Dual Vector Research Articles

Increase recombinant antibody yields through optimizing vector design and production process in CHO cells.

Chinese hamster ovary (CHO) cells are the most commonly used host cells for the production of recombinant monoclonal antibodies (mAbs) due to their several advantages. Although the yields of recombinant mAbs can be greatly increased by some strategies, such as medium formulation, culture conditions, and cell engineering, most studies focused on either upstream design or downstream processes. In the present study, we first expressed recombinant adalimumab through combination of the vector design and production process optimization in CHO cells. Bicistronic vector, monocistronic vector, and dual promoter vector were constructed, and the production process was optimized using low-temperature and fed-batch culture. The results showed that the dual promoter vector exhibited the highest yield under the transient and stable transfected cells among three different vector systems in CHO cells. In addition, low-temperature and fed-batch culture could further improve the yields of adalimumab. The purified antibody displayed tumor necrosis factor-α (TNF-α) binding activity. In conclusion, combination of expression vector design and production process optimization can achieve higher expression of recombinant mAbs in CHO cells. KEY POINTS: • The dual promoter vector is more effective for expressing recombinant antibodies. • The yields of antibodies are related to the LC chain expression level. • Low-temperature and feed addition can promote antibody production.

An innovative and efficient solution for axisymmetric contact problem between structure and half-space

In this paper, with displacement increment as variables, the scaled boundary finite element equations of the axisymmetric plate and half-space are respectively derived based on the principle of virtual work. The dual vector is introduced to solve the boundary stiffness matrix. Linear complementary contact conditions are formulated as B-differentiable equations. With discrete, static assumptions of small displacement, B-differentiable equations system was formed by combining the contact conditions and the stiffness matrix of the plate-foundation. The equations can be iteratively solved by B-differentiable Newton method. In numerical experiments, the contact stress, slip and contact status of each contact node pair are compared with the results of the augmented Lagrangian contact algorithm based on FEM. When adopting different load types, thickness-to-radius ratio, plate-foundation stiffness ratio and friction coefficient, the axisymmetric contact problems between plate and half-space are discretized by 120 one-dimensional first-order elements. The applicability, accuracy and effectiveness of B-SBFEM for axisymmetric contact problems are verified by numerical examples.

THE NON-DEGENERACY OF THE SKEW-SYMMETRIC BILINEAR FORM OF THE FINITE DIMENSIONAL REAL FROBENIUS LIE ALGEBRA

A Frobenius Lie algebra is recognized as the Lie algebra whose stabilizer at a Frobenius functional is trivial. This condition is equivalent to the existence of a skew-symmetric bilinear form which is non-degenerate. On the other hand, the Lie algebra is Frobenius as well if its orbit on the dual vector space is open. In this paper, we study the skew-symmetric bilinear form of finite dimensional Frobenius Lie algebra corresponding to its Frobenius functional. The work aims to prove that a Lie algebra of dimension is Frobenius if and only if the -th derivation of the Frobenius functional is not equal to zero. Indeed, this condition implies that the skew-symmetric bilinear form is non-degenerate and vice versa. In addition, some properties of Frobenius functionals are obtained. Furthermore, the computations are given using the coadjoint orbits and the structure matrix. As a discussion, we can investigate these results in the algebra case whether giving rise to a left-invariant K hler structure of a Frobenius Lie group or not.

Dual space vector PWM technique for a three-phase to five-phase quasi Z-source direct matrix converter

ABSTRACT This article proposes a dual space vector PWM control technique for a three-phase to five-phase quasi Z-source direct matrix converter (QZSDMC). The proposed circuit consists of a quasi-impedance source network along with a five-phase traditional matrix converter. With the observation of bidirectional operating capability and the utilization of shoot-through duty ratio, the proposed converter can ensure both buck and boost operations. This converter overcomes the voltage transfer ratio restriction and poor voltage regulation of the traditional matrix converter. Also, this converter ensures continuous input current with an absence of input LC filter components. The proposed direct AC to AC power converter gives an alternate solution to the multiphase drive system instead of the traditional voltage source inverter. The developed dual space vector PWM control technique for three-phase to five-phase voltage conversion is derived from the basic space vector PWM approach. The proposed dual space vector PWM technique utilizes 90 active states and 3 zero vector states among 243 switching states. The QZSDMC has been analysed with a dual space vector PWM approach along with the distribution of shoot-through state in the zero vector. The proposed topology along with the control strategy has been simulated using MATLAB/Simulink environment.

