s−1. For the B. cepacia fusion enzymes no IET rate was reported, but by considering the specific current densities for both enzymes, which are 20 and 8.5 nA mm−2, respectively  It is known from CDH that charge repulsion plays a big role in the interaction of the flavodehydrogenase domain and the cytochrome domain. Studies of the pH‐dependent domain interaction as already performedThe catalytic rate of the These three factors demonstrate that the selection of the cytochrome for a fusion enzyme is a difficult choice and that existing cytochromes need adaption to complement and interact with the catalytic domain, which currently restricts the generation of fusion enzymes. Although the mobility of the cytochrome is important to shuttle electrons between the enzyme and the electrode, the rate limiting step will be in most cases the correct, IET‐competent association of the domains. Protein‐protein docking and molecular dynamics (MD) simulations are the methods of choice to engineer compatible interaction surfaces that allow a fast association of the cytochrome to the catalytically active domain, but also allow a fast dissociation to transfer the electron to the electrode.9b‐type cytochrome or a c‐type cytochrome for the electron transfer. The selection of the cytochromes might reflect the necessary redox potential . In reductases the redox potential needs to be lower than the active‐site cofactor, which is achieved by a low haem redox potential between −238–20 mV vs. SHE). The smallest found redox potential difference between an active‐site cofactor and a cytochrome is 41 mV, but typically it is higher than 80 mV.Multi‐cofactor enzymes featuring a cytochrome domain or a cytochrome subunit are engaged in diverse, mostly oxidative, reactions and use other redox proteins or redox enzymes as electron acceptors/donors. All found and summarized enzymes in this review use either a b and cytochrome c domains or subunits can be found in matrix‐associated and soluble intra‐ or extracellular enzymes. The size and fold of the cytochromes differ, but has no obvious effect on IET or DET rates. No tendency of smaller cytochromes for a faster IET or DET has been found, but for many enzymes a more detailed characterization of these rates is required. For some of the classified multi‐cofactor enzymes a DET to electrodes has not yet been demonstrated. The obvious reason is that some of these enzymes are not suitable, but it is also obvious that the detection of DET also depends on a pure and active enzyme preparation, a suitable electrode, surface structure and chemical substitution. By optimizing the electrode architecture new DET‐enzymes can be discovered and the performance of enzymes with a demonstrated DET can be increased.The cellular location of an enzyme determines no prevalence of a cytochrome type and a similar number of cytochrome The fusion of cytochromes to catalytically interesting enzymes is a promising strategy to generate DET‐enzymes for new substrates/analytes. Initial research has proven the validity of this concept and it will be interesting to see the rational design of fusion enzymes tackling the need for DET‐capable biosensor catalysts. The optimization of the cytochrome domain's redox potential to be sufficient for fast IET, but not wasting to much potential difference, the obtained voltage under load of biofuel cells can be increased and biosensors with a lower polarization potential might also suffer less from interferences of electroactive physiological or pharmaceutical compounds such as ascorbate or paracetamol.Which design guidelines for the rational engineering of efficient DET‐enzymes can be derived from the existing data? The protein interface of catalytic domain and cytochrome domain has to be sterically and electrostatically complementary. The edge‐to‐edge distance of the cofactors should be as close as possible in the closed conformation. In case of a mobile cytochrome domain a balance between the factors supporting a closed conformation and the open conformation is necessary to facilitate IET as well as DET. Also the length and composition of the protein linker between the domains is important to support, but also restrict the cytochrome mobility. Future studies in this direction will certainly improve our knowledge on how to rationally design DET‐enzymes, but careful measured data are necessary to evaluate the results.The authors declare no conflict of interest.Su Ma received her PhD degree in 2013 from Ocean University of China. In 2014, she joined the Biocatalysis and Biosensing Laboratory (BBL) at BOKU – University of Natural Recourse and Life Science, Vienna (Austria) as a Marie Curie research fellow. She continued postdoctoral research for one year in an ERC Consolidator Grant Project and is now a junior PI managing different projects of the BBL in the field of protein engineering and bioelectrochemistry.Roland Ludwig received his PhD in biochemistry from BOKU – University of Natural Resources and Life Sciences, Vienna (Austria) in 2004. He worked as a postdoctoral researcher at the Research Centre Applied Biocatalysis, Graz (Austria) and at Lund University (Sweden). In 2011, he started the Biocatalysis and Biosensing Laboratory at BOKU where he studies enzymes by protein engineering, characterization and applications in biocatalytic processes and biosensors.