Lagrangian/Eulerian formulation. After a transformation of the diffusion problem onto the fixed domain, we use the formal method of two-scale asymptotic expansion to derive the upscaled model, which is nonlinearly coupled through effective coefficients. The effective model is implemented and validated using an application-inspired model problem. Numerical solutions for both, cell problems and macroscopic equations, are investigated and interpreted. We use simulations to qualitatively determine the effect of the deformation on the transport process.In this paper, we derive an effective model for transport processes in periodically perforated elastic media, taking into account, e.g., cyclic elastic deformations as they occur in lung tissue due to respiratory movement. The underlying microscopic problem couples the deformation of the domain with a diffusion process within a mixed  Heat and mass transport in materials characterized by a complex microgeometry are actively researched topic in various fields such as material sciences or geosciences as well as in the biological/medical field. Here, mathematical models can help to identify underlying mechanisms as well as complement or replace experiments by numerical simulations. This is of particular importance in the medical field, where experiments and animal studies or clinical trials are expensive if at all feasible. The main question of this work is, how transport processes are influenced by a complex microstructure of the carrier medium and its deformation. Applications are especially found in the context of diseases whose clinical picture includes a dysfunction of the lung, such as COVID-19, pneumonia or sepsis. Experimental evidence already indicates that in addition to the microstructure formed at the cellular level by pneumocytes, the periodic deformation caused by inhalation and exhalation also plays an important role in transport processes such as those for nutrients or respiratory gases, see e.g. Huh et al. .Lagrangian/Eulerian framework. The deformation process is described by a linear elasticity equation on the microscopic reference domain as opposed to the transport problem, which is initially formulated on the current, moving microscopic domain. In this form, the problem is not accessible for standard upscaling tools. Therefore, we use a transformation, which is defined by means of the solution to the elasticity problem, to reformulate the transport problem on the reference domain for the elasticity equation. Then we can exploit the formal method of two-scale asymptotic expansion in order to obtain a macroscopic model describing transport processes and deformation for a periodically perforated domain. The resulting model is formulated on a homogeneous domain and involves effective coefficient functions. These effective quantities are computed by means of solutions to so-called cell problems which are formulated on the reference cell and carry the information on the microscopic geometry. Additionally, we develop and study numerical methods for the derived effective model. A lot of work has been done concerning the upscaling of fluid–structure-interaction-models, pure diffusion or elasticity, or (linear) coupled models for heat transport and deformation. In contrast, there are hardly any papers considering modeling and numerical simulations for an elasticity-diffusion problem, where the diffusion process is formulated within the Eulerian framework and transformed into a common framework, leading to coupling through nonlinear coefficients. We discretize both, the cell problems as well as the macroscopic problem using the finite element method and the implicit Euler method for discretization in time. Afterwards, we use a application-inspired model problem with oscillating Dirichlet boundary conditions in the deformation for numerical convergence tests to validate the code. For the model problem, we investigate and interpret the numerical results. Finally, again within the scope of the model problem, we give a quantitative answer to the question how the deformation affects the transport of a diffusing substance. To this end, we perform a parameter study for key parameters involved in the deformation of the domain and analyze the sensitivity of the effective model with respect to these parameters. This is accompanied by the visualization of the homogenized coefficient functions.Unfortunately, the heterogeneous microstructure of such problems can only be resolved to a very limited extent for small domains in numerical simulations. For problems involving multiple scales, one has to rely on mathematical tools that are capable of deriving approximate problems, which are easier to solve but still incorporate the information on the microgeometry. To this end, we derive in this paper an effective model for transport processes in an elastically deformable, periodically perforated medium, where we restrict ourselves to the case of a purely elastic solid phase that is not coupled with a fluid phase. This model is suitable for applications involving low-density fluids, for example, and it is also a first step for dealing with more general problems. We approach the problem by formulating first the microscopic problem on the perforated domain in a mixed Lagrangian/Eulerian framework were considered, where the equations for the fluid flow as well as the transport equation were formulated on the deformed domain, i.e., with respect to Eulerian coordinates. Here, we mention, e.g., Collis et al.  have been extensively studied in Oleinik et al. . Transpos et al. , where as et al.  for the s et al.  for lines et al.  for hypes et al. ; Peter  i i2.1d) i problem –2.1h) i iLagrangequation  is givenIn order to make the elasticity-diffusion system accessible for standard homogenization techniques, we transform equations –2.1h) t t2.1h) tfined in . For givin Peter .\documenm in Eq.  , , 2.1e), Next, let us replace the dependent and independent variables by scaled versions:mentclass2pt{minimUsing the relations in (2.3) and dropping the daggers for the sake of an easier notation we obtainmentclass2pt{minimThe existence of a weak solution for the elasticity Eqs.  –2.5d) i i2.5d) iAlthough the domain for the elasticity-diffusion system is now fixed in time, we still have to deal with its heterogeneous microstructure. This issue is addressed by application of the formal two-scale asymptotic expansion method with the aim of deriving effective equations on the spatially homogeneous macroscopic domain Y-periodic with respect to y. For derivatives of such functions, we have by the chain ruley. However, the only periodic rigid displacements are constant functions and therefore we sett and x, which we choose equal to zero by assuming mean value zero for i-th canonical unit basis vector in y gives the macroscopic problem:Following the standard approach of two-scale asymptotic expansion, we postulate for the microscopic displacement }uε from –2.5d). . \documec and the solution of diffusion cell problems n cell problems for c and c solves the following macroscopic problem: For the upscaling of the transport problem –2.5h), , 2.5h), rding to  by usingentation  for \docthe form  with\document}cε , \documeto Eqs.  –(2.5h). to Eqs.  –2.5d) b b\documeWe now summarize the upscaled equations which we have derived previously. They consist of coupled, effective problems for the displacement and the concentration, and are complemented by cells problems for the elasticity problem and for the diffusion problem, respectively. Thus, the macroscopic problem is given byIn the following, we focus on the simulation of the upscaled model summarized in Sect. entclass1pt{minimaWe avoid having to solve a nonlinear problem by organizing the scheme according to the special structure of the system. In each time step, we solve first the quasi-static macroscopic elasticity problem independently from the diffusion problem due to the coupling being only one-sided. Next, the effective coefficients for the diffusion equation, The scheme is also organized in a way that takes advantage of the specific properties of the cell problems. To this end, the treatment of the elasticity cell problems differs from the treatment of the diffusion cell problems due to the latter depending on time and location of the cell within the macroscopic domain. It is in fact sufficient to only solve the elasticity cell problems once for each }D0 (see  and (2.1}D0 (see ). Theref}D^ (cf. ) being tdeal.II ,,\documenLamé-constants We fix now data for further investigations of the upscaled problem from Sect. entclass1pt{minimaproblems , we set entclass1pt{minimaensor in  is set tentclass1pt{minimaY, they do not lead to the same effective coefficients, as asymmetries are also reflected in For a more comprehensive study of the properties of the effective coefficients in dependence of the micro-geometry, see Fig. entclass1pt{minimac with respect to the discretization parameter h of the grid for h is the diameter of the largest quadrilateral in the triangulation. For the simulations, we use the model problem presented in the previous Sect. Before we proceed with a more detailed view on the solutions of the proposed model problem, let us first investigate the convergence of the finite element scheme. Due to the two-scale character of the problem, there are two distinct computational domains, representing m-th component, Let us now give a more detailed analysis of the numerical solution to the effective elasticity subproblem using the aforementioned data. As stated earlier, it suffices to solve the elasticity cell problems  once at nt}A∗ in . For a ventclass1pt{minima}u1, see , in the }u1, see  for \docdimnumber of time stepsnumber of elementsnumber of quadrature points per element diffusion cell problems that have to be solved numerically during the simulation. For illustration, numerical approximations of the functions Finally, we investigate the solution of the diffusion equation for the model problem from Sect. c, defined on c using the following transformationDue to the transformation of the diffusion problem onto a reference domain in Sect. M(t) is the approximation of the mass or the number of particles that resides at time t in the domain f or the amplitude a in (M(t) for different simulations we gain insights on the sensitivity of the transport of the diffusing substance with respect to the deformation parameters.Finally, we analyze the system’s sensitivity with respect to experimental parameters which we can modulate by manipulating the boundary conditions of the elasticity problem. We adopt again the setup from the model problem in Sect. (cf. Eq. ), while ude a in . By compFig. M(t) is plotted for varying amplitudes of the lateral displacement. The percentage in the legend encodes the maximal length of the domain The system clearly exhibits a higher sensitivity with respect to the amplitude of the lateral displacement, as can be seen from Fig. Y.Visualization of the homogenized quantities entclass1pt{minimaentclass1pt{minimaument}D^  transferLagrangian/Eulerian framework, an upscaled model, formulated on a fixed, homogeneous, reference domain on the macroscale, has been derived. The underlying heterogeneity is addressed via cell problems for the deformation and the diffusion process. Although the initial problem is posed with constant coefficient functions, a transformation of the diffusion problem onto the reference domain introduces a diffusion coefficient for the cell problems which depends not only on the microscopic space-variable, but also on time and on the macroscopic space-variable. Solving individual cell problems and computing effective coefficients in each time step and for each quadrature