Sphingomyelin Phodiesterase Acid-Like 3A Promotes Hepatocellular Carcinoma Growth Through the Enhancer of Rudimentary Homolog.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, with unclear pathogenesis. Sphingomyelin phodiesterase acid-like 3A (SMPDL3A) affects cell differentiation and participates in immune regulation. However, its molecular biological function in HCC has not yet been elucidated.MethodsData from 180 HCC patients were analyzed the relationship between the expression of SMPDL3A in liver cancer tissues and the prognosis of liver cancer patients. Crispr-Cas9 dual vector lentivirus was used to knock out SMPDL3A in HCC cell lines. The effects of SMPDL3A on cell viability were determined by CCK8 assay, clone formation experiment, cell cycle assay, cell scratch, TUNEL experiment and flow cytometry. Xenograft tumor assays in BALB/c nude mice confirmed that SMPDL3A promoted tumor growth and in vivo. Preliminary exploration of SMPDL3A interacting protein by mass spectrometry analysis and co-immunoprecipitation.ResultsThis study showed that the expression of SMPDL3A in HCC tissue differed from that in tumor-adjacent tissues. Moreover, the overall survival rate and tumor-free survival rate of patients with high-SMPDL3A expression were significantly lower than those with low-SMPDL3A expression. SMPDL3A expression was closely related to the level of protein induced by PIVKA-II, liver cirrhosis, tumor diameter, microvascular invasion, and Barcelona clinic liver cancer staging. Thus, SMPDL3A is an independent risk factor that affects the tumor-free survival rate and overall survival rate of HCC patients. In vitro study using Crispr-Cas9 genome editing technology revealed the knockout effect of SMPDL3A on cell proliferation, apoptosis, and migration. Cell counting kit-8 assay and clone formation experiment showed that sgSMPDL3A inhibited tumor cell proliferation and migration. Flow cytometry and TUNEL assay showed that sgSMPDL3A promoted apoptosis in tumors. Moreover, sgSMPDL3A inhibited tumor growth during subcutaneous tumor formation in nude mice. Immunohistochemistry of Ki67 and PNCA also indicated that sgSMPDL3A inhibited subcutaneous tumor proliferation in tumor-bearing nude mice. Further experiments showed that SMPDL3A interacts with the enhancer of rudimentary homolog (ERH).ConclusionsHigh-SMPDL3A expression was related to poor prognosis of patients with HCC. Knockout of SMPDL3A inhibited the proliferation and migration and accelerated the migration of HCC cells. SMPDL3A interacted with ERH to affect the tumorigenesis and progression of HCC.

The Dual Expression of Parallel Equidistant Ruled Surfaces in Euclidean 3-Space

In this study, we examine the dual expression of Valeontis’ concept of parallel p-equidistant ruled surfaces well known in Euclidean 3-space, according to the Study mapping. Furthermore, we show that the dual part of the dual angle on the unit dual sphere corresponds to the p-distance. We call these ruled surfaces we obtained “dual parallel equidistant ruled surfaces” and we briefly denote them with “DPERS”. Furthermore, we find the Blaschke vectors, the Blaschke invariants and the striction curves of these DPERS and we give the relationships between these elements. Moreover, we show the relationships between the Darboux screws, the instantaneous screw axes, the instantaneous dual Pfaff vectors and dual Steiner rotation vectors of these surfaces. Finally, we give an example, which we reinforce this article, and we explain all of these features with the figures on the example. Furthermore, we see that the corresponding dual curves on the dual unit sphere to these DPERS are such that one of them is symmetric with respect to the imaginary symmetry axis of the other.