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Caenorhabditis elegans, have been shown to have similar anti-stress systems to mammals, suggesting that analogous defense systems are widely conserved throughout the animal kingdom. Experimental evidence in lower animals provides important information beyond mere laboratory-confined utility, such as regarding how these systems transformed during evolution, which may help characterize the mammalian system in greater detail. Recent advances in genome projects of both model and non-model animals have provided a great deal of useful information toward this end. We herein review the research on Keap1-Nrf2 and its analogous systems in both mammals and lower model animals. In addition, by comparing the amino acid sequences of Nrf2 and Keap1 proteins from various species, we can deduce the evolutionary history of the anti-stress system. This combinatorial approach using both experimental and genetic data will suggest perspectives of approach for researchers studying the stress response.The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Its regulatory mechanisms, e.g., stress-sensing mechanism, proteasome-based regulation of Nrf2 activity and selection of target genes, have been elucidated mainly in mammals. In addition, emerging model animals, such as zebrafish, fruit fly and  From birth, animals are destined to fight against a variety of stressors that disrupt their homeostasis. All animal species must cope with oxidative stress generated by their own metabolism. They were also forced to evolve detoxifying systems in case of accidental encounters with toxic chemicals in the environment. Animals could not have prospered without the anti-stress mechanisms evolved by their ancestral species.Drosophila pyrene is a well-studied pro-carcinogen that forms a highly reactive intermediate after phase I metabolism and is detoxified by phase II reactions. The loss of Nrf2 was suspected to potentially weaken phase II metabolism and enhance the carcinogenicity of benzo[a]pyrene. This was experimentally demonstrated by showing the high susceptibility of Nrf2-deficient mice to benzo[a]pyrene-induced tumor formation, suggesting that Nrf2 is indispensable for intact phase II metabolism [The main function of phase II enzymes is to detoxify the highly reactive intermediate metabolites generated by phase I reactions and accelerate the excretion of toxic xenobiotics . Benzo[atabolism ,16,17. Ltabolism ,19 as wetabolism ,21, suggtabolism . The modtabolism ,24,25. TThe Nrf2 system is also activated by endogenous cues, such as endoplasmic reticulum (ER) stress, and confers defense against such stress ,27. ThisBy contrast to these beneficial effects on human health, adverse effects of the Nrf2 system have also been reported: Nrf2 is constitutively activated in cancer cells and confers resistance against chemotherapy ,32,33,34Research on the Nrf2 activation mechanism has greatly progressed since a partner protein of Nrf2, Keap1, was discovered A. Keap1 In addition to the Keap1-dependent degradation, Nrf2 protein is also regulated in a Keap1-independent manner. Salazar et al.  revealedThese regulatory mechanisms of the Keap1-Nrf2 system have been identified mainly using mammalian cells. Non-mammalian animals also have anti-stress systems analogous to the mammalian Keap1-Nrf2 system. In the next section, we will overview these analogous systems in lower animals.Saccharomyces cerevisiae, have shown that the transcription-based stress response stems from single-cell eukaryotes. Yap family proteins are a well-studied group of transcription factors that confer protection against oxidative and chemical stress [Studies of budding yeast, l stress . The Yapl stress . Unlike l stress ,51,52,53Drosophila melanogaster, is a classic model animal with substantial advantages in the fields of developmental biology and genetics. The fly homolog of Nrf2 was discovered as an important protein in the development of the cranial portion  of larvae [Drosophila small Maf protein, Maf-S, was also demonstrated [Drosophila evolved from a common ancestral system with the mammalian Keap1-Nrf2 system.The fruit fly, f larvae . Becausef larvae . Despitef larvae ,57. CncCf larvae . In addif larvae ,59, and nstrated . The tarnstrated ,61,62,63Caenorhabditis elegans is also a strong model in developmental biology, and skinhead-1 (Skn-1), an ortholog of Nrf2 in mammals, was discovered as a protein that is important for normal pharyngeal development [C. elegans does not have an authentic ortholog of Keap1 [C. elegans, the WD40 repeat protein-23 (WDR-23)/damaged DNA binding protein 1 (DDB1) complex is involved in the ubiquitination of Skn-1 under basal conditions [The laboratory worm elopment . Skn-1 helopment . Uniquelelopment . Althougof Keap1 . Insteadnditions . Howevernditions ,70,71. Onditions ,72; therC. elegans, a unique regulatory mechanism was evolved.Although the regulatory mechanism differs from that of the mammal Nrf2 system, the target genes of Skn-1 are similar to those of mammalian Nrf2. The gene expressions of phase I, II and III detoxifying enzymes, antioxidant proteins and proteasome subunits are regulated in a Skn-1-dependent manner ,68,73. TDanio rerio), an emerging model animal in medical research, the Keap1-Nrf2 system is highly conserved with that of the mammalian system. Zebrafish Nrf2 and Keap1 were first cloned in 2002 and found to be structurally similar proteins to their mammalian counterparts [gstp1 [In zebrafish  [Strongylocentrotus purpuratus) [Octopus bimaculoides) [Hydra magnipapillata [The existence of an analogous Keap1-Nrf2 system in  species . In additinalis) , sea urcpuratus) , octopusuloides)  and the apillata .Hydra) seem to have only 1 Nrf gene locus, implying the diversification of this protein family in vertebrate evolution. Of the Nrf family proteins in mammals, Nrf1 has the most similar domain structure to Nrf2 and is known to regulate the transcription of antioxidant and phase II enzymes through binding to ARE, forming heterodimers with small Mafs. Despite this similarity to Nrf2, the regulatory mechanism of Nrf1 differs substantially from that of the Nrf2 system, largely due to the localization to the ER membrane by its ER binding region in the N-terminal domain, while Nrf2 stays in the cytosol under unstressed conditions. In addition, activity of Nrf1 is regulated by the glycosylation/deglycosylation, not by a Keap1-dependent ubiquitination mechanism [Vertebrates have 4 Nrf genes—Nrf1, Nrf2, Nrf3 and NF-E2 A—while lechanism . Similarechanism ,87. Studechanism . NF-E2 iechanism ,90. Geneechanism , and genechanism ,93. In cechanism ,94, suggThe Neh1 domain, also known as the CNC-bZip domain, is an essential region for DNA binding (CNC domain and basic region) and dimerization with small Maf proteins (leucine zipper region). The amino acid sequence of this region, especially the basic region in the middle of this domain, is highly conserved among a wide range of species, suggesting that all these proteins were derived from the same origin, and that the DNA binding ability has been inherited from ancestral species. Interestingly, Skn-1 was observed to have lost the leucine zipper, an essential region for the dimerization, which may therefore explain why Skn-1 binds to DNA as a monomer.Hydra Nrf protein, but Nrf of other triploblastic animals all have this domain. Nrf1 also possess these two motifs, while NF-E2 and Nrf3 have lost the Neh2 regions. Another important point is the conservation of lysine residues between the DLG and ETGE motifs, as seven lysine residues in this region of mouse Nrf2 were shown to be ubiquitinated, which promotes proteasomal degradation [Xenopus tropicalis, fugu, medaka fish and platypus, suggesting that the regulation of the Keap1 binding affinity by phosphorylation evolved after the appearance of vertebrates, but it was subsequently lost in some species.The Neh2 domain is characterized by its interaction with Keap1, making it an essential domain for the proteasome-dependent degradation of Nrf2 . Direct radation . Althougradation . This siThe C-terminal Neh3 domain was shown to be crucial for the transactivation of target genes. Mutant Nrf2 proteins with an incomplete Neh3 domain cannot activate ARE-dependent gene expression . In partHydra or in protostomes, Drosophila or octopus Nrf, implying that these domains evolved only in deuterostomes. In sea urchins and ascidians, conserved Neh4 and -5 domains were found. Teleost Nrf2 proteins  have complete TRAM as well as a conserved Neh5 domain, although Nrf2a in cavefish does not have a conserved Neh5 domain (http:www.ensembl.org). Regarding other Nrf family proteins in mice, TRAM was incomplete in Nrf3 and was not found in Nrf1 or NF-E2. The Neh5 domain was conserved in Nrf1 and NF-E2 but not in Nrf3.The Neh4 and Neh5 domains are also transcriptional activation domains, which interact with transcriptional co-activator CREB binding protein (CBP). Transcriptional adapter motif (TRAM) (FxD/ExxxL) in Neh4 is considered to be an essential motif for this interaction . No homoHydra; therefore, the Nrf activity might be regulated by phosphorylation-dependent degradation from common ancestors of species investigated. This motif was also conserved in other Nrf proteins in mice, suggesting that the activity of these isoforms is also modulated by phosphorylation. The Neh6 domain of mouse Nrf2 has another phosphorylation site in its C-terminus (DSAPGS motif) [Hydra, fly, octopus, sea urchin or ascidian. Although this motif was found in vertebrate Nrf proteins, the conservation of the amino acid sequence was relatively weak, and the functional importance of this region is unclear.The Neh6 domain plays a role in the phosphorylation-based regulation of Nrf2 activity. The DSGIS motif in the N-terminus of