point for the effective diffusion problem are the most time consuming tasks when simulating the effective model. There is some potential for optimization by computing cell solutions in parallel or by solving fewer, representative cell problems which are chosen by an adaptive algorithm.In this work, an effective model for transport processes in elastically deformable, heterogeneous media has been derived and investigated. Starting from a microscopic description of an elasticity-diffusion problem, which contained the underlying heterogeneity of the modelled process explicitly in a mixed Investigations associated to the numerical solutions of the upscaled problem such as convergence tests and interpretation of solutions within the context of realistic experimental setups, inspired by applications, are used to verify the implemented model and give insight on the qualitative and quantitative behavior of the model. The simulations emphasize, that the deformation can significantly influence the transport processes and therefore shows the necessity to consider e.g. the effect of respiratory movement when investigating transport processes on the cellular level in the lung or related problems.Lagrangian/Eulerian framework, the effective model includes a nonlinear version of the Biot-equations for poro-elasticity. We remark that the homogenization of fluid–structure-interaction problems in porous media in Lagrangian/Lagrangian framework leads to linear Biot-equations where the effective elasticity coefficients are the same as the coefficients The present model has to be understood as a starting point for further research in different directions. As a first generalization, it is reasonable to expand the model by including also an influence of the diffusing substance on the elastic properties of the solid, leading to a fully coupled system. In the medical context, this may be especially interesting when the effect of fibrosis on the functionality of the lung has to be considered. Furthermore, until now, the effects of the perforations of the microscopic domain are only considered in the sense that they introduce the heterogeneity of the problem. From a biological perspective, the space that is not occupied by cells can be interpreted as the extracellular matrix, which, as a first approximation, can be modelled by a fluid. Starting from this, a more comprehensive model could be derived, governing also the effects of fluid–structure-interaction in porous media. Here, due to the mixed Finally, as we state in the introduction, the model is inspired by the lung and a related biomimetic microdevice that shares common features with the lung. Although the structures of these examples are based on thin layers , we chose to formulate the model for a domain that spreads in all space dimensions for the sake of simplicity.For the homogenization of a model governing an elasticity-diffusion process in a thin perforated elastic layer, an additional limit process has to be performed by letting the thickness of the layer tend to zero. There multi-scale methods for simultaneous homogenization and dimension reduction are needed. For the case of elastic perforated layers without transport such methods have been developed in Gahn et al. , see als


The MCI‐induced interfaces can effectively hinder domain continuity and modify the domain configuration. Besides, the MCI effect also results in diffused phase transitions, which is beneficial for achieving enhanced thermal stability. Modulation of chemical inhomogeneity demonstrates great potential for engineering desirable domain configuration and properties in ferroelectric ceramics. Additionally, the MCI can be easily controlled by regulating the processing condition during solid‐state synthesis, which is advantageous to industrial production.Domain engineering in ferroelectrics endows flexibility for different functional applications. Whereas the domain engineering strategy for single crystals and thin films is diverse, there is only a limited number of strategies for bulk ceramics. Here, a domain engineering strategy for achieving a compact domain architecture with increased domain‐wall density in NbO A novel domain engineering strategy is developed for ferroelectric ceramics based on the control of mesoscopic chemical inhomogeneity. Since the strategy can be easily realized via processing control, it is highly accessible to the community. This method is expected to be applicable to various ferroelectric ceramics, offering a valuable opportunity to engineer their macroscopic properties in an unexplored way. They are not only the basis of modern civilization but also critical components of the technology advancement. Ferroelectrics are featured with electrically switchable spontaneous polarization, which forms during the phase transition from a high‐symmetry para‐phase to a low‐symmetry ferro‐phase. Owing to the energy degeneracy of multiple possible polarization orientations, ferroelectrics will spontaneously form a unique domain configuration, where domains with different polarization orientations are separated by domain walls. Since the properties of ferroelectrics depend on the domain configuration, research on domain engineering1 (e.g1/3Nb2/3)O3‐PbTiO3 (PMN‐PT) poled along [001]c directions with a multiple‐domain state shows much larger longitudinal piezoelectric constant d33 than the one poled along [111]c directions with a single‐domain state. Poling PMN‐PT with an alternating‐current electric field was found beneficial for obtaining higher piezoelectricity and optical transparency. Periodically poled LiNbO3 with tunable domain widths are useful for electro‐optic applications. Different from single crystals, domain configurations in thin films are generally governed by the depolarizing field and mechanical clamping effect. Therefore, choices of substrates and modulation of film thickness are decisive to the domain configuration of grown thin films. Over the past decade, numerous fascinating topological structures have been constructed in thin films.The purpose of domain/domain‐wall engineering in ferroelectrics varies from material to material, for which, different processing techniques are required. For example, domain configurations in single crystals are sensitive to the poling conditions. Rhombohedral relaxor ferroelectric P. Similar dependency has been observed in many other ferroelectric ceramics, e.g., O3 PbO3, and TiO3, which is highly favorable for dielectric and piezoelectric applications Compared to single crystals and thin films, the number of engineering approaches in bulk ceramics seems to be limited. The grain‐size effect has been one of the most effective strategies for bulk ceramics.7 Non‐ KNN ceramics with high Curie temperature and superior electromechanical properties are promising lead‐free piezoelectrics for the next generation. Chemical engineering has been the primary strategy to promote the electromechanical properties in KNN. Construction of phase boundaries at room temperature via chemical modification is critical to the enhancement, which is explained by the flattened free energy profile that facilitates polarization rotations among coexisting phases. Meanwhile, characteristic microstructures, including dense nanodomain, slush polar region, and local structural heterogeneity, have been frequently observed in high‐performance KNN‐based materials. It has been suggested that these characteristic domain configurations might also contribute to the enhanced electrical susceptibility, though the understanding is still at a shallow level. Further enhancement of electrical susceptibility is advantageous to the development of KNN in piezoelectric applications, for which a novel engineering technique is essential.Since a wide sintering temperature window and high relative density are the prerequisites for achieving the grain‐size effect, the grain‐size effect is not an appropriate strategy for ceramic materials that suffer from poor sinterability, e.g., potassium sodium niobate (KNN).12 KNNFigureIn the present study, we proposed using mesoscopic chemical inhomogeneity (abbreviated as MCI) to engineer the domain configuration in KNN ferroelectric ceramics with fixed chemical stoichiometry Figure1. The 22.1xTa  powders were prepared by using the solid‐state synthesis , which only occur when reacting with Nb2O5 or Ta2O5. The presence of multiple DTG peaks suggests competitive reactions among raw materials occur during synthesis. The competitive reaction can result in a strong chemical inhomogeneity in the synthesized products, depending on the choices of raw materials . Even though, the chemical inhomogeneity in synthesized products can be vanished to some extent via thermal homogenization at above 950 °C, as discussed in our previous work.KNN‐s Figure . The solxTa powders were calcined at 850 and 950 °C, to prepare chemically inhomogeneous and chemically homogeneous powders, respectively. From the XRD patterns shown in Figure xTa , variation of Nb/Ta ratios will superpose on the K/Na ratios. By analyzing the d‐spacing ratio of 200(or 002)/020 (denoted as d200/020)) reflections of primary phases  and 950 °C (abbreviated as KNN‐15Ta@950) were selected for comparison, as the discrepancy of chemical homogeneity is expected to be the most significant. To ensure consistency, the samples were prepared in the same batches and same sintering conditions . The morphology of sintered ceramics was characterized by using scanning electron microscopy (SEM), as shown in Figure  was employed. Unusual domain configuration was found in the KNN‐15Ta@850 sample Figure , where t2.3FigureThe KNN‐15Ta@850 sample was characterized by using TEM, as shown in FigureThe core‐shell interface was further analyzed by using atomic‐scale imaging, as shown in  The formation of the superlattice in the KNN system has been explained by the tilting of oxygen octahedrons, which is suggested to be associated with the variation of the K/Na ratio. The observation suggests that the microstructural modification due to the MCI effect is complicated, and the influence of such microstructure requires further investigation.We discovered a unique microstructure at another MCI region, as shown in Figure 23 The2.4xTa samples was investigated, as presented in Figure In contrast, relatively sharp phase transitions were observed in KNN‐15Ta@950. The diffuseness of phase transition in KNN‐15Ta@850 can be inferred from the γ value ≈1.5 obtained from the fitting of modified Curie‐Weiss law , tetragonal, and cubic phases. Meanwhile, the phase transitions were also investigated by using the temperature‐dependent XRD  with different lattice parameters but a coherent interface was observed, we conjecture that the core and shell are influencing each other, and they tend to relieve the mismatch strain whenever possible.Even though, frequency dispersion in the typical relaxor was not observed in KNN‐Ta@850 . On one hand, the imposed local stress may prevent the phase transition of “shell”, i.e., restricting the change of its lattice structure; while on the other hand, it may facilitate the phase transition of “core”. The phenomenon will result in an enhanced TC for “shell” and a reduced TC for “core”. Namely, the ideally two separated sharp TC in a core‐shell heterostructure will merge into one but diffused TC. Schader et al. discovered that applied compressive stress can increase the first‐order nature of ferroelectric–paraelectric phase transition of BaTiO3. The increased first‐order nature observed in KNN‐15Ta@850 is likely associated with the imposed local stress due to the formation of the interface. The proposed mechanism can reasonably explain the diffused phase transition and the