Establishment of a Novel Baculovirus-Silkworm Expression System.

The baculovirus vector expression system is a well-established tool for foreign protein production and gene delivery. In this study, we constructed a recombinant baculovirus vector system. The UAS promotor region and Bombyx mori nucleopolyhedrovirus (BmNPV) polyhedrin coding region were ligated into a pFastBac Dual vector to obtain a BmBac-UPS recombinant bacmid. The recombinant bacmid BmBac-Gal4 was generated by the same strategy which has a Gal4 coding region controlled by the IE2 promoter. BmBac-UPS and BmBac-IGal4 were co-infected into silkworm BmN cells to confirm the ability of the UAS/Gal4 system to form polyhedrons in B. mori cells. Furthermore, the recombinant viruses were tested for infection efficiency and the ability to generate polyhedra in transgenic B. mori cell line BmE. The results showed that recombinant viruses have the ability to form polyhedrons and gain raised pathogenicity when orally infected B. mori larvae and are applied as the preferred tool for foreign gene delivery and expression

Single-Cell Quantification of Triple-AAV Vector Genomes Coexpressed in Neurons.

Adeno-associated viruses (AAVs) are one of the most widely used types of viral vectors for research and gene therapy. AAV vectors are safe, have a low immunogenic profile, and provide efficient and long-term transgene expression in a variety of tissues and organs targeted by a specific serotype. Despite these unique features, therapeutic applications, as well as basic research studies, of AAVs have been limited by their packaging capacity of less than 5 kb. Multiple strategies have been explored to deliver large genes. One strategy is to split large transgenes into two or three fragments and package them into separate AAV capsids, generating dual or triple AAV vectors. Combining the fragments potentially allows reconstitution of an mRNA transcript containing the complete sequence of transgene in the same cell. The success of AAVs as vectors for the delivery of large or multiple genes depends directly on the efficiency of co-transduction. Here, we describe a method to measure the efficacy of codelivery, quantifying the number of AAV vectors per cell. We detail how to calculate the average number of incoming AAV genomes in neurons, given the distribution of cell fluorescence across in vitro and in vivo experimental models. To validate the method, we simulated a triple AAV strategy using three fluorescent-protein-encoding genes. We provide a general protocol for constructing plasmids and producing and purifying AAV vectors. We also include a protocol for triple AAV vector co-transduction in primary neuronal cultures and mouse brain. The method can be applied to multiple organs and tissues for the treatment of disorders caused by mutations in multiple or large genes. These protocols will be useful for researchers working to develop and improve new gene delivery technologies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Construction of AAV plasmids and production of AAVs Basic Protocol 2: AAV transduction of primary superior cervical ganglia (SCG) neuronal cultures Basic Protocol 3: Mouse surgery, AAV injection, and tissue collection and processing Basic Protocol 4: Image analysis and AAV genome quantification.

On characteristic forms of positive vector bundles, mixed discriminants, and pushforward identities

We prove that Schur polynomials in Chern forms of Nakano and dual Nakano positive vector bundles are positive as differential forms. Moreover, modulo a statement about the positivity of a “double mixed discriminant” of linear operators on matrices, which preserve the cone of positive definite matrices, we establish that Schur polynomials in Chern forms of Griffiths positive vector bundles are weakly positive as differential forms. This provides differential-geometric versions of Fulton–Lazarsfeld inequalities for ample vector bundles. An interpretation of positivity conditions for vector bundles through operator theory lies in the core of our approach. Another important step in our proof is to establish a certain pushforward identity for characteristic forms, refining the determinantal formula of Kempf–Laksov for holomorphic vector bundles on the level of differential forms. In the same vein, we establish a local version of Jacobi–Trudi identity. Then we study the inverse problem and show that already for vector bundles over complex surfaces, one cannot characterize Griffiths positivity (and even ampleness) through the positivity of Schur polynomials, even if one takes into consideration all quotients of a vector bundle.