Neh6 is essential for the GSK-3β-dependent phosphorylation and subsequent ubiquitination by β-TrCP . This moS motif) , but no We next searched for homologous sequences to the N-terminal ER binding region of mouse Nrf1 and its vertebrate orthologs. N-terminal homology box1 (NHB1) was shown to be important for Nrf1 tethering to the ER . Nrf3 ofThe conservation of each domain is summarized in Zebrafish have the second Nrf2 homolog, Nrf2b . AlthougHydra Nrf protein lacks an Neh2 domain and possesses an ER binding domain, its activity is suspected to be regulated mainly at the binding to the ER membrane, just like Nrf1 in mammals. The rise of the Neh2 domain in triploblasts may correspond to the appearance of Keap1-dependent regulation and probably activation in response to various stressors. Data for sea urchins and ascidians have suggested that deuterostomes obtained Neh4 and -5 domains, while protostomes (fly and octopus) did not. C. elegans evolved a unique protein, Skn-1, that lacks most of the original domains, even the C-terminal half of Neh1 (leucine zipper).From the structural analysis above, we deduced the evolutionary history of the Nrf proteins . Of the Drosophila generates at least 16 variants of Cnc, one of which (CncI) has a similar structure to Nrf2 that lacks the N-terminal ER binding domain [Ciona intestinalis shows that the Nrf locus generates two variants: one with an ER binding domain and the other without it.At the appearance of vertebrates, the Nrf gene locus was diversified into four loci, which vary in domain structure. In addition to obtaining six complete functional Neh domains, Nrf2 lost the ER binding domain, thus suggesting that Nrf2 had avoided localization to the ER. Recent advances have shown that g domain . In addig domain . FurtherTaken together, these findings suggest that, at the latest, the mechanism of alternative splicing and generation of varied proteins from a single Nrf locus was already present at the appearance of triploblastic animals. In vertebrates, at gene quadruplication, varied proteins are assigned to each replicated locus. For the Nrf2 locus, a transcription factor regulated by a Keap1-dependent and ER-independent mechanism is assigned.http:www.ensembl.org). To determine the evolutionary origin of this duplication, we further consulted the genomic information of the African coelacanth  [Anolis carolinensis) [Xenopus tropicalis. Two Keap1 co-orthologs were found in the coelacanth and frog but not in the lizard. The second Keap1 is suspected to have been generated at the appearance of vertebrates and lost in amniotes.We performed a similar phylogenetic analysis of Keap1 . In zebralumnae) , green ainensis)  and XenoKeap1 is composed of three domains, broad complex-tramtrack-bric a brac (BTB), intervening region (IVR) and double glycine repeat (DGR) domains A, all of(1–3)Lx(2–4)LxL), which is important for localization at the cytoplasm [The C-terminal DGR domain is the Nrf2 binding region. A detailed structural analysis revealed the direct binding sites in Keap1 to the DLG and ETGE motifs of Nrf2 . Nrf2 biytoplasm . This siThe notable characteristic of Keap1 is its richness in cysteine residues. Given their high reactivity, cysteines can function as “sensor” amino acids . When cyRegarding other cysteines, as summarized in We attempted to clarify how Keap1 changed during evolution . Of the tert-butyl hydroquinone (tBHQ), sulforaphane, diethyl maleate (DEM) and dimethyl fumarate (DMF) [Cys-151 has been determined to be an important cysteine for the detection of electrophiles, such as te (DMF) ,120,121.te (DMF) , from thte (DMF) . The mecte (DMF) . It will2, an anti-inflammatory prostaglandin [2, implying that having either Cys-273 or Cys-288 is enough for 15d-PGJ2 sensing [Cys-273 and Cys-288 have been shown to be essential for repressing Nrf2 activity under basal conditions ,119. Howaglandin ,121. We  sensing . Acrolei sensing . Althoug sensing , zebrafi sensing  and flie sensing , and allDrosophila.Recent progress has shown that Cys-226 and Cys-613 are necessary for the sensing of hydrogen peroxide and heavy metals such as zinc, cadmium, arsenic and selenium ,125,126.In addition to the cysteines described above, Cys-257, Cys-297 and Cys-319 were determined to have highly reactive thiols ,127. CysDrosophila Cnc and C. elegans Skn-1, which have been shown to be essential factors for embryonic development, vertebrate Nrf2 may perform functions other than those related to the stress response, even though Nrf2-knockout mice and Nrf2 mutant zebrafish develop normally [pgd) is also under the regulation of Nrf2 in zebrafish [Similar to normally ,11,76. Snormally . Second,normally . We receebrafish , suggestGrowing evidence shows that endogenous stressors are involved in the pathogenicity of various diseases. Whether or not the interaction of the Keap1-Nrf system with the ER stress response and autophagy pathway are conserved in lower animals remains unclear; however, Skn-1 is known to have a close relationship with the ER stress response pathway . Determi