missing frequency dispersion in KNN‐15Ta@850. We think that the hypothesis could be further examined by using a temperature‐dependent TEM in the future.Local stress has known to be a decisive factor in the properties of phase transition in ferroelectrics.29 Let Recently, Zheng et al. proposed using a 2‐2 type multilayers composite heterostructure with gradient chemical compositions to induce a diffused phase transition in KNN‐based ceramics to achieve enhanced thermal stability. We think the strategy is very similar to the one in this work, but the heterostructure constructed herein is a 0–3 type core–shell structure. In the strategy proposed by Zheng et al., layers of multiple compositions have to be selected and stacked together. However, our work demonstrates that diffused phase transition can be easily modulated even in one fixed chemical composition, as long as we can feasibly engineer the chemical inhomogeneity. The simplicity of our strategy would be advantageous for large‐scale manufacturing.Conventionally, to achieve diffused phase transition, one may have to purposely design the chemical compositions.30 Rec The heterostructure is not only advantageous for enhanced thermal stability but also beneficial for potential enhancement of electromechanical properties, where an excellent demonstration can be found in our recent work.Since chemical inhomogeneity is a general phenomenon in materials, we expect the MCI effect can be achieved in most of the ferroelectric ceramics, but the regulation of processing parameters might have to be further optimized for each composition independently. Modulation of the core‐shell heterostructure, e.g., the core‐shell volume ratio, density of core–shell interface, and degree of lattice mismatch, can be meaningful attempts for achieving different properties.28 The3xTa ferroelectric ceramics. The MCI‐induced interface at the core‐shell heterostructure leads to the formation of a complex domain configuration with a smaller domain size and higher domain‐wall density. Meanwhile, a significant diffused phase transition was observed in the KNN‐xTa with core‐shell heterostructure, which is beneficial for enhancing thermal stability. We propose that the complex stress field that arises at the coherent interface between core and shell with different lattice parameters can account for the formation of increased domain‐wall density and the diffused phase transition, which is the key to the domain engineering via MCI. We believe that domain engineering via MCI is highly accessible and beneficial to the community, by which desirable domain configuration and properties can be obtained in ferroelectric ceramics.In summary, modification of domain configuration via MCI was successfully validated in KNN‐40.5Na0.5)O3 (abbreviated as KNN‐xTa)  powders were prepared by the conventional solid‐state reaction method. The raw materials K2CO3 , Na2CO3 , Nb2O5 , and Ta2O5  were weighed accordingly and subjected to planetary ball milling at 300 rpm for 24 h with ethanol as a dispersant. The mixtures were dried at 120 °C and a portion of each mixture was sampled for thermogravimetric analysis (TGA). Then, the dried mixtures were divided into two batches and calcined at 850 and 950 °C for 4 h. The calcined powders were subjected to the second ball milling with identical conditions to the first one. The milled powders were again dried and compacted into pellets with 10 mm in diameter and 1.5 mm in thickness under a uniaxial pressure of 150 MPa, followed by cold isostatic pressing under 200 MPa for 2 min. Finally, these green pellets were sintered in the air at 1100 °C for 4 h. with Cu Kα radiation. SEM , EPMA , and Cs‐corrected scanning transmission electron microscope (STEM)  were utilized to characterize the chemical homogeneity and domain configuration in sintered ceramics at different scales. For SEM and EPMA measurement, the samples were mechanically polished but without being subjected to any chemical or thermal etching. For TEM measurement, the sample was mechanically grounded and subjected to ion‐beam milling. The domain configuration of sintered ceramics was also characterized by using an atomic force microscope  with the PFM module. The preparation of samples for PFM measurement was identical to that for SEM and EPMA measurement. Phase structure evolution was investigated by using temperature‐dependent Raman spectroscopy . A 632 nm laser excitation was employed in the back‐scattered measurement with a spatial resolution ≈3 µm. The spot size should be able to include the core‐shell heterostructure. The phase structure evolution was also investigated by using temperature‐dependent XRD  with Cu Kα radiation, which was equipped with a temperature controller . The diffraction pattern was collected for every 20 °C increasing from room temperature to 400 °C. The diffraction patterns were analyzed by using Line‐Profile Analysis Software (LIPRAS).TGA was performed by using a synchronous thermal analyzer  from 30 to 1200 °C, with a heating rate of 10 °C min(LIPRAS).33−1 for 30 min, and cooled down with field‐on until room temperature. Temperature‐dependent dielectric properties were measured by using an impedance analyzer , which was coupled with a temperature‐regulated furnace. The quasi‐static piezoelectric coefficient d33 was measured by using a Berlincourt piezo‐meter . For d33 measurement, three pellets of the same batch were measured for each system; the samples are aged for at least 24 h before measurement.The as‐sintered pellets were ground to 1 mm in thickness, coated with silver paste on both surfaces, and then fired at 600 °C for 30 min to form electrodes. Ceramic samples were poled at 120 °C in a silicone oil bath with an applied electric field of 4 kV mmThe authors declare no conflict of interest.Supporting informationClick here for additional data file.


Setaria italic) flour to improve the contents of protein and nutrients and increase its commercial importance. FTM flour was mixed with wheat flour (Triticum aestivum) at a ratio of 0:100, 30:60, 40:50, and 50:40, and the samples were named as control, FTM30, FTM40, and FTM50 noodles, respectively. Mushroom (Pleurotus ostreatus) and rice bran (Oryza sativa L.) flour were added at a percentage of 5% to all the composite noodles . The contents of biochemicals, minerals, and amino acids, as well as the organoleptic properties of the noodles, were examined and compared with wheat flour as a control. The results revealed that the carbohydrate (CHO) content of FTM50 noodles was significantly lower (p < 0.05) than all the developed and five commercial noodles named A-1, A-2, A-3, A-4, and A-5. Moreover, the FTM noodles had significantly higher levels of protein, fiber, ash, calcium, and phosphorous than the control and commercial noodles. The percentage of lysine calculated protein efficiency ratio (PER), essential amino acid index (EAAI), biological value (BV), and chemical score (CS) of FTM50 noodles were also higher than that of the commercial noodles. The total bacterial count was nil for the FTM50 noodles, and the organoleptic properties were consistent with those of acceptable standards. The results could encourage the application of FTM flours for the development of variety and value-added noodles with enhanced level of nutrients.Noodles are a popular snack mainly produced from wheat flour; however, the low contents of protein, minerals, and lysine are a concern. Therefore, this research developed nutri-rich instant noodles by using foxtail millet (FTM) ( Triticum aestivum) is the second major food in Bangladesh and is extensively used in bakery and confectionery products. However, the local climatic conditions in Bangladesh are not very conducive for wheat farming. In Bangladesh, the consumption rate of wheat is 7.1 million metric tons (mmt) per year, whereas production is only 1.15 mmt per year [Noodles are a convenient meal because of their ease of preparation, low cost, and relatively long shelf life. Changing food habits, increasing population, and urbanization have led to increasing consumption of noodles worldwide. It is mainly produced from wheat flour, which contains 10–12% protein. Wheat  is a drought-tolerant and salt-tolerant crop with little use of herbicides for crop growth. Though Bangladesh FTM production fluctuated substantially in recent years, it tended to increase through 1972–2021 period, ending at 9616.28 tonnes in 2021 [Foxtail millet (FTM) ( in 2021 . It actsPleurotus ostreatus) are simple plant leaves found throughout the world and are a source of nutrition and medicine. It is deficient in chlorophyll, fat, and calories but rich in proteins, vitamins, minerals, folic acids, and dietary fibers. Mushrooms are cooked with other foods to increase their nutritive value, or they are used as a strengthening agent with other herbs [(Oryza sativa L.) is the outer layer of the rice kernel, a byproduct of the milling system, and is a rich source of antioxidants that reduce blood cholesterol and decrease the incidence of atherosclerosis. Rice bran is a rich source of proteins, fiber, vitamins (vitamins B1 and B2), minerals , and bioactive compounds  [Mushrooms (er herbs . Furthertrienol) . Many reBangladesh is the most densely populated country in the world, and approximately 25% of people are living in deep poverty and food insecurity. Bangladeshis follow an imbalanced diet that is dominated by cereals. Rising sea levels, loss of arable lands, frequent flooding, extreme weather patterns, and the COVID-19 pandemic pose threats to food security. Therefore, children and women in Bangladesh have extreme malnutrition and micronutrient deficiencies. However, people are becoming health conscious, and several researchers are focusing on the diversified use of multigrain flour to improve the nutritional quality of daily foods. In Bangladesh, composite flour-based noodles are unavailable to maintain a balanced diet. Consequently, this study developed highly nutritive instant noodles from FTM-based composite flour  to improve the condition of malnutrition and reduce the consumption of wheat flour.FTM was collected from Plant Breeding Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur, Bangladesh. Wheat was collected from Bangladesh Wheat Research Institute, Dinajpur, Bangladesh; mushroom was collected from National Mushroom Development and Extension Center, Savar, Bangladesh; and rice bran was collected from Bangladesh Agricultural Development Corporation, Sylhet, Bangladesh. The best-quality local commercial noodles were collected from supermarkets in Dhaka city to compare their nutritional quality with that of the prepared noodles.Whole wheat flour was mixed with FTM flour, mushroom, and rice bran flour at a ratio of 100:0:0:0, 60:30:5:5, 50:40:5:5, and 40:50:5:5 to obtain four types of flour, namely control, FTM30, FTM40, and FTM50, respectively.Noodles were prepared as described by Collins and Pangloli (1997). The ingredients for each composite flour were weighed accurately and mixed properly for dough preparation. The dough was rounded, covered with wrapping paper for fermentation at an ambient temperature, and allowed to stand for 30 min. Subsequently, it was kneaded by hand for 1 min and sheeted using a pasta machine . Then, the sheets were placed on a hand-operated pasta machine to obtain 3 mm noodle threads. The threads were transferred on a net for steaming at a temperature of 100 °C for 3 min. The steamed noodles were dried in a food-grade dryer  at 70 °C for 6–8 h. The dried noodles were cooled at room temperature, packed in an airtight plastic bag, and stored at 22–25 °C for further analysis.The contents of moisture, crude protein, fat, ash, carbohydrate, and crude fiber in the prepared flours were determined using the Approved Methods of Official Analytical Chemists . The totThe carbohydrate content (%) was evaluated as follows, according to Equation (2):Mineral contents were determined using the Official Method of the American Association of Cereal Chemists (AACC) . SamplesA microbiological examination of the noodles was performed to assess bacterial, fungal, yeast, and mold counts under laboratory conditions. Standard Plate Count (SPC), fungal, yeast, and mold counts, and total counts of coliform bacteria and Salmonella spp. in the noodles were determined using the method of the American Public Health Association . The plaOrganoleptic evaluation of the prepared noodles was performed using the method of Chauhan et al. . A 7-poiThe protein quality of FTM noodles was determined on the basis of the amino acid profiles that were determined using the Technicon Sequential Multi-Sample Amino Acid Analyzer . The samples were hydrolyzed for 22 h at 105 ± 5 °C with 6 M HCL as per the method of Jamroz et al. . Amino aThe calculated protein efficiency ratio (C-PER) was analyzed using Equation (4) developed by Hidvégi and Békés .