Antheraea pernyi silk fibroin-coated adenovirus as a VEGF165-Ang-1 dual gene delivery vector

Vascularization is a key challenge in the regeneration of tissues containing blood vessels. In this study, spermine was used for cationic modification of Antheraea pernyi silk fibroin (ASF) to synthesize cationized ASF (CASF). CASF/Ad complexes prepared by coating adenovirus (Ad) with CASF were used as delivery vectors for vascular endothelial growth factor 165 and angiopoietin-1 dual genes. The results showed that the zeta potential of the Ad was reversed from −7.75 mV to approximately +8.40 mV after CASF coating, and the sizes of the CASF/Ad complexes were 200 to 290 nm. Furthermore, human umbilical vein endothelial cells HUVECs were cocultured and infected with CASF/Ad in vitro. The results of confocal laser scanning microscopy, flow cytometry and CCK-8 assay showed that coating Ad with CASF at concentration of 20 and 50 µg/mL not only reduced the cytotoxicity of naked Ad, but also significantly promoted cell proliferation. Therefore, the CASF/Ad complexes could be beneficial to reduce the dosage of Ad and the potential toxicity risk of high doses of Ad in vivo, which has the potential of application to promote vascular network regeneration.

HIV-1 VIF and human APOBEC3G interaction directly observed through molecular specific labeling using a new dual promotor vector

Over the last few decades, protein NMR isotope labeling methods using E. coli based expression have revolutionized the information accessible from biomolecular NMR experiments. Selective labeling of a protein of interest in a multi-protein complex can significantly reduce the number of cross-peaks and allow for study of large protein complexes. However, limitations still remain since some proteins are not stable independently and cannot be separately labeled in either NMR active isotope enriched or unenriched media and reconstituted into a multimeric complex. To overcome this limitation, the LEGO NMR method was previously developed using protein expression plasmids containing T7 or araBAD promoters to separately express proteins in the same E. coli after changing between labeled and unlabeled media.Building on this, we developed a method to label the Human Immunodeficiency Virus type 1 viral infectivity factor (HIV-1 Vif), a monomerically unstable protein, in complex with CBFβ, it’s host binding partner. We designed a dual promoter plasmid containing both T7 and araBAD promoters to independently control the expression of HIV-1 Vif in NMR active isotope enriched media and CBFβ in unenriched media. Using this method, we assigned the backbone resonance and directly observed the binding of HIV-1 Vif with APOBEC3G, a host restriction factor to HIV-1.

Dual Quaternions and Dual Quaternion Vectors

We introduce a total order and an absolute value function for dual numbers. The absolute value function of dual numbers takes dual number values, and has properties similar to those of the absolute value function of real numbers. We define the magnitude of a dual quaternion, as a dual number. Based upon these, we extend 1-norm, $$\infty$$ -norm, and 2-norm to dual quaternion vectors.

Automatic classification of atrial fibrillation from short single-lead ECG recordings using a Hybrid Approach of Dual Support Vector Machine

Millions of people worldwide are affected by cardiac arrythmias currently. One of the life threatening arrythmias associated with high morbidity and mortality is Atrial Fibrillation (AFr). Electrocardiogram (ECG) is regularly used in the diagnosis and evaluation of cardiac arrythmias. The goal of the ECG is to improve the outcome as well as reduce time to diagnosis in heart disease identification. Using an ECG makes the diagnosis of a cardiac arrythmia much easier. Cardiologists depend on the ECG signal to determine the appropriate treatment method of the cardiac arrythmia. In this work, cardiac segmentation was used to further evaluate and diagnose AFr based on ECG. The issue with segmentation is that it is complicated due to similarities in amplitude, time among different ECG signals as well as noise. In this paper, a comparative study is made on detection of Atrial Fibrillation using the top five-scoring methods submitted in the PhysioNet/Computing in Cardiology Challenge 2017. A new method based on a Hybrid Approach of Dual Support Vector Machine (HA-DSVM) is used for the detection of atrial fibrillation. This proposed method is accomplished by stochastic gradient descent with cross entropy loss function. The method is tested on the dataset collected from the 2017 Physionet/CinC challenge dataset with performance evaluation on training (99.27 %). Using this technique, an F1 score and accuracy of 0.95 and 99.27% can be obtained on the validation data set. One of the advantages of the proposed technique is the high reliability and accuracy which simplifies the extraction process and removal of detecting ECG signal fiducial points and removing hand-crafted features. This provides screening for a large population with a new method for diagnosing atrial fibrillation. The proposed method can be applied to many the population with symptoms of atrial fibrillation which will improve the accuracy of diagnosis and determine the required treatment.

