Dual Vector Research Articles

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Model Predictive Torque Control for Dual Three-Phase PMSMs with Simplified Deadbeat Solution and Discrete Space-Vector Modulation

Unprecise voltage vectors applied in conventional model predictive control (MPC) would cause additional ripples in electromagnetic torque. To eliminate the problem, this article proposes an improved model predictive torque control (MPTC) based on deadbeat solution and discrete space-vector modulation (DSVM) for dual three-phase permanent magnet synchronous machines (PMSMs). First, deadbeat-direct torque and flux control (DB-DTFC) algorithm is applied and simplified, so that the computational burden can be reduced. Second, the virtual voltage vectors (VVVs) are adopted in dual three-phase voltage vector space to reduce voltage harmonics. Also, the DSVM is further proposed in this VVVs based vector space to generate more voltage vectors. After that, the simplified DB-DTFC and the DSVM scheme are artfully combined to select suitable voltage vector candidates for the MPTC, and the best one is then chosen to control the dual three-phase PMSMs. Finally, both simulation and experimental results are given to verify the effectiveness of the proposed method.

A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in Nicotiana benthamiana. We engineered sonchus yellow net rhabdovirus (SYNV)-based gene silencing vectors through expressing a sense, antisense, or double-stranded RNAs of target genes. Robust target gene silencing was also achieved with an SYNV vector expressing a designed artificial microRNA. In addition, ectopic expression of a short tandem target mimic RNA using the SYNV vector led to a significant depletion of the target miR165/166 and caused abnormal leaf development. More importantly, SYNV was able to harbor two expression cassettes that permitted simultaneous RNA silencing and overexpression of large reporter gene. This dual capacity vector also enabled systemic expression of a whole-molecule monoclonal antibody consisting of light and heavy chains. These results highlight the utility of the SYNV vector system in gene function studies and agricultural biotechnology and provide a technical template for developing similar vectors of other economically important plant rhabdoviruses.

Sensorless Field-Oriented Control for Open-End Winding Five-Phase Induction Motor With Parameters Estimation

This paper proposes a sensorless field-oriented control (FOC) of an open-end stator winding five-phase induction motor (OESW-FPIM). The FOC technique used is associated with dual Space Vector Modulation (SVM) to provide a constant switching frequency and lower harmonics distortion. Furthermore, a simple hybrid observer is proposed which combines a model reference adaptive system (MRAS) and a sliding mode (SM) observer. The examined observer is designed for the estimation of the rotor flux and rotational speed as well as for the estimation of the load torque disturbances. Lyapunov theorem is used in this paper to prove the observer's stability. The work presented in this paper aims to enhance the researched motor's sensorless control and its robustness against external load disturbances and parameters variation. In the proposed MRAS-SM observer, the reference model is replaced by a SM model which uses a sigmoid function as a switching function to overcome the chattering problem. This combination is intended to make use of the advantages of both strategies. At the same time, to preserve the high-level performance of the sensorless FOC technique and to reduce system uncertainties, an estimation algorithm is developed to identify the rotor resistance and the stator resistance simultaneously during motor operation. The parameter estimation algorithm is combined with the proposed control to improve the speed estimation and control accuracy, particularly at low-speed operation. Finally, the effectiveness of the proposed control is validated in real-time by utilizing a hardware-in-the-loop (HIL) platform.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion

Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells.To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas.VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays.Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs.A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

Zona incerta neurons projecting to the ventral tegmental area promote action initiation towards feeding.