(4)C−PEThe predicted protein efficiency ratio (P-PER) values of the different accessions were calculated from their amino acid composition by using the regression Equation (5) developed by Hidvégi and Békés .(5)P−PEThe Essential Amino Acid Index (EAAI) was calculated according to the method of Zinina et al.  and by ur et al.  and by up values of <0.05 were considered statistically significant.Data were analyzed using a one-way analysis of variance, and the Tukey honestly significant difference test was applied for the comparison of each group. Statistical analyses were performed using SPSS . Data are presented as mean ± standard deviation. The p < 0.05) were observed between the prepared and control noodles. FTM50 noodles had a significantly higher (p < 0.05) moisture content (6.61%) than the control noodles (5.11%). Moisture is the most crucial element affecting food preservation and the shelf life of grain ingredients. Moreover, FTM50 noodles had the highest ash content (2.61%); the presence of more inorganic nutrients in the noodles may be responsible for the dark color [p < 0.05) fat content (17.85%) than the control (14.32%), FTM30 (16.51%), and FTM40 (16.23%) noodles. Although the same quantity of fat was used in the preparation of the noodles, a variation was observed due to the use of different proportions of wheat and FTM flour. The fiber content was enhanced by using FTM flour instead of regular flour, with the content varying from 2.42 to 5.37%. A higher percentage of fiber was observed in the FTM50 noodles (5.37%) compared with the control noodles (2.42%). The noodles prepared entirely from wheat flour had the lowest percentage of protein (10.75%), whereas FTM50 noodles had a considerably higher (p < 0.05) percentage of protein (14.78%) because of the use of a higher percentage of FTM. The FTM50 noodles are an excellent source of dietary protein and may fulfill the daily protein requirements for humans. Moreover, the FTM50 noodles contained significantly lower (p < 0.05) percentages of carbohydrates (57.74%) than the control noodles (67.07%). Carbohydrates are organic compounds that serve as a source of energy. The low carbohydrate content in the FTM noodles has several health benefits, such as improvement of digestion in the colon and reduction in constipation from refined grain flours [The biochemical properties of the prepared noodles are listed in rk color . The fatn flours .p < 0.05) than those of the control (3.67), FTM30 (4.33), and FTM40 noodles (4.67), respectively. The incorporation of FTM and other components (5% mushroom and 5% rice bran flour) enhanced the flavor score of the FTM50 noodles (6.33) compared with the control noodles (4.33). The texture of noodles was also affected because of the increasing level of FTM flour substitution. The texture value was significantly higher (p < 0.05) in the control noodles (5.67) than in the FTM noodles (ranging from 5.00 to 4.33). Because FTM is a hardy crop, the preparation of dough was challenging. Moreover, strands of noodles tend to break due to the hardness if more than 50% of FTM flour is used [p < 0.05) in overall acceptability was observed among the FTM30, FTM40, FTM50, and control (100% wheat flour) noodles. Therefore, the acquired organoleptic scores confirmed the improvement of FTM noodles in terms of flavor and taste. The improved flavor and taste could be related to the dough fermentation. The results indicate that various forms of noodles and baked products can be produced using up to 50% of FTM flour. This may enrich the nutrition of a product and reduce the consumption of wheat flour. These results are consistent with those of Chillo et al. [The organoleptic score of the FTM noodles is displayed in  is used . No signo et al. .The nutritional quality of proteins depends primarily on the capacity of nitrogen and its essential amino acids. For the maintenance of excellent health and regular functioning of the human body, 20 distinct amino acids are required . Of thessalmonella and bacillus per gram of the FTM noodles were nil/g. The low counts of an organism in prepared foods indicate the good quality of the thermal process, raw materials, and processing conditions under which the foods are produced [The counts of microorganisms in the FTM noodles during the 6-month storage period are listed in produced .Nutrient intake at an adequate level is required for a healthy lifestyle. The percentage of nutrients per 100 g of the FTM50 noodles could fulfill the Recommended Dietary Allowance (RDA) . In termp < 0.05) amount of protein (14.78%) than all commercial noodles (ranging from 4.42 to 9.50%). The use of FTM-based composite flour improved the quality of FTM instant noodles; therefore, it can be used to alleviate protein and energy malnutrition in children, thereby improving the growth, repair, and maintenance of their bodies. Proteins act as enzymes and hormones to maintain fluid, electrolyte acid–base balance, and a strong immune system [p < 0.05) amount of carbohydrates (57.74%) compared with all commercial noodles (ranging from 66.74 to 77.98%). Carbohydrates are organic compounds that are the fundamental source of energy. However, it may cause negative health effects, such as obesity, hypertension, and diabetes. Therefore, the total carbohydrate content in the human diet must be maintained within appropriate limits. The FTM noodles also contained a significantly higher (p < 0.05) amount of energy  than the commercial noodles (ranging from 422.56 to 460.94) kcal/100 g). The amount of calcium and phosphorus (23.40 and 536.75 mg/100 g) in the FTM50 noodles was significantly higher (p < 0.05) than in all the commercial noodles. Calcium is the principal mineral contributing to strong bones and is important for muscle contraction, nerve transmission, and blood clotting [According to the proximate analysis, sensory evaluation, microbiological and amino acid evaluation of the prepared noodles, the FTM50 noodles fulfilled the standard quality features. Consequently, the FTM50 noodles were compared with commercial noodles to determine their overall nutritional quality . The FTMe system . The humclotting . The FTMp < 0.05) than those of A-2 (3.21 mg/100 g), A-3 (1.86 mg/100 g), and A-5 (0.76 mg/100 g) commercial noodles but was significantly lower (p < 0.05) than those of A-1 (7.75 mg/100 g) and A-4 (8.56 mg/100 g) commercial noodles. Iron is an essential micronutrient for the synthesis of hemoglobin (an oxygen carrier in the red blood cells), myoglobin (used in muscle contraction), and enzymes and coenzymes [Phosphorous enhances the body’s immune system, thereby reducing the risk of infections and fostering the proper functioning of the organs . The FTMoenzymes . TherefoNoodles are a popular snack among all age groups; however, those available in the market are nutritionally poor. In the present investigation, attempts have been made to develop nutrient-rich instant noodles by the addition of FTM, wheat, mushroom, and rice bran flour. Among all the formulation tried, noodle sample prepared from 50:40 flour combination (FTM50) showed a promising source of protein, fiber, and minerals. The CS, BV, C-PER, EAAI, and sensory scores of the FTM50 noodles revealed their high nutritional quality compared to that of the commercial noodles. Furthermore, the total bacterial count was nil for the FTM50 noodles, and the organoleptic properties were consistent with those of acceptable standards. Therefore, the FTM50 noodles can be used as an alternative source of protein to alleviate protein and energy malnutrition. Moreover, FTM can be a potential ingredient in household food preparation  and will be preferable for working mothers to prepare food in a short time for tiffin or in-office meetings.


Soy flour dispersions are used as adhesives for bonding interior wood laminates, but the high viscosity of these dispersions requires low solids in the adhesive formulations; the greater water content causes excessive steam pressure during hot press manufacturing. This limits the utility of soy adhesives in replacing urea–formaldehyde adhesives; thus, understanding the cause of high soy viscosities is important. Lack of literature on aqueous soy flour dispersion rheology led to our dynamic rheology studies of these dispersions to understand high viscosity and the effect of various additives. Even at low soy solids, the elastic nature outweighs the viscous properties at low shear, although increasing the shear results in shear-thinning behavior after the yield point. At even higher shear, beyond the flow point where the storage and loss moduli cross, some of the dispersions show an additional shear thinning transition. The comparison of the rheological properties of aqueous dispersions of the soy flour and protein isolate, and another natural protein, ovalbumin from egg whites, led to a better understanding of different types of rheological behaviors. The experimental observations of two observed shear thinning events for soy are consistent with the model of dispersed particles, forming clusters that then form large scale flocculants. The large size of the industry for bonded wood products has induced much research on bio-based adhesives ,2,3,4,5;Protein backbone sequences are difficult to relate to their physical and chemical properties because of the multitude of hydrophobic and hydrophilic domains dictated by the backbone sequences made from varying amounts and order of the 20 monomeric amino acids ,7. Of inThe literature is not particularly helpful for several reasons, in that the dynamic rheology of only purified and modified soy protein isolates (SPI) have been studied, and then mainly in relation to gelation for food, fiber, and film applications ,15,16,17After developing appropriate rheological analysis methods, the research focused on studying the effect of soy flour concentration, added defoamer, SMBS and cross-linker polyamidoamine epichlorohydrin (PAE) resin, and the differences between native flour, native SPI and commercial SPI. Additional information has been provided by examining the rheology of well-studied systems such as ovalbumin from egg whites and commercial synthetic wood adhesives. This research led to selecting a model for rationalizing the rheology aspects of the soy proteins and that fits with other data on soy. However, other ways of investigating the secondary and higher structures and protein surface properties are needed to reach more definitive conclusions.Soy proteins from various sources were used in this study. Commercial soy