CRISPR genome surgery in a novel humanized model for autosomal dominant retinitis pigmentosa

Mutations in rhodopsin (RHO) are the most common causes of autosomal dominant retinitis pigmentosa (adRP), accounting for 20% to 30% of all cases worldwide. However, the high degree of genetic heterogeneity makes development of effective therapies cumbersome. To provide a universal solution to RHO-related adRP, we devised a CRISPR-based, mutation-independent gene ablation and replacement (AR) compound therapy carried by a dual AAV2/8 system. Moreover, we developed a novel hRHOC110R/hRHOWT humanized mouse model to assess the AR treatment invivo. Results show that this humanized RHO mouse model exhibits progressive rod-cone degeneration that phenocopies hRHOC110R/hRHOWT patients. Invivo transduction of AR AAV8 dual vectors remarkably ablates endogenous RHO expression and overexpresses exogenous WT hRHO. Furthermore, the administration of AR during adulthood significantly hampers photoreceptor degeneration both histologically and functionally for at least 6months compared with sole gene replacement or surgical trauma control. This study demonstrates the effectiveness of AR treatment of adRP in the human genomic context while revealing the feasibility of its application for other autosomal dominant disorders.

DCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy.

Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the appropriate counterpart(s) to compensate for the missing gene function. This approach offers key advantages over conventional gene therapies because it is mutation- and gene size-independent. Here, we describe a protocol for the design, execution and evaluation of such gene therapies using dCas9-VPR. We offer guidelines on how to identify functionally equivalent genes, design and clone single guide RNAs and evaluate transcriptional activation in vitro. Moreover, focusing on inherited retinal diseases, we provide a detailed protocol on how to apply this strategy in mice using dual recombinant adeno-associated virus vectors and how to evaluate its functionality and off-target effects in the target tissue. This strategy is in principle applicable to all organisms that possess functionally equivalent genes suitable for transcriptional activation and addresses pivotal unmet needs in gene therapy with high translational potential. The protocol can be completed in 15-20 weeks.

MiR-128-1-5p regulates differentiation of ovine stromal vascular fraction by targeting the KLF11 5′-UTR

Fat content is an important index to evaluate the individual performance of livestock animals such as sheep for meat production purposes. Reducing the subcutaneous and visceral fat while increasing the intramuscular fat is a valuable goal to achieve for the meat production industry. Here, we investigated the effect of miR-128-1-5p on adipogenesis of subcutaneous fat by targeting 5’-UTR in KLF11, a rare mechanism where most miRNAs bind the 3’-UTR of mRNAs. A dual fluorescence reporter assay was conducted to validate the binding sites of miR-128-1-5p on 5’-UTR of KLF11 mRNA. Roles of miR-128-1-5p in KLF11 expression were measured through co-transfecting miRNA mimics with KLF11-expressing vectors (CDSs together with or without the 5′-UTR) into ovine stromal vascular fractions (SVF). Additionally, functional roles of miR-128-1-5p, and KLF11 in adipogenesis of ovine subcutaneous fat were investigated. Results showed that miR-128-1-5p targeted KLF11 5′-UTR, reduced the fluorescence activity of the dual fluorescent reporter vector, as well as KLF11 mRNA, and protein expression levels. During the differentiation of SVF, disturbing the expression of miR-128-1-5p and KLF11 changed the adipogenic differentiation of SVF as observed in the lipid formation, and adipogenic marker genes. This study indicates that miR-128-1-5p promotes the expression of lipogenic marker genes and the formation of lipid droplets by targeting KLF11 5′-UTR. Furthermore, overexpression, and inhibition of KLF11 indicate that KLF11 inhibited SVF differentiation. In summary, the 5’-UTR binding mechanism discovered in this study extends the understanding of miRNA functions. Key roles of miR-128-1-5p and KLF11 in the adipogenesis of sheep subcutaneous fat have potential values for improving the meat and/or fat ratio of domestic animals.

Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES.

Background:Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. In the original study, we tested this hypothesis, by placing the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods:Here we employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results:With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter's ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g., AMD1 tail inhibitory effect. We further show by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of RNA levels. Conclusions:While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of readthrough permissive sequences and of IRES elements, though these require a separate investigation.

Fault detection in closed-loop systems using a double residual generator

A double residual generator for fault detection in closed-loop systems is presented in this paper. The residual generator gives the standard residual vector and a dual residual vector as outputs. The standard residual vector can be applied directly for fault detection. The dual residual vector depends on known inputs to the system e.g. reference inputs and auxiliary inputs but decoupled from the disturbance in the system. It is also an input vector that can be applied for active fault detection (AFD). The dual residual vector can be considered as a generalized input vector in connection with AFD in closed-loop systems. This gives the possibility to use all known inputs for detection. The dual residual vector gives a link between passive and AFD in closed-loop systems. The double residual generator is given directly as the left Bezout matrix from the Bezout equation.

Development of a Ligation-Independent Cloning-Based Dual Vector System for RNA Interference in Plants.

RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism that responds to double-stranded RNA (dsRNA) by sequence-specific downregulation of target genes. The dsRNA-mediated RNAi technology has become one of the most widely used and powerful tools for functional genomic studies in diverse organisms. However, its application has been limited due to the technical difficulty of making RNAi constructs caused by the inverted repeat structure that is required for the formation of hairpin RNA. Here, we present a ligation-independent cloning-based dual vector-mediated RNAi system for silencing specific genes in plants. This approach is simple, efficient, and cost-effective and can be readily adapted to other binary vectors for functional analysis of target genes and the development of sustainable disease and pest control strategies in a broad range of plant species.

Transverse vibration responses of the in-plane-wise functionally graded piezoelectric composite plates

Aided by the scaled boundary finite element method (SBFEM), analytical precise integration solutions to the transverse free vibration responses of functionally graded piezoelectric composite plates are provided for the first time. Distributions of the functionally graded piezoelectric materials follow arbitrary mathematical formulae as concerns the in-plane unidirectional or bidirectional coordinates. The vibration behaviors of piezoelectric composite plates with various geometrical shapes, multifarious boundary constraints and any number of laminae can be explored by the introduced technique. Additionally, the shear correction factors and assumptions on the changing patterns of mechanical and electrical variables are not needed to be offered in the employed methodology. The proposed approach regards only four quantities containing three elastic displacement components and the electric potential as the primary unknowns, which can be denoted as an analytical matrix exponent in terms of the thickness coordinate. Only two-dimensional high-order spectral elements are adopted to discretize the in-plane surface of the plate with less nodes and degree of freedoms, which promotes to lowering the calculative expense and advancing the computational efficiency. The introduced scaled boundary coordinate system and the dual vector technology are helpful to simplify the basic partial differential equations of piezoelectric materials into the first order ordinary differential SBFEM governing equation, from which the highly accurate global stiffness matrix are constructed by dine of the precise integration technique (PIT). With the aid of the kinetic energy scheme and compatibility conditions between neighboring layers, the whole mass matrix for the laminated piezoelectric plates can be built. According to solving the eigenvalue equation, the precise integration solutions to free vibration frequencies can be acquired. Finally, numerical experiments are afforded to elucidate the high precision and fast convergence of the present method. Moreover, the effect of different boundary constraints, gradation parameters and aspect ratios on the transverse vibration responses of in-plane-wise functionally graded piezoelectric composite plates is revealed.