Key points The zona incerta (ZI) and ventral tegmental area (VTA) are brain areas that are both implicated in feeding behaviour. The ZI projects to the VTA, although it has not yet been investigated whether this projection regulates feeding.We experimentally (in)activated the ZI to VTA projection by using dual viral vector technology, and studied the effects on feeding microstructure, the willingness to work for food, general activity and body temperature.Activity of the ZI to VTA projection promotes feeding by facilitating action initiation towards food, as reflected in meal frequency and the willingness to work for food reward, without affecting general activity or directly modulating body temperature.We show for the first time that activity of the ZI to VTA projection promotes feeding, which improves the understanding of the neurobiology of feeding behaviour and body weight regulation. Both the zona incerta (ZI) and the ventral tegmental area (VTA) have been implicated in feeding behaviour. The ZI provides prominent input to the VTA, although it has not yet been investigated whether this projection regulates feeding. Therefore, we investigated the role of ZI to VTA projection neurons in the regulation of several aspects of feeding behaviour. We determined the effects of (in)activation of ZI to VTA projection neurons on feeding microstructure, food‐motivated behaviour under a progressive ratio schedule of reinforcement, locomotor activity and core body temperature. To activate or inactivate ZI neurons projecting to the VTA, we used a combination of canine adenovirus‐2 in the VTA, as well as Cre‐dependent designer receptors exclusively activated by designer drugs (DREADD) or tetanus toxin (TetTox) light chain in the ZI. TetTox‐mediated inactivation of ZI to VTA projection neurons reduced food‐motivated behaviour and feeding by reducing meal frequency. Conversely, DREADD‐mediated chemogenetic activation of ZI to VTA projection neurons promoted food‐motivated behaviour and feeding. (In)activation of ZI to VTA projection neurons did not affect locomotor activity or directly regulate core body temperature. Taken together, ZI neurons projecting to the VTA exert bidirectional control overfeeding behaviour. More specifically, activity of ZI to VTA projection neurons facilitate action initiation towards feeding, as reflected in both food‐motivated behaviour and meal initiation, without affecting general activity.

Free vibration analysis of functionally graded magneto-electro-elastic plates with in-plane material heterogeneity

A three dimensional elasticity analysis for the transverse free vibration characteristics of functionally graded magneto-electro-elastic plates based on the scaled boundary finite element method (SBFEM) incorporated with the precise integration algorithm (PIA) is presented. The material properties of magneto-electro-elastic plates are changing along the in-plane direction with arbitrary mathematical functions. In the proposed methodology, the strategies of only discretizing the in-plane surface and utilizing the two-dimensional spectral elements to construct diagonal coefficient matrices are adopted, which contributes to decreasing the calculation effort. The derivation process begins with the three dimensional governing equations of magneto-electro-elastic materials. Neither the plate mechanical kinematics nor invoking assumptions on the spatial distributions of electric and magnetic quantities are adopted. Built upon the introduced scaled boundary coordinates, the principle of virtual work and the technique of dual vectors, a first order ordinary differential SBFEM matrix equation for the in-plane functionally graded magneto-electro-elastic plates is obtained. Its general solution is analytically denoted as the matrix exponent. To improve the computation accuracy of the matrix exponent, the PIA is utilized to form the stiffness matrix. By virtue of the kinetic energy technique, it is convenient to construct the mass matrix of the in-plane functionally graded magneto-electro-elastic plates based on the SBFEM for the first time. Finally, comparisons of flexural frequency parameters with those from exact solutions and other numerical methods are provided. The accuracy, effectiveness, and versatility of the employed technique are validated. Moreover, additional numerical exercises are conducted to exhibit the influences of boundary conditions, material gradation functions, and aspect ratios on the free vibration behaviors of in-plane functionally graded magneto-electro-elastic rectangular, circular, and perforated plates.

Model Predictive Current Control of Dual-Permanent-Magnet Vernier Motor based on Anti-Windup Controller

Dual-permanent-magnet vernier motor incorporates the merits of high torque density and low cost, suitable for low-speed high-torque applications. Due to integral saturation and high current ripples in dual-permanent-magnet vernier motor control system, a model predictive current control strategy based on Anti-Windup PI controller is proposed. Adaptive adjustment variable structure is adopted in integral segment of the controller, which can quickly eliminate integral saturation in system. Mode predictive current control based on dual vector is adopted in current interior loop of the system, double arbitrary voltage vector can be selected to operate simultaneously in each control cycle that extends selection range of voltage vector and adjustment range of amplitude, which can realize more accurate current control. Meanwhile, considering system delay, current delay compensation is added to predictive control. The simulation results showed that proposed control strategy can effectively restrain integral saturation, reduce current ripples, thus improving dynamic and steady state performance of the control system.