flours, Prolia™, were obtained from Cargill  in three varieties: 90 PDI (protein dispersion index ), 70 PDIMixtures of soy flour were prepared by adding sufficient solid material to water to produce a 150 g mixture with the desired wt. % of soy solids, ranging from 10–30%. About 0.1 g of defoamer was added to the water first. Mixing was carried out using a stand mixer with a 2″ disperser blade stirring at 500 rpm while material was being added, followed by a further one hour mixing at 750 rpm. In cases where sodium metabisulfite was used as an additive, a fixed amount of 0.12 g for all mixtures was added along with the flour. In cases where PAE was used as an additive, the volume was calculated so that the solid mass of the PAE matched the set fraction of the solid mass of the soy flour, ranging from 1–10%. The PAE was added to the mixture before the flour and, after mixing, small volumes of 1.5 M NaOH were used to adjust the pH to approximately 6.2, the pH of soy flour adhesives without PAE. For mixtures of soy protein isolate, the total mass was reduced to 75 g in order to save material due to the effort required to obtain this material.For the insoluble fraction from the purification of the native SPI , the mat® were placed across the top of the cup to cover it as much as possible without contacting the stem of the bob. After the temperature stabilized, the instrument carried out a 25-point oscillatory amplitude sweep by ramping the shear strain from 0.01% to 1000% logarithmically at a constant frequency of 1 Hz.After mixing, a sample of 25 mL of the mixture was loaded into the rheometer . The measuring configuration used was a Cylindrical Peltier Thermal Device (C-PTD200) and 27 mm diameter concentric cylinder. After inserting the bob, the sample temperature was equilibrated at 26.7 °C for five minutes. To minimize evaporation at the surface, strips of Parafilm MOur research, spanning numerous years, has shown that consistent viscosity values for soy adhesives are hard to obtain on a traditional laboratory rotational viscometer without a detailed procedure to control prior shear and heat history and a specified time in the viscometer under shear. This is because similar to most protein dispersions, soy proteins are shear thinning, both in respect to time and shear rate. In contrast, dynamic rheology allows for studies under varying shear conditions, but only the measurements in the linear viscoelastic (LVE) region, where the material behaves in strictly in an elastic manner, can be evaluated by standard rheological theory. The oscillatory LVE is determined by starting at very low shear strains with the shear amplitude being gradually increased past the yield point (YP) at the end of the LVE. Outside this LVE region, additional information is found, such as the flow point (FP) where the storage modulus is equal to the loss modulus, which indicates that the material behavior is changing from solid-like to liquid-like properties.A dynamic shear rheometer in the oscillatory mode can determine both the storage modulus (G′), which is the in-phase strain response to the applied stress, and the loss modulus (G″), which is the out-of-phase strain response to the applied stress. For a solid-like viscoelastic material, the elastic component  is greater than the viscous component  at very low shear strains. An example of the viscoelastic behavior of elastic materials is shown in The use of the common parallel plate method did not work well for our protein because of the wide range of viscosities, shear thinning caused exuding of the samples in some cases, and the cone-and-plate configuration was not viable due to particle sizes being too large. Thus, the cup-and-bob method was used for this soy rheology work.The first step was to make sure that rheological measurements started out in the LVE where standard rheology theories apply. This is performed by starting at very low shear strain in the oscillatory mode, and then increasing the amplitude of the oscillation until the final set point is reached. This is important because the soy flour has a linear behavior up to the yield point when shear thinning begins. The shear thinning is important for better mixing and flow through the applicator of “high viscosity” soy dispersions.This research focused on mainly the flour of 90 protein dispersibility index (PDI) since this flour is considered the native structure due to limited heating during processing. Of course, the soy adhesives are more than just soy flour in water; thus, the effect of additives also needed to be investigated. A defoamer is used to minimize the air from both the solid soy flour and the wetting out process from being incorporated into the dispersion. The dispersion density is lowered with an increasing amount of air incorporation, and prior Brookfield viscosity work showed an undesired increase in viscosity as the amount of air incorporated increased. Since defoamers decrease surface tension, there is the potential for defoamer to influence the measured soy rheology. In In addition to the G′ and G″, the rheological data allow the calculation of the complex viscosity. As shown in Having established the procedure for preparing the samples for dynamic rheology, we determined the effect of the soy flour concentration of the commonly used 90 PDI soy flour on the rheological curves. Under the experimental conditions, the values of the LVE region, the YP, and FP can be determined. Due to the magnitude values, the data are plotted on log–log scales with amplitude change being plotted as the more intuitively meaningful shear stress value, rather than the applied amplitude change.As expected, Proteins receive the most attention as soy flour adhesives because they are the main adhesive component for dry and wet strength, but they are only about half of the flour composition. The other half consists of equal portions of soluble and insoluble carbohydrates. These are isolated in the protein purification process in which the proteins are separated from the insolubles (higher molecular weight carbohydrates and insoluble proteins) by centrifugation of an aqueous 90 PDI dispersion at a pH of about 8, and then collected as solids after lowering the pH to about 4.5 to separate it from the solubles . The solubles should not influence significantly the rheology since they are mainly low molecular weight sugars, such as sucrose, raffinose, and stachyose, and low molecular weight proteins . On the Although the native 90 PDI flour can be used for making wood adhesives, there are two other common soy flours, the denatured 20 PDI and 70 PDI. As discussed previously, there are multiple drawbacks with using soy flour in adhesive applications. In addition to the wet strength of the bond not being as high as required ,27,28, tAnother route to lower aggregate size is to break the disulfide bonds by adding thiols or sodium metabisulfite (SMBS) ,31, withThe effect of the SMBS addition becomes more important at lower soy flour solids as shown in Currently, most commercial soy adhesives for wood bonding use polyamidoamine-epichlorohydrin (PAE) resin to cross-link the soy particles ,32,33. TBecause the flour and soy protein isolate are mixtures of very different protein structures ,11,34, oFor understanding soy adhesive rheological behavior compared to some typical adhesives, the amplitude shear stress curves are shown in Soy proteins behave very differently than typical wood glues and this needs to be considered in applications, because a simple rotational viscosity measurement initially shows that the proteins have much higher viscosity than the phenolic, amino, polyurethane, or polyvinyl acetate adhesives. However, the mixing and application to wood involve high shear rates, which causes the protein adhesives to flow readily due to shear thinning, unlike other wood adhesives. This naturally leads to the question as to why the soy flours and their components are shear thinning. To understand this issue, it is valuable to understand what the oscillatory shear experiments are actually measuring. The predominant phase is water, but there must be high association between molecules in the soy flour to produce an elastic response. Newtonian behavior is true of most polymer dispersions, including most wood adhesives, where the dispersed particles interact more with the aqueous phase rather than each other. Deviation from this pattern indicates some other phenomena exists, such as deformation of the particle shape from spherical to elliptical to allow less resistance to the oscillating water flow past the dispersed particle. However, with protein dispersions, the particles have significant interaction with each other ,12,35; tProteins are complex structures with hydrophobic, non-ionic hydrophilic, cationic, and anionic groups. At the isoelectric point (pI), the cationic and anionic charges are balanced, and the protein particles have high hydrophobic interaction with each other and often precipitate. For soy dispersions, most of the proteins have a pI around 4.5; at this point, these proteins precipitate and the viscosity is minimal. Away from the pI, inter-particle electrostatic force becomes important in relation to the hydrophobic forces, causing the protein particle to disperse by better interaction with the aqueous phase. However, there is still a strong hydrophobic interaction between soy proteins . The soyOnly when the pH is above 10 are the repulsive forces great enough to overcome the hydrophobic attraction. This knowledge led to development of protein adhesives in the early 20th century, when proteins were dispersed at high pH using monovalent bases that were then displaced by polyvalent cations as cross-linkers during the bonding process  which alIn the case of ovalbumin, the electrostatic repulsion keeps the hydrophobic domains buried and there is little interaction between individual proteins . Thus, tHowever, soy proteins are known to form aggregates, such as glycinin and conglycinin, under dilute conditions ,11,39. OHowever, at this point, there is not a clear explanation in the literature of why soy flour has two shear thinning transitions. One possibility is that there are larger flocs of the clusters that are more easily broken, and the second transition is breaking up of the clusters . The larThere is abundant literature of the soy protein isolate rheological gelling properties, especially the denatured jet-cooked version because of its importance to food products ,16,41,42The effect of adding defoamer to limit the air incorporation, which reduces dispersion density and viscosity increase, is minimal. However, adding sodium metabisulfite to cleave the disulfide linkages between protein chains had a much larger reduction in the storage and loss moduli although it did not eliminate the two transitions. In contrast, the rheology of ovalbumin from egg whites, with its surface hydrophilic groups dominating its hydrophobic tendencies, is similar to that observed for typical synthetic polymeric adhesive dispersions, which are visco-elastic fluids. In addition, high levels of ovalbumin become thicker, showing that steric crowding is not the reason that soy dispersions are shear thinning. While typical organic polymers behave similar to cooked spaghetti, forming entangled structures, the soy proteins are globules with their strong protein–protein interaction similar to sticky rice and the ovalbumin with its low protein–protein interaction behaves similar to non-sticky rice. The rheological properties of soy flour dispersions can be explained by formation of pre-gel clusters and flocs, with the loosely bound flocs yielding first, followed by breaking up of the clusters at higher shear forces .


The essential role of drunkenness on the healthy development of Chinese Baijiu was studied in this research. This study revealed the effects of Baijiu on the behaviors of mice and evaluated the degree of drunkenness of soy sauce-, strong-, light-, and light and soy sauce-flavored Baijiu. The parameters obtained from the open field test were transformed into the behavioral drunkenness index by mathematical statistical analysis and the drunkenness-associated key compounds of Baijiu were analyzed. The results showed that strong- and light-flavored Baijiu presented higher levels of drunkenness and sobriety than soy sauce-flavored Baijiu. Interestingly, light and soy sauce-flavored Baijiu showed low drunkenness but a high sobriety degree. Specifically, the degree of drunkenness was positively correlated with fusel alcohol and aldehydes but negatively correlated with esters and acids. This study will enrich references for Baijiu behavior studies and lay a foundation for the research and development of healthy Baijiu. There are key drunkenness compounds in Baijiu that can induce or reduce the degree of drunkenness, which lead to the differences in behavioral characteristics of mice after drinking Baijiu.  As a characteristic of traditional Chinese liquor, Baijiu has become an important product in daily life. Since 2016, the Baijiu industry has shown a trend of “volume reduction and efficiency increase” and has made a significant contribution to the economy . The chaPrevious studies have shown that animal models and behavioral tests are the classical methods used to study and evaluate the drunkenness of Baijiu. For example, Wu et al. established a linear regression model between comprehensive drunkenness and the behavior abilities of mice to evaluate the drunkenness of Baijiu . Xie et Our previous study found that the flavor characteristics and quality of Baijiu were systematically correlated with the kinds and contents of compounds by exploring the flavor structure of Baijiu . In thisA total of 36 Baijiu samples produced in 2019 were purchased from 10 famous companies, including 9 samples of SSB ; 9 samples of SB ; 9 samples of LB ; and 9 samples of LSSB . Based on the sensory flavor, characteristic volatile compounds structure, and price range of Baijiu  to obtain 12 representative samples  were acquired from Wuhan Servicebio Technology Co., Ltd., China [Permission no. SYXK (Hubei) 2018-0101]. All mice were provided with a 12 h light/dark cycle (08:00–20:00) under specific pathogen-free conditions of controlled temperature (20–25°C), relative humidity (50–55%), and noise < 60 dB. Standard basic feed  and sterile water were used for adaptive feeding for a week. The body weight and food intake of the mice were recorded once a day at 09:00 a.m., and all mice were fasted for 12 h before gavage. All animal procedures were strictly performed according to the legislation for the care and use of laboratory animals of China and ratified by the Ethical Committee for Animal Experimentation of Guizhou University. The animal experiments were conducted in Human Provincial Laboratory Animal Center, Human Center for Safety Evaluation and Research of Drugs, Changsha, Hunan, People’s Republic of China.n = 10 per group), including 3 control groups: CN (no treatment), CW (water), and CEt , and 4 Baijiu groups: SSB-M (Soy sauce-flavored Baijiu-Low), SB-M (Strong –flavored Baijiu-Middle), LB-M (Light-flavored Baijiu-Middle), and LSSB-M (Light and soy sauce-flavored Baijiu-Middle). The CW, CEt, and Baijiu groups were given the same gradient dosages .According to the safe alcohol dosage for adults prescribed by the World Health Organization  and the The behavior and death situations of mice were observed by loss of righting reflex (LORR), and the rates of drunkenness and death within 5 h were calculated , 11. Drun = 12 per group), including 3 control groups: CN (no treatment), CW (water), and CEt , and 12 Baijiu groups  or after gavage , and the behavioral parameters within 5 min were recorded from the 2nd min. These parameters included total movement distance, central movement distance, central activity time, central stagnation time, and climbing wall standing time. After the behavioral test at each time point, the blood of 25% of the mice was immediately collected through eyeball extirpation. After the blood was incubated at 4 °C for 2 h and centrifuged at 3,000 r/min for 15 min, the upper serum was taken and stored at –80°C for further analysis.The behavioral drunkenness index (BI) of mice was calculated according to the methods described in the literature and adjusted appropriately , 8, 11.CBts and CBtn represent the comprehensive behavioral capacity parameters of mice in the gavage groups  and the no treatment group (CN), respectively.EC represents the exercise capacity of mice .CC represents the cognitive capacity of mice (arithmetic mean of the anxiety parameter ratio (RA) and the exploration parameter ratio (RE) before and after gavage). Among them, the anxiety parameter is the central average velocity, and the exploration parameter is equal to the arithmetic mean of the central stagnation time and the climbing wall standing time.P < 0.05) were evaluated with one-way analysis of variance and multiple comparisons of least-significant differences by SPSS Statistics 26. The classification prediction model was established by OPLS-DA using SIMCA 14.1. The variable importance for projection (VIP) was calculated according to the model, and the key variables were selected with VIP > 1 and P < 0.05 , and the climbing wall standing time of all mice decreased (22.20 ± 0.83 s to 8.20 ± 1.53 s). A total of 58% of the mice died at the 0.3 mL/20 g⋅bw dose, and the mice that did not die lost the capacity to climb the wall and stand after 1.5 h. Generally, the 0.2 mL/20 g⋅bw dose resulted in a high drunkenness rate (67%) without death, which could effectively reflect the drunkenness effect of mice with different flavors and quality levels of Baijiu. Thus, 0.2 mL/20 g⋅bw was determined to be the optimal dosage for subsequent experiments.As shown in P < 0.05, At a 0.2 mL/20 g⋅bw dose, the climbing wall standing time was used to select the time points with significant behavioral differences in mice . The resP < 0.01, the lowest was 4881.86 ± 715.85 cm, the highest was 14407.39 ± 1025.18 cm), and the movement locus was chaotic and varied. Therefore, it could be indicated that the impacts of different flavors Baijiu on mouse behavior were mainly experienced at 0.5–2 h. The average total distance at 5 h was low (6302.64 ± 768.01 cm), but it showed the characteristics of uniform locus and wall-sticking activity, which was consistent with the observation of normal rats in the open field by Whishaw  higher than those of the other Baijiu groups, explaining why SB had the strongest inhibitory effect on the exercise capacity of mice among the 4 flavors of Baijiu. In particular, the total distance of the LB-L group (13830.73 ± 631.434 cm) was close to that of the CEt group (14266.72 ± 1144.90 cm) at 0.5 h, which initially indicated that LB-L had a high similarity with CEt. Interestingly, the characteristics of LSSB were similar to those of SSB; LSSB-L showed a large circular movement at 0.5 h , and the inhibitory effect of the SB group was stronger than those of the SSB, LB, and LSSB groups. In terms of the inhibitory effect on the exercise capacity of mice, low-quality Baijiu and CEt showed higher levels than high-quality Baijiu, which may be related to the key compounds that could reduce the effects of alcohol in the complex components of high-quality Baijiu.Briefly, 4 representative flavors Baijiu had significant differences in the exercise capacity of mice from 0.5 to 2 h  and exploration behavior  of mice during the drunken period (0.5–5 h) are shown in The results showed that the central movement distance and activity time of CEt  were significantly higher than CW  and CN  increased, and the mice tended to move in circles in the central region  higher times than the CEt group (1.17 ± 0.33 s), while the Baijiu groups were between the CEt and normal groups . Research has shown that exposure to external stimuli can increase anxiety-like behaviors. Stimuli have complex effects on open field behavior, with physical stimuli reducing activity and emotional stimuli increasing activity  and CN (1.62 ± 0.68 s) groups. This result suggested that Baijiu could weaken the exploration behavior of mice. With regard to the climbing wall standing time , the CW activity . These rThese results indicated that SSB, LB, and LSSB might positively promote the cognitive capacity (anxiety and exploration) of mice, while SB had the opposite inhibitory effect, which was in agreement with previous works on the cognitive capacity of mice , 20. In P < 0.01) different from the CEt, SB, and low-quality groups. The effect on exercise capacity of mice was mainly reflected after 0.5–2 h, with most of the mice returning to normal capacity at 5 h. In addition, the quality of Baijiu was negatively correlated with the injury degree of exercise ability. However, the SSB-H group was still drunk at 5 h, and the inhibition of exercise capacity of mice in the SSB-H group was higher than those in other groups, indicating that substances in SSB-H prolong the duration of drunkenness. As shown in To further reveal the difference in drunkenness time, this study tracked the dynamic changes in the main behavioral parameters of mice before (0 h) and after gavage , as shown in The anxiety of mice was represented by the changes in central movement distance and central activity time . The resP < 0.05) increased at 0.5 h (14.99 ± 8.60 s) and 2 h (15.68 ± 8.87 s), and the climbing wall standing capacity decreased at 2 and 0.5 h, respectively. These results indicated that there were significant differences in drunkenness time among the 4 representative favors of Baijiu.The exploration of mice was reflected by the central stagnation time  and the P < 0.05).The BI value was calculated according to the behavioral characteristics of the mice, and BI = 1.0 indicated a normal level in the CW and CN groups . The resAmong the SSB groups, the BI value of SSB-L showed a trend of increasing first and then decreasing with time, and the corresponding BI values were 1.43 ± 0.18 0.5 h), 1.79 ± 0.19 (2 h), and 0.97 ± 0.02 (5 h), respectively . Compare h, 1.79 The BI values of the SB groups showed a gradient downward trend over time . At 0.5 The variation tendency of the LB-L, LB-M, and LB-H groups was consistent with that of the CEt group; the BI value peaked at 0.5 h  and then gradually decreased . It is kP < 0.05). The results showed that the drunkenness of LSSB was relatively slow (mainly occurring at 2 h). The BI values of the LSSB-L, LSSB-M, and LSSB-H groups  were lower than those of the other Baijiu groups at 5 h, which fully indicated that the LSSB groups showed slow drunkenness and rapid sobriety. Compared with the previous results, LB had the characteristics of fast digestibility with fewer drunken compounds, while SSB had more decelerating drunken compounds and slow drunkenness. Interestingly, LSSB had not only the characteristic of quick sobriety of LB but also the common characteristic of complex compounds of SSB, thus enabling the slow drunkenness and fast sobriety of mice.The changes in the BI values in the LSSB groups are shown in The most obvious finding to emerge from the analysis was that the SSB groups during 2–5 h had low degrees of drunkenness and sobriety. The SB groups were drunk at 0.5 h and returned to normal at 2 h, indicating high degrees of drunkenness and sobriety. The duration of drunkenness in the LB groups was 0.5–2 h, with characteristics of high drunkenness and sobriety degree. The LSSB groups were drunk at 2 h and had gradually sobered up at 5 h, with significant characteristics of low drunkenness but a high degree of sobriety.A strong relationship between drunkenness and metabolism has been reported in the literature, exhibiting differences in the regulation of anxiety and stress . This diThe results of clustering analysis showed that the BI values of the different quality Baijiu groups were similar at 0 and 5 h, indicating that the drunkenness of mice was relatively low at 5 h . The lowCorrelation analysis revealed that the drunkenness index of 4 representative Baijiu flavors had good correlations with key drunken compounds, among which SSB, SB, and LSSB were obviously divided into 3 categories. However, there were no compounds in LB that distinguished the degree of drunkenness .The OPLS-DA model was used to further calculate the VIP value, and a total of 28 characteristic compounds were identified that contributed significantly to the distinction of drunkenness . The resBased on the above characteristic compounds, we further analyzed the correlations between these compounds and the drunkenness degrees of the 4 representative Baijiu flavors. In this study, SB was positively correlated with “fusel alcohol” and other drunkenness factors. It is known that fusel alcohol (such as 2-methyl-1-propanol) in Baijiu might stimulate and anesthetize nerves, which could significantly increase drunkenness and even dizziness , 25. TheThis study found that fusel alcohol and aldehydes were positively correlated with SSB and LSSB, which may be related to the high levels of drunkenness of SSB and LSSB at 2 h. Clinical trials have proved that fusel alcohol and aldehydes have slow metabolism and long accumulation, which could cause a state of drunkenness characterized by blushing, dizziness, and unstable standing , 25, 27.In particular, the BI values were found to be high at 5 h in the SSB, LSSB-L, and LSSB-M groups. Since ethyl lactate might promote alcohol to stimulate the cerebral cortex and produce excitement , in thisIn summary, the degrees of drunkenness of representative Chinese Baijiu were analyzed based on the behavioral characteristics of mice at different times after intake. Both SB and LB presented higher levels of drunkenness and sobriety than SSB, while LSSB showed low drunkenness but a high sobriety degree. Meanwhile, the degree of drunkenness was negatively correlated with the quality of Baijiu. The correlations between drunkenness and key compounds showed that drunkenness was significantly affected by fusel alcohol, aldehydes, esters, and acids in Baijiu. Based on these effects, the types of drunkenness could be divided into Type I , Type II (compounds-induced), and Type III (compounds-reduced). This study provided new directions for the potential optimization and theoretical research of low-drunkenness Baijiu production.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This animal study was reviewed and approved by the Ethical Committee for Animal Experimentation of Guizhou University.YH and XG conceived the idea of the study. XG analyzed most of the data and wrote the initial draft of the manuscript. YH and YC contributed to refining the idea. All authors discussed the results and revised the manuscript.