Direct matrix converter based on improved quasi‐Z source network

This study proposes a three-phase direct matrix converter (DMC) topology based on an improved quasi-Z source (QZS) network. The proposed topology shows the following features: (i) Improve the voltage transmission ratio of traditional DMC. (ii) The voltage and current on the output side can be affected by the input side to the minimum, which minimises the harmonic content of the output current. (iii) Due to the existence of the improved QZS network, the three bidirectional switches of each output phase bridge arm are turned-on at the same time, which improves the reliability of the system. (iv) The improved QZS network has an input LC filter function, and no additional filter circuit is required on the input side. This study analyses the topological structure of the improved QZS-DMC and elaborates its boost working principle. On this basis, an improved dual space vector modulation strategy with shoot-through zero vector is proposed, which can effectively reduce the loss of the switch tube and improve the output waveform quality of the converter. Simulation and experimental results verify the proposed topology and strategy.

A discrete method for the initialization of semi-discrete optimal transport problem

Semi-discrete optimal transport setting is a very important formulation in the computation of Wasserstein distance, as it is an approximation form of the continuous setting of optimal transport. However, initialization process of dual weight vector for the dual problem in this setting is required for the computation of the first and second order methods, since the Laguerre cells associated to the dual weight vector ought to have positive mass during the calculation. Although there are approaches to initialize the weight vector, systematic computing procedure has not been available. To resolve this problem, we discretize the domain of source distribution of this problem, approximate the Laguerre cells with respect to the weight vector, and finally employ the local perturbation method (Jocelyn, 2019) combined with boundary method to force all measures of the Laguerre cells great than zero. In addition, we study the corresponding theoretical results of the computation process and provide the algorithm, ensuring that the problem can indeed be efficiently computed.

Bending behaviors of the in-plane bidirectional functionally graded piezoelectric material plates

The transverse bending behaviors of in-plane bidirectional functionally graded piezoelectric material (FGPM) plates are semi-analytically investigated by the scaled boundary finite element method (SBFEM) in association with the precise integration method (PIM). The proposed scheme is able to explore the structural characteristics of FGPM plates with the material coefficients obeying arbitrary form of mathematical functions according to the in-plane coordinates. The present methodology selects only four quantities consisting of three translational displacement components and the electric potential as the fundamental unknowns. Additionally, variations of the four primary variables across the thickness direction are expressed as an analytical exponential matrix. In the developed approach, plates are regarded as a kind of three dimensional structure. But, an arbitrary in-plane surface discretized with two-dimensional spectral elements is set as the research domain. The practice is conducive to cut down the computational effort and increase the calculation efficiency. The SBFEM governing equations are formulated from the three-dimensional basic equations of piezoelectric materials without any assumptions on the plate kinematics and distributions of electromechanical components. By means of the scaled boundary coordinate system and the dual vector methodology, the key partial differential equations of piezoelectric materials are conveniently converted into the easily-solved first order ordinary differential SBFEM governing equation. The stiffness matrix is constructed from the analytical exponential matrix aided by the highly accurate PIM to predict the changing patterns of mechanical and electric quantities. To further improve the precision, the technology of dividing the plate into two parts with equal thickness is exploited. Finally, numerical exercises of square, rectangular and triangular piezoelectric plates are provided to validate the accuracy and fast convergence of the developed technique and reveal the effect of geometrical shapes, gradient functions, types of external loadings and thickness-to-span ratios on the static flexure of FGPM plates owning the in-plane bidirectional stiffness.