Amyloid precursor protein (APP) is a transmembrane protein that plays critical role in the pathogenesis of Alzheimer's disease (AD). It is also involved in many types of cancers. Increasing evidence has shown that the tyrosine phosphorylation site Y682 in the intracellular tail of APP is crucial for APP function. Here, we report that Vav2, a guanine nucleotide exchange factor (GEF) for Rho family GTPase, is a novel interaction partner of APP. We found that Vav2-SH2 domain was able to bind directly to the Y682-phosphorylated intracellular tail of APP through isothermal titration calorimetry and NMR titrating experiments. The crystal structure of Vav2-SH2 in complex with an APP-derived phosphopeptide was determined to understand the structural basis of this recognition specificity. The interaction of APP and Vav2 in a full-length manner was further confirmed in cells by GST pull-down, co-immunoprecipitation and immunofluorescence staining experiments. In addition, we found overexpression of Vav2 could inhibit APP degradation and markedly increase the protein levels of APP and its cleavage productions in 20E2 cells, and this function of Vav2 required a functional SH2 domain. It is best known for its role in the pathogenesis of Alzheimer’s disease (AD)3. AD is a neurodegenerative disease and is the most often found form of dementia among elderly people. Currently, there is no effective treatment for this disease4. The pathological hallmark of AD is the presence of amyloid plaques in the brain of AD patients6. APP is proteolytically cleaved by β- and γ-secretase to generate the amyloid β peptide (Aβ), which is the major component of amyloid plaques9. In addition, APP has been found to be overexpressed in various cancers, including breast cancer and can promote cancer cell migration and invasion11.Amyloid precursor protein (APP) is a type-I transmembrane protein that is crucial for neuronal development and homeostasis12. All three forms share a similar architecture with a large ectodomain, a single transmembrane domain and a short intracellular tail14. The short intracellular tail of APP is essential for APP function16. Numerous cytosolic proteins are found to bind directly to the intracellular domain of APP17. It is 47 amino acids in length and contains several functional motifs and phosphorylation sites, including Y682, whose phosphorylation level has been shown to be significantly increased in AD brains19. Most interactions of APP involve the Y682 phosphorylation site. Y682 modulates the interactions of APP with different proteins through its phosphorylation and dephosphorylation20. Some proteins, such as X1123, Fe6526, and JIP-127 interact with APP when Y682 is not phosphorylated, whereas others, such as Grb229, Shc30, only when Y682 is phosphorylated. Increasing evidences show that these interactions play key roles in the regulation of APP processing and function, and in AD35. For example, Mint and Fe65 family modulate cellular trafficking and the processing of APP and affect Aβ production37. JIP-1 facilitates APP axonal trafficking and regulates APP-dependent axonal transport of synaptic vesicles38. Notably, a recent study showed that targeting the APP-Mint2 protein–protein interaction with a peptide-based inhibitor reduced amyloid β formation, which may present an alternative strategy in the pursuit of new therapeutic approaches in AD treatment39.In human, there are three main isoforms of APP derived by alternative splicing: a 695-residue form expressed primarily in the central nervous system and 751- and 770-residue forms that are ubiquitously expressed41. Vav2 is broadly expressed in human tissues and is involved in regulating various biological processes, including cell spreading and migration, neuronal development, angiogenesis, and cancer cell motility47. Vav2 consists of multiple domains, including a calponin homology (CH) domain, an Acidic (Ac) region, a catalytic Dbl homology (DH), a pleckstrin homology (PH) domain, a zinc finger (ZF) domain, a Src homology 2 (SH2) domain and two Src homology 3 (SH3) domains48. Among all Ras superfamily GEFs, only Vav family proteins possess an SH2 domain, a common protein interaction module that specifically recognizes phosphotyrosine motif50. Through its SH2 domain, Vav2 can bind to the tyrosine-phosphorylated cytoplasmic domains of several membrane receptors and then mediate different extracellular signals to intracellular responses53.Herein, we report the identification of Vav2 as a novel interaction partner for APP. It is a guanine nucleotide exchange factor (GEF) for Rho GTPases belonging to the Vav family Our research demonstrated that Vav2 can interact with APP through its SH2 domain which binds directly to the Y682-phosphorylated APP intracellular tail. A crystal structure of Vav2-SH2 domain in complex with the APP-derived phosphopeptide APP-pY682  was determined at 2.45 Å resolution, which revealed a conserved recognition mechanism. The interaction of APP and Vav2 in a full-length manner was further confirmed by GST pull-down experiments, co-immunoprecipitation and immunofluorescence staining. Moreover, we found that overexpression of Vav2 significantly increased APP protein level and promoted Aβ40 generation in 20E2 cells, an AD cell model. We further show that Vav2 overexpression inhibited APP protein degradation. This function of Vav2 requires its SH2 domain. Together, these findings uncover a novel interaction between Vav2 and APP and a regulatory role of Vav2 in APP turnover.55. We found that the tyrosine phosphorylation site Y682 in the intracellular domain of APP, within the sequence of YENP, exactly matched this consensus motif. Therefore, we speculated that Y682-phosphorylated APP might be able to bind the SH2 domain of Vav2. To explore the possibility, we performed NMR titration and isothermal titration calorimetry (ITC) experiments using the recombinantly expressed Vav2-SH2 protein and a synthesized phosphotyrosine peptide derived from residue Y682 in APP   Fig. . As a co95) Fig. , and the1H–15N HSQC spectra56 of 15N-labeled Vav2-SH2 domain at a series of protein to peptide ratios were collected, respectively. As shown in Fig. 1H–15N HSQC spectra of Vav2-SH2 domain. In contrast, titration with the phosphopeptide APP-pY682 caused substantial chemical shift perturbations (CSPs) in the protein, indicating direct binding , a central β-sheet (βB–βD), two α-helices (αA and αB) and a small β-sheet (βD’ and βE) . GST pull-down assays were performed to test the binding of APPswY682A to Vav2-SH2 domain using HEK293 cells lysate expressing APPswY682A. HEK293 cells lysate expressing wild type APPsw was used as a positive control. As expected, the results showed that APPswY682A abolish its binding to Vav2-SH2  and then performed GST pull-down assays682 Fig. . In the 682 Fig. . The GST682 Fig. A. To invSH2 Fig. B. To tesSH2 Fig. D. Taken To investigate whether Vav2 affects APP metabolism, 20E2 cells were transfected with Vav2 plasmid or empty vector (as control). 48 h after transfection, we measured the levels of FL-APPsw as well as its cleaved products C99 and C83 by western blot. As shown in Fig. R680A). As shown in Fig. R680A, the increase of FL-APPsw and its productions was significantly diminished, compared with Vav2WT. This observation suggests that the SH2 domain is important for Vav2 to increase the levels of APPsw and its productions. In addition, to test whether the GEF activity of Vav2 is involved in the APPsw levels increased by Vav2 overexpression, we constructed a GEF activity-dead mutant of Vav2 (Vav2E205A)57. Similar to the overexpression of wild type Vav2, overexpression of Vav2E205A mutant in 20E2 cells also led to a significant increase in the levels of APPsw and its productions, suggesting that the GEF activity is not required in this process.Given that Vav2 binds to Y682-phosphorylated APP via its SH2 domain, we then analyzed whether a functional SH2 domain is required for Vav2 to upregulate the levels of APP and its productions. We designed a R680A mutant of Vav2  chase assay. 20E2 cells were transfected with Vav2 and empty control respectively and then chased with cycloheximide. In CHX chase assay, APPsw degradation rate in Vav2 transfected 20E2 cells was markedly disrupted comparing to control cells Fig. A,B. Thes59. The interaction between Vav2 and APP is mediated by the SH2 domain of Vav2 and the Y682 phosphorylation site in the intracellular domain of APP. Y682 has been shown to play a crucial role in modulating the binding and unbinding of APP to specific cytosolic proteins through its phosphorylation state.In the present work, we have identified that Vav2 is a novel APP-interacting protein. It is a ubiquitous guanine nucleotide exchange factor (GEF) for Rho family GTPases. Vav2 is reported to interact with several tyrosine-phosphorylated cell surface receptors through its SH2 domain and is involved in regulating a wide range of biological processes29. The reported binding Kd of Grb2-SH2 to Y682-phosphorylated peptide is 0.29 μM which is comparable to the Kd of Vav2-SH2 binding to APP-pY682, suggesting that Vav2-SH2 and Grb2-SH2 bind to Y682-phosphorylated APP with similar affinities. However, there are obvious differences in the recognition mechanism of the phosphopeptide by Vav2-SH2 and Grb2-SH2. In Grb2-SH2 complex, the phosphopeptide does not adopt an extended conformation as that seen in the Vav2-SH2 complex, but presents a folded “U” shaped structure. Both the residues Y + 2 and Y + 4 are found to contact directly with Grb-SH2. However, in the Vav2-SH2 complex, no interaction of