Analysis and Design Guidelines for Current Control Loops of Grid-Connected Converters Based on Mathematical Models

Having a method for analyzing and designing regulators of controls that contain many current loops such as active filters is not a trivial task. There can be many parameters of regulators and filters that must be carefully selected in order to fulfill certain desired requirements. For instance, these can be stability, dynamic response, robustness under uncertainty of parameters, and rejection capability to switching harmonics. Hence, this paper provides general analysis guidelines for designing current control loops by using mathematical models in an αβ reference frame. Then, by using the proposed modeling tool, a multi-objective tuning algorithm is proposed that helps obtain all the control loops’ regulator and filter parameters, meeting all the desired requirements. Thus, the proposed analysis and design methodology is illustrated by applying it to three different controls conceived in a dq rotating reference frame with PI (Proportional Integral) regulators. The first control presents two current loops (simple dq current control), the second control uses four current loops (dual vector control, for unbalanced loads), while the third control presents eight current loops (active filter controlling current harmonics). Several experimental and simulation results show the effectiveness and usefulness of the proposed method. Since the mathematical model employed is in the αβ reference frame, it can also be easily applied to controls conceived in a αβ reference frame using resonant regulators, providing also a common comparative framework.

Complete phenomenon of reflection at the plane boundary of a dissipative anisotropic elastic medium

SUMMARYA complex slowness vector governs the 3-D propagation of harmonic plane waves in a dissipative elastic medium with general anisotropy. In any sagittal plane, this dual vector is specified with phase direction, propagation velocity and coefficients for attenuation. A generalized reflection phenomenon is illustrated for incidence of inhomogeneous waves at the stress free boundary of the medium. Each reflected wave at the boundary is characterized by its propagation direction, propagation velocity, inhomogeneity, amplitude ratio, phase shift and energy flux. These propagation characteristics are exhibited graphically for a numerical example of anisotropic viscoelastic medium.

Dual Membership Fuzzy Support Vector Machine Algorithm Based on SVDD

In the case of excessive overlap between positive and negative samples in data set, the deviation in the category of reconstructed sample points will lead to unsatisfactory discrimination of SVM, no matter what methods are used to reconstruct the sample set. A dual membership fuzzy support vector machine algorithm based on support vector data domain description was thus proposed, followed by a simulation analysis of common data set. Experimental results show that the proposed algorithm can work well in classification when the sample set is overlapped.

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 (AMD1 tail). To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression governed by 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 assay and western blotting to explore the effects of AMD1 tail and control sequences on reporter 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. The decreased reporter activity that appears to be induced by the readthrough sequence occurs only in reporters containing EMCV IRES. Monocistronic reporters with the same readthrough context sequence exhibit only a modest reduction in reporter activity. Furthermore, in monocistronic vectors, the disproportionate reduction of reporter levels greatly increased when AMD1 tail was translated as a result of readthrough. Such readthrough-mediated reduction was not observed when AMD1 tail was substituted with unrelated sequences in agreement with our original hypothesis. 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.

Convergence of Weak*-Scalarly Integrable Functions

Let (Ω,F,μ) be a complete probability space, E a separable Banach space and E′ the topological dual vector space of E. We present some compactness results in LE′1E, the Banach space of weak*-scalarly integrable E′-valued functions. As well we extend the classical theorem of Komlós to the bounded sequences in LE′1E.

Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing.

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo. We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vivo.

CRISPR-mediated Transfection of Brugia malayi.

The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.

Efficient Boolean Keywords Search over Encrypted Cloud Data in Public Key Setting

Searchable public key encryption- (SPE-) supporting keyword search plays an important role in cloud computing for data confidentiality. The current SPE scheme mainly supports conjunctive or disjunctive keywords search which belongs to very basic query operations. In this paper, we propose an efficient and secure SPE scheme that supports Boolean keywords search, which is more advanced than the conjunctive and disjunctive keywords search. We first develop a keyword conversion method, which can change the index and Boolean keywords query into a group of vectors. Then, through applying a technique so-called dual pairing vector space to encrypt the obtained vectors, we propose a concrete scheme proven to be secure under chosen keyword attack. Finally, we put forward a detailed theoretical and experimental analysis to demonstrate the efficiency of our scheme.

A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation.

Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR-mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.