these two residues to Vav2-SH2 domain was observed. It should be noted that although our biochemical and structural data using APP phosphopeptide has established that the Y682 phosphorylation site of APP can act as a docking site for Vav2-SH2 domain, it is unclear whether other regions of APP as well as the membrane environment would affect this interaction. Further study should be carried out using full-length APP protein under a proper model membrane system.Our ITC and NMR data showed that Vav2-SH2 domain can interact directly with the Y682-phosphorylated APP peptide APP-pY682. The complex structure revealed that this phosphopeptide bound to the Vav2-SH2 domain adopted a typical extended conformation. Previous study has shown that Grb2 can also bind to Y682-phosphoryled APP through its SH2 domain and the crystal structure of Grb2-SH2 in complex with Y682-phosphorylted peptide is availableY682A mutant. These results may suggest a potential role of Vav2-APP interaction in the regulation of APP level.In this study, we have also showed that Vav2 overexpression can inhibit APP degradation and thus lead to a significantly enhancement of the levels of APP and its productions in both 20E2 cells and HEK293 cells. Using the SH2 domain mutant (R680A) and GEF-dead mutant (E205A), we found that the SH2 domain but not the GEF activity is required for Vav2 to elevate APP level. Moreover, Vav2 overexpression has no effect on the level of APPsw19. The high levels of APP Y682 phosphorylation may enhance the interaction between Vav2 and APP in AD patient. The potential role of APP-Vav2 interaction in AD need to be further studied. In addition, APP is found to be overexpressed in multiple cancers, such as breast cancer62. It has been shown recently to promote cancer cell migration and invasion11. However, the underline mechanism is not clear. It is well known that Vav2 is also overexpressed in most human cancers and promotes cancer cell migration and invasion in several types of human cancer65. Therefore, the identification of the interaction between Vav2 and APP may open up a novel avenue for further research on the role of APP in cancer.Notably, the phosphorylation level of Y682 is significantly elevated in AD patient54, generating a fusion protein with an N-terminus 6 × His tag. For GST pull-down experiments, the Vav2-SH2 was subcloned into pGEX4T-1 vector. The APP expression plasmid pAPPsw and the Vav2 expression plasmid pCMV5-myc-Vav2 were constructed as previously described66. Mutants were generated by PCR mediated site-directed mutagenesis. All the constructs were verified by DNA sequencing.For NMR and ITC experiments, the DNA fragment encoding the SH2 domain of Vav2 (residues 659–771) was cloned into a pET28a (+) (Novagen) plasmid as described previouslyEscherichia coli BL21 (DE3) (Novagen) strain. Cells were grown at 37 °C up to an A600 nm of 0.8, and then were induced with 0.5 mM IPTG at 25 °C for 8 h. For the production of uniformly 15N-labeled samples, cells were grown in minimal medium using 15NH4CL (0.5 g/L) as the sole nitrogen source. 15N-NH4Cl was purchased from Cambridge Isotope Laboratories, Inc. The His-tagged proteins were purified by a chelated-nickel column followed by Thrombin protease treatment to remove the tag as previous described54. The GST and GST fusion proteins were purified using immobilized glutathione. All proteins were further purified on a Superdex75 gel-filtration column . The purity of proteins was confirmed by SDS–PAGE. Protein concentrations were estimated with absorbance spectroscopy using the molar absorption coefficient.The recombinant plasmids harboring His-tagged or GST-tagged Vav2-SH2 were transformed into The phosphotyrosine peptide APP-pY682 (QNG-pY-ENPT) corresponding to residues 679–686 of APP695 with Y682 phosphorylated and unphosphorylated peptide APP-Y682 (QNGYENPT) were synthesized by GL Biochem Ltd. (Shanghai).15N-labeled Vav2-SH2 (0.1 mM) mixed with phosphotyrosine peptide APP-pY682 (8 mM) or APP-Y682 (8 mM) which were dissolved in the same NMR buffer and the solutions were adjusted to pH 7.0 using NaOH. A series of 1H–15N HSQC spectra were recorded as the titrant gradually titrated into the protein solutions using the Bruker hsqcfpf3gpphwg pulse program. NMR experiments were carried out at 293 K on a Bruker Avance 600 MHz NMR spectrometer equipped with cryoprobes. NMR data were processed with NMRPipe67 and analyzed using Sparky3 . The assignment of Vav2-SH2 was extracted from a previous study55.The NMR sample of the apo Vav2-SH2 contained 0.1 mM of protein was dissolved in NMR buffer: 20 mM Tris/HCl with 100 mM NaCl, 3 mM DTT in 90% H2O, 10% D2O (pH 7.0). D2O was purchased from Cambridge Isotope Laboratories, Inc. For the SH2/peptide complex samples, the ITC experiments were performed at 298 K on a PEAQ-ITC titration calorimeter . Vav2-SH2 domain wild type and mutants were dialyzed extensively against the titration buffer containing 10 mM Tris/HCl, 100 mM NaCl (pH 7.4). Peptides were dissolved in the same buffer and the solutions were adjusted to pH 7.4 using NaOH. 0.75 mM peptides were titrated into Vav2-SH2 domain wild type or its mutant (30 μM). Control experiments were performed under the same condition by titrating same peptide into buffer alone. ITC data were analyzed with a single-site binding model using Microcal PEAQ-ITC software.2·6H2O and 0.1 M Tris/HCl, pH 8.4. Before flash-freezing crystals in liquid nitrogen, crystals were soaked in a cryoprotectant consisting of 85% reservoir solution and 15% glycerol.For crystallization experiments, purified Vav2-SH2 protein was concentrated to 15 mg/mL in a buffer containing 10 mM Tris/HCl, pH 7.4, 100 mM NaCl and 5 mM β-mercaptoethanol. Purified protein was mixed with pY682 peptide at a 1:1.2 molar ratio and crystallized using the sitting-drop vapour-diffusion method at 16 °C after mixing 0.5 μL of the protein solution with 0.5 μL of the reservoir solution. Crystals were obtained with a reservoir solution containing 25% PEG 3350, 0.2 M MgCl68 and programs in the CCP4 suite69. The structure of the Vav2-SH2 domain complexed with pY682 was solved by molecular replacement with co-ordinates from PDB entry 3MXC29 and the program PHASER70. All the structural models were subsequently refined by programs REFMAC571, PHENIX72, and COOT73. Crystallographic parameters are listed in Table X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beam line BL18U. The data were processed with HKL2000HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% (V/V) fetal calf serum and penicillin/streptomycin. The 20E2 cell line, a Swedish mutant APP stable HEK293 cell line was maintained in complete DMEM supplemented with 100 μg/mL Zeocin. Cells were transfected using Lipofectamine™ 3000 (Invitrogen) reagent according to the manufacturers’ protocols.74. Primary antibodies used are: anti-myc tag mAb , C20 antibody, anti-β actin mAb (Sigma-Aldrich), anti-Phospho-Tyrosine mAb . Detection was performed with the Li-Cor Odyssey imaging system and quantitated with ImageJ software.For immunoblotting analyses, 20E2 cells were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Science). Immunoblotting was performed as described previouslyR680A for 2 h at 4 °C in GST binding buffer . The beads were then washed 4 times with GST binding buffer and incubated for another 2 h with 0.2 mg indicated lysates of 20E2 cells stable expressing the indicated APPsw constructs. After washing 4 times with GST binding buffer, beads were boiled in SDS sample buffer, run on 12% SDS–PAGE gel and analyzed by Coomassie or immunoblotting.One hundred microliter glutathione-agarose beads were incubated with 1 mg purified GST or indicated GST-SH2 fusions and the mutant GST-SH2HEK293 cells were co-transfected with pAPPsw and pCMV5-myc-Vav2. For co-IP, cells were harvested after 48 h and lysed in 1 mL of 1% NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (Roche Applied Science). Cell lysates were then incubated with primary antibody and protein A/G-agarose beads  at 4 °C overnight. Mouse or rabbit IgG  was performed with protein A/G-agarose beads as negative controls. After washing 3 times with PBS, beads were boiled in SDS sample buffer, run on 8% SDS–PAGE gel and analyzed by immunoblotting with indicated antibodies.75. Primary antibodies used were C20 antibody and anti-myc tag mAb . Secondary antibodies were CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG and CoraLite594-conjugated Affinipure Goat Anti-Mouse IgG . Images were captured by LSM 880 fluorescent microscope  and analyzed with ZEN software.Immunofluorescence was performed as previously describedWT, Vav2R680A, Vav2E205A and empty control, respectively. Forty-eight hours after transfection, the level of Aβ40 in the supernatant was measured using the Aβ1–40 Elisa kit  according to the manufacturer’s protocol.20E2 cells were transfected with Vav2WT and empty control, respectively. Forty-eight hours after transfection, cells were treated with 150 μg/mL of CHX  and harvested after 0, 3, 6 and 9 h, respectively. The protein level of FL-APPsw was detected by western blotting and quantitated with ImageJ software.The 20E2 cells were transfected with Vav2t test was performed for differences between two groups. One-way or two-way ANOVA with Bonferroni’s multiple comparisons post hoc test was applied for multigroup comparisons. All analyses were performed with GraphPad Prism 9 software (GraphPad). Differences were defined to be statistically significant at p < 0.05.Data are presented as means ± SEM from three to five independent experiments. Student’s Supplementary Information.